Decker Genex-ErgänungFA

Diese Fragenausarbeitung wurde von mir nach besten Wissen und
Gewissen verfasst. Trotzdem kann es natürlich sein, dass sich

Fehler eingeschlichen haben. Ich hoffe einigen von euch damit
helfen zu können.
Um Sonnbert zu zitieren:
„If you have paid for this – you have been ripped off“
In diesem Sinne viel Spaß beim Lernen! 
Kleinotti
14.12.2013
Eine ChIP-Analyse zeigt in der Nähe des Transkriptionsstarts eines Gens "X", dass Histone
trimethyliertes Lysin 4 (K4) haben. Was schließen Sie daraus über die Transkription dieses Gens X?
Welche Modifikationen erwarten Sie noch im Promotorbereich?
Histonmodifikationen:
Are found on the N-Termini of histones, as those are long extended (therefore they are accessible for
proteins modifying them), unstructured and rich in a i oa ids that ould e odified → L si e,
Serine, Threonine
Lysine:
 Acetylation
 Methylation
Serin, Threonine:
 Phosphorylation
Histone H3 is the one, that is known to have the largest amount of amino acids modified. H4 carries
the 2nd most modifications, H2B 3rd most and H2A the fewest.
ChIP: Chromatin-Immunoprecipitation:
1) Cross-li k h o ati so that it does ’t fall apa t oss-linker: e.g. formaldehyde)

2) Cut it mechanically by ultra sound: It breaks it (chromatin) into little pieces (the little pieces
hold together due to the cross-link)
The pieces contain:
- Proteins associated with the histone octamere
- Histones (may carrying N-terminal modifications)
3) Use / Add antibodies that recognise a certain histone, if it carries a distinct modification (e.g.
Antibody against H3K4me3)
4) Added A ti od i ds if K e histo es H a e p ese t → I u op e ipitatio → possi le
to isolate the precipitated histones / pieces out of the rest.
There are different ways to read out a ChIP:
1) Isolate the DNA immunoprecipitated -> de-cross-li k p otei s a d DNA → deg ade p otei s.
Amplify DNA by PCR (to do that at least a presumption of the DNA sequence isolated is
necessary, because if not, have no clue which primer-sequence to use).
This is possible if the question wanted to answer is like following: Does promotor of gene XY
contains – under certain conditions – acetylated histons?
2) ChIP-Seq.:
- Precipitate all e.g. K4me3 histons → all DNA bound to K4me3 is precipitated too
- Sequence the whole precipitated DNA (Next Generation Sequencing)
- As esult get: Reads of “e ue es → the o espo d to the DNA se ue e apped
a ou d the K e histo es → use ioi fo ati s to dete i e e e specific region of
the ge o e a d o pa e ith the eads fou d → alig the eads
ChIP analysis showed that Histon (H3) K4me3 modification often correlates with H3K2me (and
H3K36me3). Further it showed that K4me3 and K27me3 correlate with binding of CTCF = a
t a s iptio fa to , o ol ed i fo atio of a ti e h o ati hu s → the efo e I ould a gue that
a p o oto o tai i g a K e odifia tio ould e o elated ith a K e → hi h is ofte
correlated with binding of CTCF leading to formation of an active chromatin hub!
(Reicht das, oder muss man noch mehr auf die chromatin hubs eingehen??)
Beschreiben Sie anhand von STAT, NFkB und beta-Catenin, wie Phosphorylierung die Aktivität
eines Transkriptionsfaktors beeinflussen kann.
JAK-STAT-Pathway:
Note: STAT is the only TF that is activated by Tyrosinkinases

by Tyrosinphosphorylation.
STAT = Signal Transducer and Activator of Transcription
If there comes no signal from extracellular (via a cytokine

binding to its receptor in membrane) STATs are inactive and
not phosphorylated.
If cytokine binds to receptor (cytokines are present because

of immune response) in cell membrane. Binding activates
Jaks (=Janus Kinasen), Tyrosine kinases that are bound to
the intracellulat domain of receptor. Activated JAKs
phospho lates “TAT. → Phospho lated “TATs fo a
dimer (Phospho-Tyr interacts with SH2 domain of another
“TAT ole ule → Di e is i po ted i to u leus, he e it
binds to the promotor of its target genes, stimulating their
transcription.
In this case phosphorylation leads to dimerization, which is

necessary to enter nucleus and stimulate transcription!
NFkB-Pathway:
I po ta t path a i i u e s ste → ithout NFkB path a oi u e espo se ould e

possible!
NFkB… Nu lea Fa to k B, o sisti g of su u its p a d p , hi h is ade as a p e u so =

p105), if its not activates, additional IkB is bound to NFkB.
If ell e ei es a sig al → liga d i ds to
receptor, activating the receptor, which
activates IkB kinase = IkK.
IKK phosphorylates Inhibitor NFkB = IkB,

which is leads to ubiquitination of IkB and
afte a ds to deg adatio of it → NFkB’s
nuclear localisation signal is not covered
a o e IkB.→ e te s u leus →
stimulates transcription of its target
genes.
Phosphorylation leads to degradation of

IkB, which covers the nuclear import
sig al of NFkB → Phospho latio of IkB
leads to import of NFkB into nucleus,
where it then stimulates transcription.
Further IKK phosphorylates CBP (a HAT), which leads to increases activity of NFkB:
Phospho latio of CBP i eases its affi it to NFkB → CBP i ds to NFkB

a d a et lates p → A et latio leads to i eased a ti it of NFkB.
Fu the phospho latio of CBP de ease its affi it to p → phospho -

CBP only binds to NFkB, but NEVER to p53. This is an elegant way, to inhibit
one pathway and activate another one at the same time.
Phosphorylation of CBP leads to increased activity of NFkB → i eases t a s iptio al a ti it .

Wnt-betaCatenin Pathway:
Developmental pathway, regulating cell growth.
Red coloured proteins prevent

a ti atio of the path a →
target genes are not transcribed.
For transcription, TF TCf needs to
be activated: it gets activated,
when it interacts with Smad4
and beta-Catenin.
Red proteins lead to

phosphorylation of beta-catenin,
which then leads to its
ubiquitination and degradation.
In this case phosphorylation

leads to transcriptional
repression.
25.10.2013
Drei TFs nennen (und ein bisschen auf sie eingehen), welche erst durch Interaktion mit anderen
Proteinen aktiv werden/an die DNA binden können. Zwei Methoden nennen, wie man solche
Proteininteraktionen nachweisen kann. (Stichworte)
“TAT → hi h is o l i po ted to u leus, if it is a di e . See above.
MyoD: its activity is regulated by the formation of different heterodimers:
cIf a progenitor muscle cell should

differentiate into a muscle cell, then MyoD
forms a dimer with E12 (another TF). They
interact via their helix-loop-helix domains
forming a coiled-coil. Both contain a basic
region, allowing interaction with the DNA,
stimulating the transcription of genes
necessary for differentiation into a muscle
cell.
If p oge ito should ’t diffe e tiate, M oD

forms a dimer with Id (inhibitor of MyoD),
which contains a helix-loop-helix domain,
ut isses the asi egio → affi it is too
lo → a ’t i d to DNA → ge es e essa fo diffe e tiatio a e ’t t a s i ed.
TGF- eta Path a : = a liga d i di g to its e epto → a ti ates
the Ser-Thr-Kinase-domain of the receptor → phospho lates
Smad-R. Phospho lated “ adR i te a ts ith “ ad → Di e is
allo ed to e te the u leus → i ds a d sti ulates the
transcription of its target genes.
Wnt-Beta-Cate i Path a → see a o e
Methods to detect such interactions:
1) Yeast-2-Hybrid System:
Idea: TF are built up on Modules : They contain one module for interaction with other TF/protein s
= Transactivating Domain (TAD). Further they have modules to stimulate stranscription (this one is
located on the 2nd TF/ Protein, that the one with the TAD interacts with).
Principle: Fuse the TAD of the FF of interest with the DNA-binding domain of yeast TF stimulating
transcription of Gal4 = BAIT DOMAIN! This construct can bind to the promoter region of the reporter
gene. But it alone is not sufficient to stimulate transcription of the reporter gene.
To analyse which TF is additional necessary for stimulation of transcription a so called FISH DOMAIN
is created: Therefore potential interaction partner of the TF of interest are fused with a TAD that
functions in yeast.
Only if the TAD of the TF of interest (=bait domain) interacts with the fish domain (= potential
interaction partner) the reporter gen (Gal4) is transcribed.
Bait and Fish domain are located on 2 different plasmids.
2 ways of doing it:
A) If have already candidates identified → do as des ied a o e

B) If have no clue what the interaction partner cloud be:
Insert bait domain
Use many different fish domains: fuse a DNA li a ith east TAD→ o l those ill grow
that received the right will grow.
2) Proteomic Aproach:
Definiere epigenetische Genregulation. Nenne damit in Verbindung gebrachte Komplexe, welche

aktivierend/reprimierend sein können. Was sind die Hauptauswirkungen oder so... (Stichworte)
Die Histon-Modifikation bzw. das Histon-Remodeling ist ein wichtiger Teil der epigenetischen
Ge egulatio . → Die DNA odie t fü P otei e, die letztli h ei e A t de Regulatio du hfüh e
können, die DNA-Sequenz-u a hä gig ist → ä li h das Ch o ati Re odeling.
Epige etis he Ge egulatio edeutet das Ch o ati zustä de e e a si d → de )usta d, de

durch das Chromatin festgelegt wird, entweder von Mutterzelle auf Tochterzelle oder von
Mutterorganismus auf Tochterorganismus übertragen werden kann.
Die Epigenetik führt dazu, dass bei identischer DNA in unterschiedlichen Zellen und Organen
Unterschiedliche, mitotisch stabile Genexpression erzeugt wird. Daher ist es möglich zu sagen, dass
ursprüngliche Ursache für die Existenz von unterschiedlichen Zelltypen(haben unterschiedliche
Expressionsmuster) in der Epigenetik liegt.
Dabei vererbten Chromatinstruktur werden im wesentlich durch (unterschiedliche) DNA-

Methylierung bestimmt wird.
Grundbegriffe der Epigenetik sind Eu- und Heterochromatin, die Zustände des Chromatins
bezeichnen, die aus heutiger Sicht entweder transkribierbar oder nicht transkribierbar sind.
Au h i diese Fall spiele T a sk iptio sfakto e ei e i htige Rolle → “ie kö e au h das

Chromatin verändern:
 durch die Assoziation mit Komplexen von Histon-modifizierenden oder -demodifizierenden
Enzymen
 in dem sie Histon-Remodeling-Komplexe rekrutieren
 in dem sie Aktivatorkomplexe bilden oder auch Silencing-Komplexe.
Aktivatorkomplexe wirken aktivierend, hingegen Silencing Komplexe Reprimierend
Es gibt zwei wichtige Typen von Komplexen, die die Chromatinstruktur verändern, einerseits die
Chromatin Remodeling-Komplexe, also Komplexe, die unter ATP-Verbrauch Nucleosomen
verschieben oder auflösen können, und andererseits solche Komplexe, die enzymatische Aktivitäten
enthalten und Modifikationen an Histonen vornehmen.
Histonvarianten:
Variante Histone sind ihren „normalen Histonen nahe verwandt (unterscheiden sie sich um ein paar
Aminosäuren):
Histon 3.3 ist dem Histo ah e a dt → e de au h gege ei a de ausgetaus ht. H . ist

esse t a sk i ie a → sei Ei au e folgt i t a sk iptio ell akti e Ge e .
Es gibt aber auch Varianten, die eher mit negativer Genregulation korreliert sind, z.B. die
H2A.ZVariante.
Man findet sie in Promotoren oder Boundary Elementen. Seine Aufgabe ist es, zu verhindern, dass
sich Heterochromatin nicht in die Bereiche von transkriptionell aktiven Genen erstreckt.
Der Austausch von herkömmlichen gegen variante Histone erfolgt durch Chromatin Remodeling
Complexes.
Die varianten Histone haben also Eigenschaften, die mit der biologischen Aktivität des jeweiligen
DNA-Abschnitts korreliert sind und diese unterstützen.
Alle Histone haben gemein, dass ihre N-Termini gut zugänglich sind und Ziel vieler Modifikationen
sind, die wichtig für die Transkriptionskontrolle sind:
 Acetylierung an Lysinen: erhöht die Transkribierbarkeit der DNA, da die positive Ladung des K
du h die Ei füh u g ei e egati gelade e G uppe aufgeho e i d → ka icht mehr
it de e e falls egati gelade e DNA → Auflo ke u g
Durch Histon-A et lt a sfe ase HAT ei gefüh t → Gege spiele = Histo -Deacylasen
Die HATs sind also eindeutig mit positiver Genregulation assoziiert, die HDACs mit negativer
Regulation.
 Die Methylierung von Histonen ist schwer zu interpretieren. Die Konsequenz ist
unterschiedlich, je nachdem, welches Lysin innerhalb eines Histons methyliert wird. H3K4-
Methylierung ist z.B. mit Transkriptionsaktivität korreliert, H3K9-Methylierung mit
transkriptioneller Repression.
Bei der Methylierung von Lysinen kann nicht nur eine Methylgruppe eingeführt werden,
so de is zu → ka also o o-, di- oder trimethyliert sein. Dadurch können sehr viele
unterschiedliche Methylierungsmuster eingeführt werden, die alle unterschiedlich wirken.
 Phosphorylierung von Serinen
 Ubiquitinierung ist eine vergleichsweise seltene Modifikation, die ausschließlich in den
Histonen H2A und H2B vorkommen und dort auch nur an jeweils einem bestimmten Lysin
(H2A: K119, H2B: K120).
12.07.2013
Wie e irkt die Phosphorylieru g die Akti ieru g ei es Tra skriptio sfaktors i NFκB-, wnt- und
Stat- Signaling Pathway?
See above 
Just to mention: in case of Wnt beta- ate i → phospo latio of eta-catenin leads to
ubiquitination, whi h is follo ed it’s deg adatio the p oteaso → as o eta-catenine is
present (due to phosphorylation), it can’t i te a t ith “ ad a d TCF → o o ple is fo ed →
TCF is ot a ti ated → NO transcription is sti ulated → Phosphorylation REPRESSES t a s iptio ‼
Nennen Sie 2 Chromatin-remodellierende Komplexe. Beschreiben Sie die biochemischen

Mechanismen von Chromatin-remodellierenden Komplexen. Woraus beziehen sie die dafür nötige
Energie?
Nennen Sie 2 Chromatin-remodellierende Komplexe. Beschreiben Sie die biochemischen

Mechanismen von Chromatin-remodellierenden Komplexen. Woraus beziehen sie die dafür nötige
Energie?
Chromatin Remodeling Complexes (CRCs) cause changes in the chromatin structure:
 Define Eu- and Heterochromatin

 Move, remove histone-octamers
 Can insert variant histone-molecules: thereby not the whole octamere is changed, but single
histone molecules are replaced
CRCs have a primordial (urspr.) function and are probably as old as histones themselves. CRCs are
o se ed → all important subunits are present in all different CRCs in different species (eg. Human,
yeast, drosophila):
 ATPase domain: All reactions catalysed by CRCs need energy, which is provided by ATP-
hydrolysis.
 Domain for interaction with histones: There are different ones, which recognise differently
modified histones:
- Bromodomains: recognise acetylated histones
- Chromodomains: recognise methylated histones
- SANT domain
- PHD finger
Examples for CRC families:
 SWI/SNIF
 ISWI
 CHD
 INO80
Biochemical activity of CRCs:
Nucleosome SLIDING (a):
Red and blue: TF-binding sites. To make them accessible the DNA needs to be slided relative to the
relative to the histone-octamere. The e the u leoso e is ’t removed; just the DNA wrapped
around it has changed. Red TF binding site is now part of the linker DNA and the blue one is at the
e d of the u eloso al DNA → its a essi ilit is i eased too.
In vivo and in vitro.
Nuclesome TRANSFERE (b):
In a DNA loop one part is wrapped around a histone octamere, while another part is histone –free.
The nucleosome is transferred from one position to the other one. Those TF-binding sites are then
accessible.
Observed in vitro.
ROTATIONAL POSITIONING:
The blue TF bindin site is oriented towards the histo e o ta e e → i a essi le. To make it
accessible, the nucleosome is just moved a bit, so that the TF- i di g site is the o the outside → TF
can bind to it.
CRCs bind around DNA of nucleosome: 1st CRC translocates itself: it moves itself bit for bit, thereby
loosening the DNA (this happe s e ause du i g o i g, CRC is still ou d to DNA. → pa tial
u appi g of histo e o ta e e→ p o ess goes o a d o it pe a e tel eeds e e g fo
o e e t . A ulge “Chleife is ge e ated → e t the histo e o ta e e is o ed i to the ulge
and the DNA tightened around it.
(or in brief: CRCs use ATP (hydrolysis) to displace DNA from histone octamere, thereby creating a
ulge. The histo e o ta e e is o ed i side the ulge a d DNA gets apped a ou d it losel . →
Nucleosome position has changed.
Activity of CRCs is influenced by TFs!
 TFs cause modifications of histones, allowing CRCs to bind.

 TFs can interact directly with CRCs
→ TFs a ause i se tio , epositio i g of u leoso es i o de to a ti ate o ep ess t a s iptio

of certain DNA regions
22.03.2015
Wel he der folge de A t orte ist ri htig a kreuze → hier fett arkiert .
1) Der Initiationskomplex der Transkription besteht aus Polymerase-associated factors (PAFs)

(Transkriptionsfaktoren, bilden den Initiationskomplex der Transkription)
2) Die Reihenfolge mit der generelle (basale) Transkriptionsfaktoren an einen Promoter
binden wurde im electrophoretic mobility shift assay (EMSA) ermittelt.
3) Aktivierte Promotoren enthalten Nukleosomen mit K4-trimethyliertem H3
4) Histonacetylierung inhibiert die Bindung von transkriptionellen Repressoren
5) TAFs sind Bestandteile des TFIID
6) TFIIH enthält eine Histonacetyltransferase
7) TBP reguliert Promotoren für alle 3 eukaryoten RNA Polymerasen
8) TFIIB verbindet TFIID und RNA Polymerase
9) TFIIB erleichtert die Strangtrennung bei der Bildung eines offenen Promoterkomplexes
10) Phosphorylierung der Polymerase II CTD ist für die mRNA capping Reaktion wichtig
Nennen Sie 2 Beispiele von chromatin remodelling Komplexen. Welche biochem. Aktivität kann
diesen Komplexen zugeordnet werden? Woher kommt die Energie für das remodelling?
See above
25.01.2013
Ja/Nein Fragen zu den Themen:
EMSA
Shows specific binding of an TF to DNA or specific

binding of a protein to a TF bound to DNA:
DNA fragment (restriction fragment,

oligonucleotide) end labelled with P32 is mixed
with an extract containg the Protein (TF) that is
thought to bind to it. Then separate it by gel-
ele t opho esis → a s a e dete ta le ause of
the P la eli g → use X- a fil ! → Co pa iso
with sample containg only DNA: If the proteins
has bound to DNA, it will be retarded, because of
its gain in size = electrophoretic mobility shift
Nuclear Run On Assay

Is used to analyse whether a certain gene (e.g. A) is transcribed (more or less) if a certain cell type is
put on a defined stimulus: Think that gene a is transcribed with a higher transcription rate ( the
transcription rate is defined as the number of RNA-Pol molecules, which are transcribing one gene at
the same time) in liver cells under lysine starvation (e.g.).
Afte isolatio , u lei a e put o i e → f eezi g stops RNA-pol, but they remain bound to DNA. Add
P la eled u leotides a d put the app oa h u de a o ditio s → RNA-Pols finish
t a s iptio → i se t P la elled NT i g o ing mRNA. mRNA is isolated and hybridized to filter
containing to A complementary oligo- t → RNA of i ds to it → use a X a filte to dete t the
sig al → the o e RNA of A is p ese t, the igge a d da e the spotted dot ill e.
Nuclear run on assay is a method, that allow analysis of some genes, but not possible to analyse a
hole ge o e ith it → eeded to e de eloped → Ge o i Ru o “e ue ing = Gro-Seq: The
whole mRNA associated with chromatin (= this includes every pre-mRNA that is transcribed at the
poi t of isolatio is isolated → add la elled Nt. → h idize it ith hip → us e ioi fo ati s fo
evaluation = transcription rate of every gene transcribed under a certain condition. It is a true
transcriptome anaylsis, as it seperates RNA stabilit , p o essi g,… .
Mit ChIP sind basenspezifische Kontakte nachweisbar
Mit ChIP-Seq ist spez. RNA-Spezies nachweisbar
ChIP: Chromatin Immuno-Preciptiation:
To determine, to which chromatin sequence a TF is binding:

Transkriptionsfaktoren haben unterschiedliche funktionelle Domänen
Bei der Primer Extension Methode findet exponentielle Amplifikation statt etc:
Pimer extension and S1

mapping are methodes
to determine the
transcription start of
certain gene.
If sequence of an mRNA
is known, but it is ’t
sure, that this mRNA is
e ti e → the efo e ot
sure if mRNA contains
the trsnciption start:
A short sequence
complementray to
mRNA is used as a
p i e → RT is added →
reverse transcription
u til the ’e d =
transcription start.
S1-nuclease mapping:
Needs DNA, that not only includes transcriptio sta t, ut the p o oto too.→ Ha e lo g pie e of
DNA and know that somewhere in this sequence the transcription start and promotor are located.
Use RNA a d h idize it to the DNA pie e → RNA-DNA h id → Use S1 nuclease to digest all
single stranded RNA/DNA. Only double-stranded sequenc e is protected. Use it to determine length
and transcription start.
Definieren Sie die positive und negative Kontrolle der Genexpression. Geben sie dies bezügl.
Beispiele und Skizzen eubakterieller Operone.
Negative regulation: a gene is under basal/ normal conditions not transcribed, due to binding of a
transcriptional repressor to the transcription start. To allow transcription an inducer is necessary:
Inducer binds to represso , leadi g to a o fo atio al ha ge → a ’t i d to DNA a oe
(allosteric effect).
Positive Regulation: a gene that is under basal / normal conditions not expressed, but has no
repressor bound. For transcription an activator is necessary.
Example: La Ope o → it is egative and positive regulated!
In basal state a repressor is bound to its transcriptional start. First have to remove the repressor, but
anyhow removal of lac-repressor is not sufficient to stimulate transcription:
Structure of lac-operon:
 Promoter
 Operators (O1, O2, O3)
 CAP-binding site
 Coding sequence (LacZ, 15acy, lacA)
Lac-repressor is encoded by lacI gene, which is always expressed. LacI binds to operator of lac
operon: it binds as a dimer to a palindromic sequence. Lactose is the inducer → if lactose is present
in cells, it i ds to ep esso → ha ges its o fo atio a ’t bind to operator anymore. Repression
is maximal if O1 and O2 or O3 are bound by repressor molecules. Binding of lac-repressor prevents
building of an open promotor conformation.
07.12.2012
10 Ja/Nein Fragen, u.a:
1) H3.3 findet man in transkriptiv aktiven Regionen

2) codierende DNA ist immer nukleosomfrei
3) Chromatin Remodelling Proteins brauchen ATP
4) Lac-Repressor formt Repressionsloop → CAP forms the loop structure: in absence of
glucose and lactose, CAP binds to CAP-binding site and allows formation of an loop, which
i gst he ope ato s i lose o ta t → sta le o fo atio → respression = max!)
5) Chromatinimmunpräzipitation ist ein Weg um Transkriptionseffizienz nachzuweisen
6) Topoisomerasen bestimmen die Twist-Zahl
Die Bedeutung von sigma-Faktoren erklären und wie würden Sie nachweisen, dass ein Gen von
sigma70 reguliert wird.
Depending on presence or absence of a sigma factor, the RNA-Pol is distingushed in the Holoenzyme
(with sigma factor bound) or in the coreenzyme (w/o Sigma factor). Holo- and Coreenzyme have
different abilities:
Coreenzyme can bind to DNA via diffusion, but to distinguish between a random DNA sequence and a
promotor, precence of sigma factor is necessary: Holoenzymes have a higher affinity to promotor
sequneces than to random sequences. Transcription itself and termination are done by the
Co ee z e → “ig a factor only necessary for finfing of a promotor.
Sigma factors have the ability to detect promotor sequnces, as they differ from other DNA sequnces:
In E.coli promotor sequnces contain specific consensus sequnces:
 -35 region: TG rich

 (extended) -10 region: TATA box
2 ways to determine, that -35 and -10 region define a promotor:
1. Site-directed mutagenesis: mutate a promotor of interest or search for natural mutations of

it → o pa e a ou t of ge e p odu t of a ge e of i te est et ee WT a d the o e ith
the mutated promotor.
2. DNA-Footprinting: use DNA, that is thought to o tai a p o oto → add RNA-Pol → the
add he i als that odif the DNA → see hi h pa ts of the DNA a e ot odif led →
protected because RNA-Pol was bound to them. Instead of using chemical that modify the
u ou d Nt. Use DNase → ill digest DNA that is u p ote ted e ause o RNA-Pol is
bound to it).
Add DNase 1 in limited amounts (so much, that stochastically every DNA-molecule is cut one)
→ sepa atio ia gel ele t opho esis. I the sample that not only contains DNA and DNase1
(and RNA-Pol o o ti ual ladde ill e di eta le → the e ill e a window where DNase
ould ’t ut, e ause RNA-Pol was bound. = Footprint
Sequence the bars, that are missing the samples containg RNA-Pol.
→ iologi al a d io he i all proofed that those regions defined a promotor in eubacteria.
Different sigma factors stimulate transcription of different target genes:
 “ig a → house keepi g ge es

 “ig a → heat sho k ge es
 “ig a → o ilit a d hemotaxis
To anayse whether a gene is regulated by a Holoenzyme containing sigma 70, I would do an in vitro
experiment: isolate DNA sequence conating the gene / operon of interest, RNA-Pol a d sig a → if
the gene / opern of interest is expressed it is regulated by sigma 70. As control use 2 additional
conditions:
 W/o any sigma factor (negative control)

 With another sigma factor (eg. Sigma 32)
19.1.2012
10 Aussagen zum Ankreuzen wenn es zutrifft. Es ging um:
Serin 2
Serin 5
TFIIB, Chromatin
sigma-Faktor
CTD
Welche Strukturen der TF für die Bindung an DNA benötigt werden, Charakteristika, je ein Beispiel
1. BASIC DOMAINS:
a) Helix-loop-helix motif:
form dimers via interaction of amphipathic helices
→ fo oiled-coil structures. The amphipathic
helices, which interact, are separated by a loop
structure.
The helix-loop-helix domain is coupled with a basic
region for DNA interaction.
E.g.: MyoD
b) Leucin zippers:
Again from coiled-coils via interaction of amphiphatic
helices, but the dimerization domains are not
separated by a loop structure: the helices contain a
la ge a ou t of Leu i egula i te als → i te a t
due to entropic reasons. 2nd region of a leucine zipper
is a basic region for interaction with the DNA: contains
many amino acids that are positive charged under
physiological conditions.
E.g.: CREB
2. ZINK FINGER:
Are composed of 1 alpha-helix and 2 beta-sheets. The helix contains a histidine at a critical
position, while the sheets contain critical Cys- residues. They form the zink finger, by
coordinating a Zn2+, which
stabilizes the structure.
a) C2H2 Zn-finger: 3 or more
fingers bind DNA as
monomers (rare)
b) C4: 2 fingers, that form a
dimer (common)
3. HELIX-TURN-HELIX:
Helix 1 and 2 are seperated by a
st u tu e alled tu → it ha ges
the orientation of the helix (from up
to down or vice versa). Helix 3 =
e og itio heli → i se ts i to the
major groove of DNA via specific
interactions. Further the N-terminus
of (next to helix 1) contacts the
adjacent minor groove.
E.g.: LacI, Trp-Repressor
a) homeo domain
b) paired box
c) Fork head
d) Tryptophane cluster
4. Beta-scaffolds:
Co ta t the i o g oo e of DNA a e → ajo it i te a ts ith ajo g oo e .
E.g.: STAT1, p53, TBPs
07.07.2012
Richtig-Falsch Fragen (nur so ungefähr, hab fast alle schon wieder vergessen, war aber viel zu
epigenetik und modifikationen und so):
1) H3K27 Trimethylierung ist wichtig für epigenetische Genregulation

2) CTCF erkennt Histonmethylierungen
3) Polycomb Komplexe haben Kinasefunktion Nei → U i uit ie e
bHLH Proteine können als inaktive und aktive Dimer wirken. Erklären Sie die strukturellen und
mechanistischen Hintergründe. + Beispiel
HLH… asi Heli -Loop-Helix
dimerisaion occurs via the helix-loop-helix

do ai → fo atio of a oiled oli
Example: transcription factor MyoD
Is necessary activate / represse

differentiation of progeny muscle cells into
us le ells → if he sti ulates
differentiation or not, despends on the 2nd
protein MyoD interacts with:
If M oD fo s a di e ith E → a othe
TF, containing a HLH domain for dimerization
a d a asi egio → o tai s a lot of a i o a ids ith a positi e ha ge → DNA is egati el ha ge
due to the phosphate- a k o e → ele t ostati i te a tio , leads to i di g of M oD-E12 dimer to
DNA → sti ulates t a s iptio of ge es ecessary for differentiation into muscle cells.
If no further muscle cells are necessary > progenitor cells should stay progenitors. To realise this
(MyoD is permanently present) it forms a dimer with Id (inhibitor of MyoD), which contains of course
a HLH do ai → /o it di e izatio ould ’t e possi le , ut la ks the asi egio → o l o e
asi egio the of M oD is ot suffi ie t fo i te a tio ith DNA → M oD-Id di e a ’t i d to
the t a s iptio sta t of ge es e essa fo diffe e tiatio → the o ’t e t a s i ed.
Alles noch etwas genauer erklären‼
13.01.2012
10 Richtig-Falsch-Fragen zB:
1) Y2H als Nachweis für Bindung des Transkriptionsfaktors an seine DNA Sequenz
(Eher ChIP???, EMSA????)
2) Realtime PCR ist eine semiquantitative Methode zur RNA Bestimmung
(naja wenn ich sie zuerst in cDNA umschreibe?? :/ ander frage: ist die rt PCR semquantitativ
oder quantitative^^)
3) Phylogenetisches Genomscreening für Transkriptionsfaktor-Bindesequenzen bei
unbekannten Promoter
4) DNA-Microarray ermöglicht Expressionskontrolle mehrerer Gene gleichzeitig
5) Sigma-faktor
In Stichworten beschreiben: Aufbau und Funktion von TFIID, TFIIH und Mediatorkomplex
TFIID: large complex of 12 subunits (1x TBP = TATA-binding protein, 11 – 14x TAFs = TBP Assosiated
factors). It is the 1st general TF binding to promotor of a gene that should be transcribed! From all
basal TF, necessary for formation of the (pre-) initiaton complex, it has the largest regions for
interaction with DNA. Further it provides a large surface, for
binding of additional / different TFs. TFIID therefore contain
several different transactivating domains (TADs), allowing
TFIID interaction with various TFs, mediator complexes, co-
a ti ato s,… e.g. such that recruit HATs.
TFIIH: general TF containg 9 subunits, part of the IC. It

contains a kinase domain: Cdk7, that is regulated by cyclin T.
It phosphorylates the CTD of RNA-Pol II at serine 5, leading to
promotor clearance. Is has a core, containg XPD, XPB (named
afte the disease the a e i ol ed i → utatio s i those
Subs lead to Xenodermia), p44, p34, p62, p52. Further it can associate with additional subunits.
Depending on the subs TFIIH is important for promotor clearance or DNA repair. If it associates with
Cdk a d li T → p o oto lea a e due to phospho latio of “e i e at CTD of RNA – Pol =
HoloTFIIH. For DNA repair, associates with repairs proteins.
Mediator: large complex of several subunits → it p o ides a la ge su fa e, for binding of different TFs
→ the efo e o tai se e al diffe e t t a sa ti ati g do ai s TADs , allo i g i te a tio ith
a ious TFs, e.g. su h that e uit HATs. Most TFs do ot o ta t the i itiatio o ple di e tl →
they interact with the mediator complex, which then interacts with the IC. → Mediato ediates the
interaction between TFs binding somewhere up- or downstream of the transcription start and the IC.
Via binding to mediator, it allows TFs to
communicate with the IC.
Important: the mediator does not bind

directly to DNA only to other proteins! It
does help recruiting the RNA-Pol, but
does not physically link the RNA-Pol to the
Mediator complexes do not always

o tai the sa e su u its → so e a e
always present, some not:
The Core-Mediator contains:
 Head domain
 Tail domain
 Middle domain
Additional:
 MED1/MED26
 Kinase-modules: contains CdKs and Cyclin
Note: it was found that the core-mediator and the kinase-module are able to function w/o each
other.
26.11.2011
Mc Fragen 10 stück:
TEFIIb bewirkt die phosphorylierung von CTD. (Achtung, das war eine falle TEFIIb war falsch
geschrieben )
3 Pathways wo die Phosporylierung eine rolle spielt. Dazu den mechanismus des Pathway
ausschreiben
See above 
e.g.: Stat-Pathway, NFkB Pathway, TNF-beta Pathway, Wnt Pathway
08.10.2011
Ca. 10 Aussagen (vor allem Nachweismethoden); welche davon treffen zu? (ohne Begründung, nur
ja oder nein)
Proteinkomplexe die bei epigenetischer Modifikation eine Rolle spielen + Hintergrund dazu
HISTONE MODIFICATIONS:
Histone Acetyltransferases (HATs): add acetylgroups to Lysine residues of N-termini of histones.

The e eut alizi g the positi e ha ge of l s → eake s the i te a tio et ee histo es a d DNA
→ DNA e o es o e accessible. → Acetylation of histones is the only modification of histones, that
exclusively correlates with transcriptional activity.
Histon-acetylation is reversible!
Examples: SAGA → GCN , P300/CPB → P /CPB
Histon Deacetylasen (HDACs): e o e a etl g oups f o histo es → o e ts the h o ati i to a

transcriptionally more unfriendly state.
E a ples: NuRD → HDAC &
Kinases: Phosphorylation of histones is possible at serine and threonine residues.
Phosphatases remove the phosphastes again
Histon Methyltranserases: eth late a gi i s, l si e → depe di g o the esidue, o o-, di- and
trimethylations are possible. Methylgroups do not neutralize the charge of lysine.
Examples: SUV39
The interpretation of histone modifications is difficult: depending on the modification of which
histone at which position and depending on the other present modifications, they can either lead to
transcriptional activation or repression.
This is calles the HISTONE CODE. There are proteins, that have the ability of reading and interpreting
it = READERS and such that can add modifications = WRITERS. Some proteins have both abilities.
For reading/ recognition of distinct modifications certain domains are necessary:
 Bromodomain: Acetylations
 Chromodomain: Methylations
 SANT domain
 PHD fingers
The most modifications are found in H3, second most in H4, 3rd most in H2B and the fewest in H2A.
DNA METHYLATION:
Correlates with silencing of the region: DNA o tai s so alled CpG isla ds → ajo it of CpG isla ds
is methylated in mammals.
DNA methylation occurs during genomic imprinting, X-i a ti atio . → a e i po ta t fo sta le

silencing of retrotransposons, retroviruses and transgenes. Rarely used for reversible gene silencing.
It is necessary to distinguish between De nuovo Methylasen (DNMT3a/b) and Maintenance

Methylasen. (DNMT1).
Maintenance Mathylasen: During replication the methylation state of the DNA needs to be
transferred to the daughter cells. → the t a sfe e the –CH3 pattern to the new strand. Knock out of
DNMT is lethal → o –Ch3 patterns cloud be transferred
De nuovo Methylasen: in germ line the –Ch patte s a e e ased → f o ge ell a e o ga is

needs to be created: Needs to build up all t pes of ells → e l i se t –Ch3 patterns w/o a
template. → othe sig als e essa to i di ate hi h C should e eth lated. A e esse tial → /o
→ lethal lead to totall dis upted/ o g i p i ti g, i a ti atio , et ot a sposo s ju p too u h,
…
No eth lase a s oth fu tio s → eithe ai te a e o de ou o → use totall diffe e t
mechanisms.
Reicht das so??
01.07.2011
Falsch - Richtig über TF, bzw. Transkriptionsregulation
Writers - Readers und der Effekt
Writers = Histone modifying enzymes: They introduce the histone code: The histone code is the sum
of all histone modifications. It determines the biological activity of a certain DNA region.
E.g.: HATs, Kinases, Histone methylases
Readers = Proteins recognising the histone modifications: They are able to interact with modified
histo es → have to carry a domain that allows the interaction specific interaction between a distinct
modified histone and the reader. There are different domains that recognise different modified
histones:
 Ch o odo ai → CH
 B o odo ai → A et l
 SANT domain
 PHD finger
Biological output:
Every reader correlates with a specific biological response:
 Transcriptional activation
 -,,- repression
 Gene silencing
 Elongation
 Behaviour during mitosis / meiosis
 …
Further there are protein complexes that contain both – a reader and a writer domain. They
recognise certain modifications and introduce further ones (if the already excisting ones →
introduced by other writers, encode for further modifications). SAGA complex whi h is a HAT →
writer), contains a bromodo ai → o l i ds a d a et lastes if al ead so e spe ifi acetylations
are present.
NuRD →HDAC also o tai s a h o odo ai → e og ises eth latio s.
If a writer does not contain a reader domain, it can be brought to the DNA by TFs: TFs thereby
indirect influence the activity of a DNA region.
11.12.2010
Beschreiben Sie die CTD der eukaryoten RNA Pol 2. Welche AA werden bei der Initiation der
Transkription phosph.? Was ist die Bedeutung der Phosph.?
The carboxy-terminal domain of RNA polymerase II typically consists of up to 52 repeats (humans) of

the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Proteins often bind the C-terminal domain of RNA
polymerase in order to activate and regulate RNA-polymerase activity. It is the protein domain that is
involved in the initiation of transcription, the capping of the RNA transcript, and attachment to the
spliceosome for RNA splicing → se es as a platfo , that all p otei s taki g pa t i the e tio ed
processes can bind to.
Regulation of the transcription cycle occurs via phosphorylation of the CTD: Either serine 5 or serine
2 is phosphorylated (meaning that, then the majority of all serines at position 5/2 in the heptade
repeats are phosphorylated). For promotor clearance (RNA-Pol II transcribes some Nt., then stopps
again) phosphorylation of ser5 is necessary. This phosphorylations is introduced by TFIIH, which
o tai s a ki ase su u it → Cdk , egulated li T . Du i g pausi g afte t a s iptio of
some Nt.) the capping reaction occurs, leading to a higher stability of the newly transcribed mRNA.
The proteins performing it are bound to the CTD!: Then to let RNA-Pol 2 know, that capping reaction
is finished and elongation possible, P-TEFb phosphorylates serine 2, which causes the RNA-pol2 to
move on and elongate. P-TEFb contains cdk9, regulated by cyclin C
→ Phospho latio is e essa to egulate t a s iptio a d RNA p o essi g.
Welches Prot. muss stabilisiert werden, damit Signaltransduktion durch Wnt Liganden zu einer
transkriptionellen Antwort führt? Nach welchem Mechanismus erfolgt die Stabilisierung?
See above 
Beta catenin must be stabilized: to activate TF TCF, it must form a complex with Smad4 and beta-
catenin. The pathway consists of counteracting protein complexes: Some want to stabilize it, others
lead to its degradation: for degradation beta catenin 1st gets phosphorylated, then ubiquitinated and
then degraded by the proteasome. Those who want to protect beta-catenin inhibit the kinase
phosphorylating it, if they get a certain stimulus.
Mechanism? By prevention of the phosphorylation (which in this case leads to degradation), beta
catenin gets stabilized.
28.09.2009
Wie ist das Nukleosom aufgebaut (Skizze)! Durch welche Komplexe werden sie bewegt und durch
welche chemischen Veränderungen wird das bewirkt?
Contains a histone core and DNA wrapped around it:
Synthesis of histone core: Is a regulated process, always happening in the same order:
 Dimerization of H3&H4
 H3-H4 dimer interacts with DNA
 2nd H3-H4 dimer is formed and included in the structure. = Tetrasome
 1st H2A-H2B dimer is added = Hexasome
 2nd H2A-H2B dimer is added = Octamere / Nucleosome
Asse l p o ess does ’t o u a do a d spo ta eousl , ut eeds p e e e of p otei s =

Histone Chaperons. For example FACT (a histone chaperone) removes one dimer of H2A-H2B,
thereby making transcription possible.
Around each histone core 146bp of DNA are wrapped. Between two nucleosomes a 20 – 60 nt long
DNA sequence is found, which could be bound by histone 1 = linker. Binding of H1 causes a more
o pa t st u tu e → fi e .
Histones mainly contain alpha helices (histone fold) and long extended, unstructured N-termini
containing many amino acids that could be modified (by histone modifiers = writers). Further
histo es o tai a la ge u e of positi e ha ged a i o a ids → e essa fo i te a tio ith the
DNA → ele t ostati i te a tio !
During replication and transcription histone chaperones are needed too: To make both processes
possible, one H2A-H2B dimer needs to be removed.
Movement of histones:
They are moved by Chromatin Remodeling complexes – ATP hydrolysis is necessary to provide
enough energy for it. It is o l possi le if the i te a tio et ee histo es a d DNA is eake ed →
need acetylation of histones, as it neutralizes the positive charge of Ly-residues.
Chromatin Remodeling Complexes (CRCs) cause changes in the chromatin structure:
 Define Eu- and Heterochromatin

 Move, remove histone-octamers
 Can insert variant histone-molecules: thereby not the whole octamere is changed, but single
histone molecules are replaced
CRCs have a primordial (urspr.) function and are probably as old as histones themselves. CRCs are
o se ed → all i po ta t su u its a e p ese t i all diffe e t CRCs i diffe e t spe ies eg. Hu a ,
yeast, drosophila):
 ATPase domain: All reactions catalysed by CRCs need energy, which is provided by ATP-
hydrolysis.
 Domain for interaction with histones: There are different ones, which recognise differently
modified histones:
- Bromodomains: recognise acetylated histones
- Chromodomains: recognise methylated histones
- SANT domain
- PHD finger
Examples for CRC families:
 SWI/SNIF
 ISWI
 CHD
 INO80
Biochemical activity of CRCs:
Nucleosome SLIDING (a):
Red and blue: TF-binding sites. To make them accessible the DNA needs to be slided relative to the
relative to the histone-o ta e e. The e the u leoso e is ’t e o ed; just the DNA apped
around it has changed. Red TF binding site is now part of the linker DNA and the blue one is at the
e d of the u eloso al DNA → its a essi ilit is i eased too.
In vivo and in vitro.

Nuclesome TRANSFERE (b):
In a DNA loop one part is wrapped around a histone octamere, while another part is histone –free.
The nucleosome is transferred from one position to the other one. Those TF-binding sites are then
accessible.
Observed in vitro.
ROTATIONAL POSITIONING:
The blue TF bindin site is oriented towards the histo e o ta e e → i a essi le. To ake it
accessible, the nucleosome is just moved a bit, so that the TF- i di g site is the o the outside → TF
can bind to it.
CRCs bind around DNA of nucleosome: 1st CRC translocates itself: it moves itself bit for bit, thereby
loose i g the DNA this happe s e ause du i g o i g, CRC is still ou d to DNA. → pa tial
u appi g of histo e o ta e e→ p o ess goes o a d o it pe a e tel eeds e e g fo
o e e t . A ulge “Chleife is ge e ated → e t the histone octamere is moved into the bulge
and the DNA tightened around it.
(or in brief: CRCs use ATP (hydrolysis) to displace DNA from histone octamere, thereby creating a
bulge. The histone octamere is moved inside the bulge and DNA gets wrapped around it closel . →
Nucleosome position has changed.
Activity of CRCs is influenced by TFs!
 TFs cause modifications of histones, allowing CRCs to bind.

 TFs can interact directly with CRCs
→ TFs a ause i se tio , epositio i g of u leoso es i o de to a ti ate o ep ess t a s iptio

of certain DNA regions
Welche Methoden um mRNA quantitativ nachzuweisen kennen Sie? Welche liefern exakte
quantitivative Werte?
19.09.2008
Was ist ein Nucleosom und was ist seine wichtigste Aufgabe in Bezug auf die Genregulation?
Welche Modifiktaionen beeinflussen die Genregulation?
See above 
Variante histones: are closely related to the basic form , but differ in some amino acids. Those
exchanges lead to the specialized fuctions of variant histones. H3.3 for example is found in
nucleosomes that are transcriptional active. H2AZ is found in nucleosomes of promotors or boundary
elements (= important DNA regions separating eu- and heterochromatin.
Wie beeinflusst die CTD Phosphorylierung den Transkriptionszyklus von RNA Pol II?
See above  it regulates the transcriptional process.
31.05.2008
Welche DNA Sequenzen sind wichtig für epigenetische Regulation + kurze Beschreibung
CpG islands: → ajo it of CpG islands is methylated in mammals.
DNA methylation occurs during genomic imprinting, X-i a ti atio . → a e i po ta t fo sta le

silencing of retrotransposons, retroviruses and transgenes. Rarely used for reversible gene silencing.
It is necessary to distinguish between De nuovo Methylasen (DNMT3a/b) and Maintenance

Methylasen. (DNMT1).
Maintenance Mathylasen: During replication the methylation state of the DNA needs to be
t a sfe ed to the daughte ells. → the t a sfe e the –CH3 pattern to the new strand. Knock out of
DNMT is lethal → o –Ch3 patterns cloud be transferred
De nuovo Methylasen: in germ line the –Ch patte s a e e ased → f o ge ell a e o ga is
eeds to e eated: Needs to uild up all t pes of ells → e ly insert –Ch3 patterns w/o a
te plate. → othe sig als e essa to i di ate hi h C should e eth lated. A e esse tial → /o
→ lethal lead to totall dis upted/ o g i p i ti g, i a ti atio , et ot a sposo s ju p too u h,
…
No methylase carrys both fu tio s → eithe ai te a e o de ou o → use totall diffe e t

mechanisms.
BOUNDARY ELEMENTS: define boarders between eu- and heterochromatin
Insulators: define chromosomal domains → i sulato s a e e essa to disti guish et ee

transcribale regions and not transcribable regions. Note! Not all genes in a region that is
transcribable are really transcribed, but (if needed) they could be transcribed.
Locus control regions: they define which genes (in transcribable regions) are actually transcribed.
Reicht das so??
Genomic imprinting:
Imprinted genes, are genes their

products somehow correlate with
cell growth and differentiation.
Silenced loci carry many

methylations. Imprinted genes are
regulated by Imprinting Control
regions (ICRs). ICRs are binding sites
for CTCF (a TF necessary for long
range interactions). Binding of CTCF
depends on the methylation state of
ICR → CTCF o l i ds, if ICR is NOT
methylated! Binding of CTCF leads to
interaction with an enhancer that
stimulates transcription of H1 →
chromatin hub is formed) and it
prevents transcription of Igf2.
(maternal).
If CTCF cant bind to ICR due to

methylation of ICR, enhancer acts to stimulate transcription of Igf2. (paternal)
DNA methylation determines, whether CTCF can bind or not. CTCF determines (by binding or not),
which part of the region is transcried and which will be stable silenced by formation of
heterochromatin.
All 3 genes are expressed maternally and not paternally. In paternal genome in return a long non
coding RNA = Air expressed, which regulates transcription.
If Air is expressed:
1. Promotor occlusion occurs: Igf2R and Air compete for all factor necessary to initiate
t a s iptio → i this ase Ai wins → all fa to s i d to its p o oto → t a s i ed
and Ifg2R not.
2. It recruits Histon Methylases that convert Sic22a3 and Sic22a2 into heterochromatin.
As today its known how imprinting works, it is possible to create bi- ate al o ga is s → i e :
Need one imprinted mature maternal genome and one that lacks those modificatio s → i i s
paternal genome.
Welche Methoden um mRNA quatitativ nachzuweisen kennen Sie? Welche liefern exakte
quantitative Werte? Kann man damit Transkription nachweisen (+Begründung)?
29.02.2008
Was bewirkt die Modifizierung der CTD der RNA-PolII während der Transkription?
See above 
Welche Unterschiede bei Steroidrezeptoren gibt es...
1. ...hinsichtlich der Bindung an die DNA?

2. ...hinsichtlich der Lokalisation in der Zelle?
3. Wie kann ein Ligand so einen Rezeptor aktivieren (bzw. was passiert durch den Liganden)?
Overview Nuclear hormone receptors:
Steroid receptors are nuclear hormone receptors. Which associate with glucocorticoids/cortisol (GR)
o se ho o es ER; PR, AR, …
Ad 1:
Nuclear hormone receptors can either bind to:
 Inverted repeats (= palindromes): Nuclear Hormone

Receptors forming homodimers bind to palindromes (e.g.
GR, ER)
 Direct repeats: are bound by nuclear hormone receptors
forming heterodimers (e.g.: TR/RXR, RAR/RXR)
→ steroid receptors (GR, ER) form homodimers, therefore bind to

palindromes.
Ad 2:
GR: in absence of glucocorticoid GR is found in the cytoplasm. It is

bound by Hsp90, which inhibits GRs import into nucleus (??). In
presence of Glucorcorticoid (it is a cholesterol derivate, therefore it
can pass th ough the ell e a e it i ds to GR → o fo atio al
ha ge → Hsp a ’t i d a o e → i po t i to u leus. There GR
interacts with a co-activator, leading to transcription of its target
genes.
Verstehst du diese Folie???
24.11.2007
mindestens 2 methoden zum nachweis der physischen interaktion zweier TF, 1 methode um zu
beweisen dass der eine TF wichtig ist für die initiation der transkription
2 methodes to determine whether 2 TFs physically interact with each other:
1) Yeast-2-Hybrid System:
= Transactivating Domain (TAD). Further they have modules to stimulate stranscription (this one is
Principle: Fuse the TAD of the FF of interest with the DNA-binding domain of yeast TF stimulating
transcription of Gal4 = BAIT DOMAIN! This construct can bind to the promoter region of the reporter
gene. But it alone is not sufficient to stimulate transcription of the reporter gene.
To analyse which TF is additional necessary for stimulation of transcription a so called FISH DOMAIN
is created: Therefore potential interaction partner of the TF of interest are fused with a TAD that
functions in yeast.
2 ways of doing it:
C) If ha e al ead a didates ide tified → do as des ied above

D) If have no clue what the interaction partner cloud be:
Insert bait domain
Use a diffe e t fish do ai s: fuse a DNA li a ith east TAD→ o l those ill g o
that received the right will grow.
2) Proteomic Aproach:
One method to analyse whether a TF is part of the initiation complex:
Binding of TF to transcription start cloud be detected by EMSA, Footprinting.
To dete i e hethe p ese e of a TF is e essa to sti ulate t a s iptio → epo te ge e assa

ith TF a d /o → k o k out or mutated) and compare between both.
genomic imprinting (allgemein gefragt, was ist das etc.)
is necessary because only presence of 2 genomes is not sufficient to develop into a whole organism.
→ ate al a d pate al a e e essa → the ust so eho diffe → due to imprinting,
meaning that the a e diffe e tiall odified = i p i t → so e ge es a e o l ate al and some
o l pate al e p essed → esse tial fo de elop e t into a functional organism and homeostasis.
After fertilization the imp i ts e ai → a e i he ited f o o e ge e atio of ells to the e t. Only

in germ line the imprinting marks are erased and new ones are established: They are sex- elated →
both genomes in germ line receive either male (paternal) or female (maternal) imprints.
Examples:
Imprinted genes, are genes their

products somehow correlate with
cell growth and differentiation.
Silenced loci carry many

methylations. Imprinted genes are
regulated by Imprinting Control
regions (ICRs). ICRs are binding sites
for CTCF (a TF necessary for long
range interactions). Binding of CTCF
depends on the methylation state of
ICR → CTCF o l i ds, if ICR is NOT
methylated! Binding of CTCF leads to
interaction with an enhancer that
sti ulates t a s iptio of H →
chromatin hub is formed) and it
prevents transcription of Igf2.
(maternal).
If CTCF cant bind to ICR due to

methylation of ICR, enhancer acts to stimulate transcription of Igf2. (paternal)
DNA methylation determines, whether CTCF can bind or not. CTCF determines (by binding or not),
which part of the region is transcried and which will be stable silenced by formation of
heterochromatin.
All 3 genes are expressed maternally and not paternally. In paternal genome in return a long non
coding RNA = Air expressed, which regulates transcription.
If Air is expressed:
1. Promotor occlusion occurs: Igf2R and Air compete for all factor necessary to initiate
t a s iptio → i this ase Ai wins → all fa to s i d to its p o oto → t a s i ed
and Ifg2R not.
2. It recruits Histon Methylases that convert Sic22a3 and Sic22a2 into heterochromatin.
As today its known how imprinting works, it is possible to create bi- ate al o ga is s → i e :
Need o e i p i ted atu e ate al ge o e a d o e that la ks those odifi atio s → i i s
paternal genome.
10.11.2007
Wie würden Sie in Bakterien einen Promoter identifizieren und seine Funktionsfähigkeit
nachweisen?
Beschreiben Sie mindestens 2 Wege epigentischer Regulation durch Methylierung.
See above 
07.09.2007
Steroid Rezeptoren... unterscheide und wie sie funktionell aufgebaut sind.
See above 
Posttranslationale modifikationen an TF
Phosphorylation:
1. Having a positive effect on TF and transcription: Phosphorylation of TFs (e.g. STAT, R-

Smad, CREB, TCF,… leads to thei a ti atio .
In case of STAT and R-“ ad phospho latio leads to di e izatio → di er is imported
i to the u leus → sti ulates t a s iptio of ta get ge es.
CREB is bound to DNA, but only can stimulate transcription, if gets phosphorylated (by
protein kinase A)
2. Having a negative effect on TF and transcription:
Ci (cubitus interruptus) is phosphorylated if no hedgehog signal is present. -
Phospho latio of Ci leadst to its p oteol ti lea age → o e pa t e ai s that fu tio s
as a t a s iptio al ep esso → e te s nucleus and inhibits transcription. (if hedgehog
i ds to its e epto , Ci does ’t get phospho lated → ot lea ed → hole o ple
enters nucleus and functions as a transcriptional activator.
Phospho latio of eta ate i leads to its deg adatio → a not activate TCF.
If Akt is activated it i hi its FOXO → a ’t i d to its ta get ge es a o e.
Acetylation:
Of NFkB by CBP (a HAT, that can not only acetylate histones, but other proteins too), causes higher
activity of NFkB.
02.03.2007
Womit interagieren folgende Proteinstrukturen:
a) SH2 Domäne:
Interact with Phospho-t osi es → Di e isatio : “TAT gets phospho lated JAK (tyrosine
kinase) if receptor is activated by binding of a cytokine. “TAT di e the e te s u leus →
transcription of target genes
b) Chromodomäne: found in readers = proteins that can recognise histone modifications. Via
chromodomains methylated histone residues are recognised and bound.
c) Bromodomäne: found in readers = proteins that can recognise histone modifications. Via
bromodomains acetylated histone residues are recognised and bound.
d) Leucin Zipper: a kind of basic helix-loop-helix motive: one domain contains (in specific
intervals) leu- esidues → a a phipathi heli is fo ed → ia i te a tio s ith a othe leu-
zippe ia the leus^^ a oiled oil is fo ed → has additio al o asi egio s →
interaction with DNA.
Common motiv in TFs.
Geben sie eine Definition für 'genomic imprinting'. Welche Modifikation ist damit verbunden und
welches Makromolekül wird modifiziert.
See above 
15.12.2006
Wie führt man eine Chromatin-Immunopräzipitation (ChIP) durch und welche Erkenntnisse kann
man daraus gewinnen?
ChIP: Chromatin-Immunoprecipitation:

The pieces contain:
3) Isolate the DNA immunoprecipitated -> de-cross-li k p otei s a d DNA → deg ade p otei s.
4) ChIP-Seq.:
- Precipitate all e.g. K4me3 histons → all DNA ou d to K e is p e ipitated too
a ou d the K e histo es → use ioi fo ati s to determine every specific region of
Skizzieren oder beschreiben sie, wie sich Phosphorelierung der CTD der eukaryontischen RNA Pol II
im Laufe des Transkriptionszyklus auswirkt!
See above 
Skizze:
13.10.2006
Nennen Sie zwei unterschiedliche Mechanismen der epigenetischen Genregulation. Welche

Modifikationen von Proteinen und / oder Nukleinsaeuren spielen hierbei eine Rolle?
See above 
Beschreiben oder skizzieren Sie den Mechanismus der Genregulation durch cAMP in Prokaryoten
und Eukaryoten
Eu a te ia: → la ope o is fo e a ple egulated the p ese e / a se e of AMP → AMP is a

se so , that tells a te ia i hi h shape the a e → AMP is p ese t, if ell la ks glu osis → sig al
for energy lack!
La Ope o → it is egati e a d positi e egulated!
Negative Regulation: In basal state a repressor is bound to its transcriptional start. First have to
remove the repressor, but anyhow removal of lac-repressor is not sufficient to stimulate
transcription:
Structure of lac-operon:
 Promoter
 Operators (O1, O2, O3)
 CAP-binding site
 Coding sequence (LacZ, 39acy, lacA)
Lac-repressor is encoded by lacI gene, which is always expressed. LacI binds to operator of lac
ope o : it i ds as a di e to a pali d o i se ue e. La tose is the i du e → if la tose is p ese t
i ells, it i ds to ep esso → ha ges its o fo atio a ’t i d to ope ato a o e. Rep essio
is maximal if O1 and O2 or O3 are bound by repressor molecules. Binding of lac-repressor prevents
building of an open promotor conformation.
Positive Regulation: depends on CAP (= catabolite activated protein). In absence of glucose and w/o
ability to catabolise another energy source (because necessary operons are repressed), intracellular
ATP levels d op! → AMP is ade, hi h the a ti ates CAP. B a ti atio of CAP, a te ia E. oli
gain the ability to metabolise another energy source (e.g. lactose), by activation of the lac-operon.
CAP is responsible for a phenomena called glucose depression.
Different possibilities:
1. Glu ose a d la tose a e p ese t: No egati e egulatio , as la is p ese t → i ds la I →

a ’t i d to ope ato a o e. But: as glu ose is p ese , e ough ATP is pe es t → o AMP
→ CAP = i a ti ated
This leads to basal levels of transcription of the lac operon. (No negative and no positive
regulation)
2. Glu is present, but no lac: repressor is bound to ope ato , o AMP → egati e egulatio a
d o positi e egulatio → NO t a s iptio of la ope o
3. Glu is a se t, ut la is p ese t: la I is i a ti ated, AMP is p ese t → CAP = a ti ated →
transcription of lac operon
4. No glu a d o la → a solutel o t a s iptio of la ope o → la I p´ ou d to ope ato s
a d CAP a ti ated → ep essi e loop is fo ed!
In eukaryotes cAMP is used as a second messenger, that is synthei´tised by adenylaat cyclase, which
is activated by a large Gprotein.
07.04.2006
Skizzieren Sie ein prokarytiotisches Zweikomponenten-System. Wie wird der Response Regulator
modifiziert, um sein Gen zu aktivieren?
2-component systems are often used for regulation of

genes expression.
E.g.: the phoR-phoB 2 comp. system is used for

regulation of genes maintaining the intracellular
phosphate concentration / levels:
PhoR = sensor: a transmembrane protein, measuring the

Phosphate concentration: If enough is present in
pe ispla s ati spa e, phosphate is ou d to PhoR → if
le els d op, o phosphate is ou d to PhoR → PhoR is
a ti ated → its ki ase do ai phospho lates PhoB =
response regulator) using ATP. Phosphorylation
a ti ates PhoB, leadi g to t a s iptio of pho ope o →
phosphate levels increase again.
Another 2-component system regulates nitrogen fixation: it requires glutamine synthase which uses
organic N2 and ATP to generate glutamine.
NtrC = sensor
NtrB = response regulator
Wieviele RNA-Polymerasen gibt es bei Eukaryoten? Was sind ihre Produkte und wie sehen ihre
typischen Promotorregionen aus?
In eukaryotes 3 different RNA-Pols exist:
RNA-Pol I:
Localized in the nucleoplasma and it transcribes the genes encoding for rRNA (except the 5S rRNA)
Their promoters contain a core-promoter and a proximal upstream regulatory sequence (URS).
RNA-Pol II:
Localized in the nucleoplasm, where it transcribes pre.mRNA and sn RNAs.
Promotors may contain a TATA-box, proximal upstream regulatory sequneces and distal enhancer
elements
RNA-Pol III:
Localized in the nucleoplasm, where it transcribes tRNAs, 5SrRNA, sn RNAs and other ncRNAs.
Promotors vary: may be farly simple, or complex. Some promotors have typical regulatory sequnces
inside the coding region. Some are quite similar to promotors transcribed by RNA-Pol II.
Comparison between the 3 eukaryotic RNA-Pols:

Schematic drawing of the promotors transcribed by the 3 different polymerases:
20.01.2006
Skizzieren Sie die Ereignisse die von der Bindung eines Wachstumsfaktors (z.B.: EGF) an seinen
Rezeptor zur Induktion von Genexpression führen. Benennen Sie Komponenten und relevante
Modifikationen die an der Signaltransduktion beteiligt sind.
Worin unterscheiden sich Nucleosomen und Enhancosomen? Welche funktionelle Bedeutung hat
die Bildung des Enhancosoms?
Enhanceosome: many TF and other proteins

taking part in regulation /initiation bind either to
the p o oto o the e ha e → distal f o
promotor, offering binding sites for many
diffe e t TF/ p otei s → o u i atio
between the proteins bound to the enhancer and
promotor is necessary: binding of proteins to DNA
auses the DNA to e d → p otei s ou d to
enhancer and promotor can now interact and
communicate = enhanceosome
Picture shows the enhancesome formed to allow transcription of the INF-beta gene: A gene
necessary for immune response. → the fo atio of e ha eso es is e essa to fo t a s iptio
of some genes.
Nuclesome: e essa fo pa ki g of DNA → it o ati s a histo e o e a d DNA p apped

around it (beads on a string = 10nm fiber). Li ke histo e H a pa k the o e losel →
fi e → fo o e i fo: see a o e 
23.09.2005
Bedeutet der Phosphorylierung von PMII
Was soll PMII sein ???????
Biochem. Funktion von Chromatin Remodelling Complexes; Nennen sie min. 1 Beispiel indem ein
TF die Transkription durch Chromatin Remodelling pos. beeinflusst
Me ha si → see a o e 
Example of TF that recruits a CRC, therby stimulating transcription of a target gene:
13.05.2005
DNA Microarray (in Stichworten Ablauf). Was sind die Vorteile gegenüber anderen Methoden?
Microarray ist eine Sammelbezeichnung für moderne molekularbiologische Untersuchungssysteme,

die die parallele Analyse von mehreren tausend Einzelnachweisen in einer geringen Menge
biologischen Probenmaterials erlauben. Es gibt verschiedene Formen von Microarrays, die manchmal
auch als „Genchips oder „Biochips bezeichnet werden, weil sie wie ein Computerchip viele
Informationen auf kleinstem Raum enthalten können.
Herstellung:
Biochips
A=2 cm2
Trägermaterial: beschichtetes Glas oder

Kunststoff
10.000-30.000 Positionen/cm2
Spot-Größe: 50-
Auftragen von 0,25-30 nL Lösung
Viele Analysen pro Zeit
Gut automatisierbar
Protein-Microarrays und DNA-

Microarrays:
Prinzip:
Hat 2 Zellkulturen (1x Krbszellen, 1x „normale Zelllinie ) -> aus beiden Isoliert man mRNA. Die mRNA
wird mittels Reversen Transkriptase in cDNA umgeschrieben und beide (also sowohl cDNA aus
Tumorzellen, als auch jene aus den gesunden Zellen) werden mit einer Floureszenzproteunsequenz
fusioniert (NICHT mit derselben! Z.B. Rot an cDNA aus Tumorzellen und grüne an die gesunden)
In jedem Spot des Chips werden nun die cDNAs von krank und gesund zusammengebracht -> man
schaut ob diese mit einander hybridisieren -> dies erkennt man an dem jeweiligen Floureszenz-signal
(grün -> mehr gesunde cDNA als kranke, rot -> mehr krank als gesund oder gelb -> karnk und gesund
in gleichen Maßen enthalten)
3 verschiedene Mechanismen der positiven Transkriptionskontrolle durch Transkriptionsfaktoren?
Was ist damit gemeint??
27.01.2005
Welche Fragenstellung beantworten Sie mit ChIP? Wie funktioniert dies?
ChIP= Chromatine Immunoprecipitation: used for analysis of a proteins binding to DNA or histone
odifi atio s → the dete i e the DNA se ue es he e su h Proteins are bound /
modificatiosnare found.

The pieces contain:
5) Isolate the DNA immunoprecipitated -> de-cross-link proteins and DNA → deg ade p otei s.
6) ChIP-Seq.:
- Precipitate all e.g. K4me3 histons → all DNA ou d to K e is p e ipitated too
a ou d the K e histo es → use ioi fo ati s to dete i e e e spe ifi egio of
Aktivierung von Adenylalcyclatase, welche andere Genexpressionswege werden auch durch
Adenylatcyclatase aktiviert?
In eukaryotes adenylate cyclase (a membrane bound protein)

activated by trimeric/large G-proteins. It synthesizes cAMP form
ATP. cAMP functions as a second messenger, which regulates
gene expression:
Binding of ligand to receptor causes the exchange of GDP to GTP

i the t i e i G P otei → leads to i te a tio of of the
“UBs ith AC → AC is a ti ated → uses ATP to ake AMP.
cAMP binds to regulatory SUB of protein kinase A, leading to
dissociation of the regulatory and catalytic subunit. The catalytic
Sub then enters the nucleus and phosphorylates CREB (=TF),
hi h al ed ou d to DNA, ut a ’t sti uate t anscription
w/o phorphorylation: when phosphorylated, it can recruit CBP
(= HAT). CBP has a domain, that recognises phosphorylated
proteins.
AMP i p oka otes sig alises the shape of the a t ia → AMP is p ese t if ATP le els a e lo →
cAMP activates CAP (=Catabolite Activated Protein), that stimulates transcription of operons,
allowing metabolisation of other energysources than glu.
19.11.2004
1. a) Welche Phasen lassen sich bei der Initiation der Transkription unterscheiden (in Stichworten)?
(Pre-)Initiation: (pre) Initiation complex is formed to position the RNA-Pol at transcription start. →
the (pre)initation complex has the same function as bac. Sigma function (+ additional functions)
P otei s i ol ed i fo atio of IC a e ge e al o asal TFs → TFII, as they are specific for RNA-Pol II:
1st TFIID i ds: it is a p otei o ple o tai i g TBP = TATA i di g p otei → i ds TATA o , if
o e is p ese t i the p o oto a d TAFs = TBP asso iated fa to s → p o idi g a la ge su fa e,
with many TADs, allowing interaction with TFs, mediator complexes, co-a ti ato s,… . Next TFIIA and
TFIIB bind. TFIIB bridges the interaction of TFIID and RNA-Pol II, which binds next. RNA-Pol I has TFIIF
bound. This is important, because if RNA-Pol II lacks TFIIF, TFIIH and TFIIE do not bind to the IC! TFIIH
o tai s a o e a d additio al “UBs → Cdk a d li T to phospho late the CDT of RNA-Pol II at
se i e → auses p o oto le e e.
Promotor clearance: RNA-Pol moves off the
t a s iptio sta t → clears the promotor
then stops again after transcription of a few Nt.
= pausing RNA-Pol
b) Lässt sich das Auffinden eines Promotors durch RNA Polymerase durch freie Diffusion
beschreiben? Welche alternativen Erklärungen kommen in Frage?
NO! Of course it would be possible that RNA Pol would by change bind to a promotor. How often this
ould happe as sto hasti all al ulated → would happen ways to few and to slow.
→ “ ie tist eeded to come up with new ideas:
 RNA Pol binds somewhere by chance to DNA and slides alonge it, until it finds a promotor (no
evidence for that model)
 RNA-Pol binds to DNA by random diffusion, but then moves by popping from DNA region to
the e t → so RNA-Pol is able to scan DNA fast enough for promoters. Calculations for this
model showed, that I wold be fast enough.
c)Wie beeinflusst der Sigma-Faktor die Assoziation der prokaryoten RNA Polymerase mit
Promotoren?
it allows the RNA-Pol to distingush between random DNA and a promotor: promoters differ from all
other DNA sequneces:
 (extended) – 10 region: TATA box
 -35 region
If RNA_pol has ou d a sig a fa to = HOLOe z e → e essa to fi d a p o oto . RNA-Pol w/o a

sig a fa to is alled COREe z e: it a i d to a do DNA a d is the t a slati g fo → sig a
fa to s o l e essa to fi d p o oto → afte a ds the disso iate.
Diffe e t sig a fa to s e og ise diffe et p o ote s: sig a → house keepi g ge es. “ig a →
heat shock genes; sigma 28 chemotaxis and mobility genes.
Erklären Sie, wie entfernt von der Initiationsstelle der Transskription bindende
Transkriptionsfaktoren die Bindung der RNA Polymerase beeinflussen können.
Via formation of enhaceosomes:
Enhanceosome: many TF and other proteins

taking part in regulation /initiation bind either to
the p o oto o the e ha e → distal f o
promotor, offering binding sites for many
diffe e t TF/ p otei s → o u i atio
between the proteins bound to the enhancer and
promotor is necessary: binding of proteins to DNA
auses the DNA to e d → p otei s ound to
enhancer and promotor can now interact and
communicate = enhanceosome
Picture shows the enhancesome formed to allow transcription of the INF-beta gene: A gene
e essa fo i u e espo se. → the fo atio of e ha eso es is e essa to fo t a s iption
of some genes.
CTCF a TF, mediating long range interactions:
E.g.: hae oglo i lo us → e odes fo diffe e t he oglo i s. The a e i po ta t du i g

development processes: in different phases of embyrogeisis / development, different hemoglobins
are produ ed e o i , fetal, adult hae oglo i → eeds egulatio : Ch o ati hu s
Insulator elements ate bound by CTCF, which forces the chromatin into a loop structure = hub. LCRs
(= locus control regions) define which genes in which hub (isolated from each other) are active 
transcribed. This is possible due to proteins, bound to LCR and such bound to enhancers of the
individual genes. Interaction is possible, because of hub!
B e th oid p oge ito ell → ell that a e o e a e th o te is p epared for transcribing
hae oglo i ge es, ut the a e ot t a s i ed et → last odifi atio of hu is ’t done yet.
(C ) erythroid cell: transcribes haemoglobin genes.
(D) brain ell → has ’t fo ed a hu i this egio → ot e essa , e ause a ai ell ill e e
transcribe haemoglobin genes!
The difference between B and C is that, in B proteins that will bind to LCR are missing! CTCF is already
bound to the insulator in B like i C , aki g lo g a ge i te a tio s possi le → that’s h the hu
like structure is already formed.
Binding of CTCF does not lead to direct interaction between the different DNA regions, but recruits
cohesins, that is then positioned around the DNA → loops DNA together.
Binding sites for CTCF are mostly (in that case) calles HS something . That’s histo i , e ause these
egio s he e fi st a al sed Additio of DNase → egio s e essa fo t a s iptio e.g. to
egulate it a e less o pa ted → DNase a atta k su h egio s ette → uts the o e ofte
than such that o elate ith i a ti e egio s → o e o pa t → ot su h a essi le fo DNase .
24.09.2004
1. a) Was sind die speziellen Funktionen von TFIIH Komplexen? b) Welche Proteinkomplexe
vermitteln die Interaktion zwischen RNA-Polymerase II und Transkriptionsfaktoren, die spezifische
Bindungsstellen eines Promotors erkennen?
See above 
Welche enzymatischen Aktivitäten von Zelloberflächenrezeptoren kennen Sie (Beispiele)? Wie sind
die Signaltransduktionswege beschaffen, die diese enzymatische Aktivität letztlich in veränderte
Genexpression übertragen (jeweils ein Beispiel in Form einer Skizze!)?
Some receptors gain – if activated – a kinase function: phosphorylate then their target TF (STAT, TGF-
eta path a → R-Smad) or another kinase that transduces the signal further (MAPK pathway).
Receptor activates a kinase that phosphorylates a inhibitor of TF, which gets – due to
phosphorylation – ubiquitinated and then degraded (NFkB, Wnt)
Other receptors interact (their intracellular domains) with large G-Proteins: If receptor is activated by
liga d i di g, it gets a ti ated a d auses the G p otei to e ha ge GDP to GTP → auses “UBs of
G p otei to i te a t ith AC ade late lase → AC s thesises AMP f o ATP → AMP a ti ates
PKA → PKA üphospho lates CREB.
02.07.2004
Y2H a) am Beispiel c-Fos---c-Jun skizzieren, wie schauen die Fusionsgene aus? b) Kann man mit Y2H
Interaktion von STATS nachweisen + Begruendung?
cJun-cfos sind doch nicht mehr teil der VO???
Ad a) Therefore use R-Smad and Smad 4 as an example.
= Transactivating Domain (TAD). Further they have modules to stimulate transcription (this one is
Fuse the TAD of R-Smad with the DNA-binding domain of yeast TF stimulating transcription of Gal4 =
BAIT DOMAIN! This construct can bind to the promoter region of the reporter gene. But it alone is
not sufficient to stimulate transcription of the reporter gene.
To analyse whether R-Smad interacts with Smad4 to stimulate transcription a so called FISH DOMAIN
is created: Therefore Smad4 with a TAD that functions in yeast.

STAT? good question ^^
a)Wie bzw von welchem Co-Faktor wird CREB aktiviert + von welchem Pathway stammt das Signal?
b) welcher Co-Aktivator ist erforderlich + wie wirkt dieser?
See above 
19.03.2004
Beschreiben Sie kurz den Aufbau eines Nukleosoms

(Skizze!) - Beschreiben Sie die Aktivität des chromatin
remodelling complexes - Was hat die (De-)Acetylierung der
Histone zur Folge?
See above 
Skizzieren Sie den kompletten Initiationskomplex bei

Transkription durch RNA Polymerase II! Welche dieser
Proteine assoziieren mit DNA-bindenden Faktoren?
(Pre-)Initiation: (pre) Initiation complex is formed to position

the RNA-Pol at transcription start. → the p e i itatio
complex has the same function as bac. Sigma function (+
additional functions)
Proteins involved in formation of IC are general or basal TFs

→ TFII, as they are specific for RNA-Pol II: 1st TFIID binds: it is
a protein complex containing 1 TBP (= TATA binding protein
→ i ds TATA o , if o e is p ese t i the p o oto a d
TAFs = TBP asso iated fa to s → p o idi g a la ge su fa e,
with many TADs, allowing interaction with TFs, mediator
complexes, co-a ti ato s,… . Ne t TFIIA a d TFIIB i d. TFIIB idges the i te a tio of TFIID a d
RNA-Pol II, which binds next. RNA-Pol I has TFIIF bound. This is important, because if RNA-Pol II lacks
TFIIF, TFIIH and TFIIE do ot i d to the IC! TFIIH o tai s a o e a d additio al “UBs → Cdk a d
cyclin T) to phosphorylate the CDT of RNA-Pol II at se i e → auses p o oto le e e.
Promotor clearance: RNA-Pol o es off the t a s iptio sta t → clears the promotor then stops
again after transcription of a few Nt. = pausing RNA-Pol
28.11.2003
Wie führt man eine Chromatin Immunopräzipitation (ChIP) durch? Welche Frage versucht man mit
einem ChIP Experiment zu beantworten?
See above 
Skizzieren Sie den Mechanismus der Aktivierung des Enzyms Adenylatcyclase durch G-
Proteingekoppelte Hormonrezeptoren. Welche weiteren Proteine und Mechanismen führen zur
Änderung der Genexpression durch die Aktivität der Adenylatcyclase?
See above 
03.10.2003
Erklären sie die begriffe positive und negative regulation der transkription & nennen sie je ein
beispiel!
See above 
inwiefern unterscheiden sie die drei eukaryontischen rna-polymerasen (struktur, biochemische

funktion, promoterstrukturen...)
see above 
25.10.2002
Welche spezifischen Fragestellungen beantwortern Sie mit folgender Methodik:
Nuclear run on assay: is the amount of mRNA regulated by transcription control?
e.g. gene A -> want to know, whether the amount of mRNA A is changed, if cells are put under a
certai sti ulus e.g. sta atio , heat,… . Isolate ul ei → put o i e → stops t a s iptio , ut RNA-
Pols do ot fall off the DNA. Add adioa ti e → P la elled Nt → warm up → RNA-Pols finish
t a s iptio → isolatio of RNA → h idise it to a filte o tai g o ple e t a DNA → isualise
it by a X ray filter.
Real time RT PCR: is a gene regulated by a certain stimulus?
Southern blot: is a gene regulated by a certain stimulus?
Primer extension: localisation of promotor
DNase I footprint: determination of TF binding sites
Hefe 2 Hybrid system: do those 2 TFs interact?
Welche Mechanismen zum Abschalten eines Gens auf Ebene der Transkription kennen Sie in pro-
und eukaryoten Organismen? (in Stichworten beantworten und Beispiele nennen)
(1) Binding of a transcriptional repressor, inhibiting transcription by either preventing binding of

RNA-Pol or by preventing of formation of an open promoter conformatio → la I of la ope o
(2) Formation of an repressive loop: compact structure that prevents binding of RNA-Pol → i
a te ia: e.g. la ope o : if glu a d la a e a se t → Cap i ds to Cap i di g site → DNA is e d
i a a that opeato s a i te a t ith ea h othe → st o g i te a tio → sta le
(3) Fo atio of hete o h o ati → DNA eth latio a d histo e odifi ation lead to higher
compactness making interaction with transcription machinery impossible (e.g. imprinted genes
→ kann ich das hier als Bsp nennen??)
(4) Not fo atio of a h o ati hu e.g. ai ells a d hae oglo i ge es → ha e o hu → o
transcription of haemoglobin genes)
Was fällt euch noch ein????
05.07.2002
Isolieren eines Promotors aus Säugerzelle (!) und Analyse desselben (notwendige Schritte in
Stichworten!)
GENOMIC CLONING in OLD DAYS (Historic): promotors where isolated and then used to form
genomic libraries: DNA was cut into ~20kb large fragments. Those fragments where inserted into a
e to = la da . E. oli the gets i fe ted la da → Ha e li a ies. See infection due to plaque
formation. Use a it o ellulose filte → la das a e t a sfe ed to it. “o e phages ill o tai the
DNA of i te est =p o ote → h id it to a p o e → auto adiog aph → see hi h pal ues o tai
the DNA of interest. As now is known which phages contain the promoto → isolate la das fo i g
this / those plaques. Let the g o agai o a E. oli la → a plifi atio of DNA of i te est →
hybridize it again
Toda : ge o es a e se ue ed → if I a t a spe ifi p o oto , I ge e ate spe ifi p i e s a d

amplify it via PCR.
P o le : Too la ge p o oto s → a t use PCR → eed BACs = acterial artificial chromosomes):
Used to generate Fragments of human DNA up to 300kb. Those BACs often derive from F plasmids.
Further such large fragments are possible to handle by classical genetic recombination!
→ Need: RECOMBINEERING: it works in vitro: amplify sequence of interest, including the

e o i atio sig al → ha e DNA f ag e ts o tai i g e o i atio sig al, that is also p ese t i
BACs. → put the togethe i a test tu e ith recombinase of la da → e o i atio ‼
If defi ed p o oto s a e e essa → ha e to fi d the i BACs ia “ u lease appi g o p i e

extension:
Pimer extension and S1 mapping are methodes to determine the transcription start of certain gene.
If sequence of an mRNA
is k o , ut it is ’t
sure, that this mRNA is
e ti e → the efo e ot
sure if mRNA contains
the trsnciption start:
A short sequence
complementray to
mRNA is used as a
p i e → RT is added →
reverse transcription
u til the ’e d =
S1-nuclease mapping:
Needs DNA, that not

only includes
transcription start, but
the p o oto too.→ Ha e lo g pie e of DNA a d k o that so e he e i this se ue e the
transcription start and promotor are located. Use mRNA and hybridize it to the DNA pie e → RNA-
DNA h id → Use S1 nuclease to digest all single stranded RNA/DNA. Only double-stranded sequenc
e is protected. Use it to determine length and transcription start.
Before recombineering was invented, recombinase reaction was performed in vivo in E.coli, if the
right recombinase is present in E.coli. it requird 2 steps:
1) BACs get the right ones (containg Promotor / DNA of interest)

2) Need a asset → lo e asset ith e o i atio sites
Weil das vll nicht so verständlich war:

1) Isolieren eines Promotors aus Säugerzelle und Analyse desselben (in
Stichworten):
Isolierung des Promotors:
1. Als Klo ie ehikel die te de -Phage. De -Phage

ist genetisch so veränderbar, dass man ihn ihm bis
zu 20 Kilobasen (20.000 bp) große DNA vermehren
kann.
2. Genom wird durch Restriktionsenzyme in 20.000 bp
große Stücke zerteilt. Dabei überlappen sich die
20.000 bp große DNA-Stücke.
3. Diese DNA-F ag e te e de u i de -Phagen
hineinkloniert  genomische DNA-Bibliothek.
Promoter wird durch Hybridisierungs-Verfahren
gefunden:
4. De -Phage mit der genomischen DNA-Bibliothek
wächst auf einem e.coli Rasen  die -Phagen
egi e die e. oli zu l sie e u d es e tstehe -
Phage-Plaques (mit toten e.coli).
5. Die Plaques werden auf einem Filter übertragen, 
-Vi io e gehe auf u d die DNA de -Phagen bleiben auf dem Filter kleben.
6. Mittels einer Genspezifischen Sonde (radioaktiv markiert) wird getestet welcher dieser Plaques
enthält eine genomische DNA die dem gewünschten Gen entspricht.
7. Mit einem Autoradiogramm wird festgestellt wo sich die DNA befindet die den Promoter für
das Gen enthält
Rekombinante DNA-Technologie (B.S.644 ff.)
Die zu klonierende DNA wird aus der Zelle isoliert und von Restriktionsenyzmen in spezifische DNA-
Fragmente geschnitten. Die DNA-Fragmente werden über einen Vektor in die Wirtszelle transferiert.
Das rekombinante DNA-Molekül repliziert sich dort, so entstehen viele Klone dieses Moleküls. Die
klonierte DNA kann transkribiert und in ihre mRNA übersetzt werden.  Untersuchung von
Genorganisation und –funktion. Die Technik der rekombinanten DNA dient zur Isolierung großer
Mengen spezifischer Gene.
Voraussetzung für Rekombinante DNATechnologie ist, dass es die Schnittstelle nur EINMAL gibt
(Plasmide), also gezielt aufgeschnitten und inseriert werden soll. Bei einem 300kb großen Genom ist
es nicht mehr möglich nur mit einem Enzym zu schneiden,  Recombineering.
Recombeneering:
Das DNA-Fragment, welches eingebracht werden soll, wird über PCR in der Phage amplifiziert und
zwar so dass die PCR auch gleichzeitig die homologen Regionen für die Rekombination erzeugt. 
Transformation es läuft eine Plasmid/Phagen mediierte homologe Rekombination ab. In vitro oder in
E.coli-Stamm findet die homologe Rekombination statt und man erhält einen genetisch veränderten
BAK (Bacterial Artificial Chromosome).
Wichtig für die Herstellung transgener Mäuse, Analyse des Transgens. Analysierung des Promoters
über Reportergene.
Analyse des Promotors:
Promotor Bashing
Bei der Promotor-Bahing-Methode geht man davon aus, dass der Promoter upstream des
Transkriptionsstartes liegt. Diese Methode wird auch als Deletionskartierung bezeichnet, dadurch
können funktionelle, wichtige Sequenzbereiche des Promoters definiert werden.
Vorgehen: Der ermittelte Promotor wird mehrfach in Plasmide kloniert, wobei die Promotorsequenz
bei jedem Plasmid von beiden Enden her sukzessive weiter von einer Exonuklease verkürzt wird. Die
Promotorsequenz dient als Promotor für ein Reportergen, das sich ebenfalls auf dem Plasmid
befindet. Die einzelnen Plasmide werden in die Zellart eingebracht, in der er vermutlich reguliert
wird z.B. Leberzellen. Je nachdem ob das Reportergen sehr gut, weniger gut oder gar nicht mehr
exprimiert wird, kann man erkennen, ab wann bei den verkürzten Sequenzen regulatorische
Sequenzen dabei waren, die für die Funktion des
Promotors von Bedeutung sind.
Reportergen – Assay:
Dabei wird der Promoter mit einem Reportergen

fusioniert. Je mehr dann von diesem Genprodukt
gebildet wird, desto besser funktioniert der Promoter.
Um die Promoter-Aktivität zu messen, muss dieser

natürlich erst zurück in eine Zelle gebracht werden,
die das betreffende Gen tatsachlich transkribieren
kann.  transiente Transfektion. Kann die Zelle den
Promoter tatsachlich transkribieren, wird das
Reportergen produziert, die Zelle kann im Anschluss
lysiert werden und man kann herausfinden wie viel
von dem Genprodukt entstanden ist. Damit das
einfach gemacht werden kann, muss das Genprodukt,
das das Reportergen codiert, eines sein, das in der
Zelle, in der der Assay durchgefuhrt wird, ursprunglich
nicht vorkommt.
Beispiel: Chloramphenicol-Acetyltransferase (CAT)

Es ist ein bei E. coli auf Resistenzplasmiden codiert,  Enzym, das das Antibiotikum Chloramphenicol
detoxifiziert, indem es mithilfe von Acetyl-CoA eine Acetylgruppe an das Chloramphenicol hangt,
wodurch dieses inaktiviert wird. Dieses Gen existiert in Saugerzellen natürlich nicht und kann dort als
Reportergen eingesetzt werden. Gibt man nach Lyse der Zelle radioaktiv markiertes Chloramphenicol
und Acetyl-CoA zum Zellextrakt, wird das Chloramphenicol in diesem Extrakt acetyliert und man kann
das acetylierte Chloramphenicol durch Dünnschichtchromatographie von dem nicht-acetylierten
Chloramphenicol trennen. Je mehr acetyliertes Chloramphenicol dabei entsteht, desto aktiver ist der
Promoter.
Weitere Reportergene:
• Luciferase (aus Glühwürmchen, erzeugt Licht bei der Substratumsetzung)

• Green fluorescent protein (GFP; kann zur Fluoreszenz angeregt werden, Expressionsanalyse auch
in lebenden Zellen möglich)
• β-Galaktosidase (oNPG-Farbreaktion)
Regulation des Schilddrüsenhormons TR: Struktur der DNA Bindedomäne/Struktur des

TR/Dimerisierung/Mechanismus der Genaktivierung und -repression?
Grundstruktur der Rezeptoren
Die Rezeptoren haben alle eine relativ nah verwandte DNA-bindende Domäne. Die Linganden-
Bindedomänen sind ebenfalls sehr eng verwandt. Am N-Terminus befindet sich die Domäne mit der
größten Variabilität zwischen den
verschiedenen Rezeptoren.
DNA Bindungsstellen von Transkriptions-

faktoren der intrazellulären
Hormonrezeptorfamilie:
– können entweder inverted repeats

(Palindrome) oder direct repeats bilden
– inverted repeats binden homodimere
Rezeptoren (z.B. GR, ER)
– direct repeats binden Heterodimere eines
Rezeptors (z.B. TR) mit einem weiteren
Mitglied der Rezeptorfamilie, RXR. Dabei
entscheidet die Anzahl an Basenpaaren zwischen den repeats über die Spezifität.
Dabei bleibt die DNA-Bindesequenz bei „direct repeats immer gleich (AGGTCA), woher kommt die
Spezifität der Bindungsstellen?: Ursache für die Spezifität liegt in der Anzahl der Nukleotide zwischen
den Bindungs-Sequenzen. Der Abstand zwischen den direct repeats erzeugt die Spezifität für ein
bestimmtes Heterodimer.
Die Rezeptoren binden als fertige TF an die DNA (permanent): in Abwesenheit von Liganden binden
sie HDACsa  remprimierender Komplex, in Anwesenheit von Liganden HATsa  Aktivierender
Komplex. Thyroidhormonrezeptoren reigulieren die Genexpression durch Bindung an Hormon-
Response-Elemente (HREs) in DNA, entweder als Monomere, Heterodimere mit Retinoid-X-Rezeptor
(RXR) oder als Homodimere.
Beispiel: In Abwesenheit von Retinsäure entsteht ein Komplex mit HDACs (A). Die Bindung des
Liganden (B) Retinsäure (RA) an den Rezeptor (RAR; gleiches gilt für den T3 Rezeptor) bewirkt die
Dissoziation eines Korepressor /HDAC-Komplexes und die Assoziation eines Koaktivator/HAT
Komplex.
In Abwesenheit des Hormons, TR im Komplex bindent mit Ko-Repressor Proteinen an HREs in einem
transkriptionell inaktiven Zustand. Die Bindung vom Hormon führt zu einer Konformationsänderung
in TR welche Corepressor vom Rezeptor / DNA-Komplex und die Rekrutierung von Co-Aktivator-
Proteine verdrängt. Der DNA / TR / Coaktivator-Komplex rekrutiert dann RNA-Polymerase, die
downstream von DNA in mRNA und Protein, das schließlich zu einer Veränderung der Zellfunktion
führt, transkribiert.
NACHTRAG: ALTFRAGEN Ab Jänner 2014
31.01.2014
Sie haben mittes DNA-Microarray nachgewiesen, dass die Zugabe von einem Wachstumsfaktor die
Expression von 120 Genen erhöht.
a) Wie können Sie nachweisen, dass tatsächlich Transkriptionskontrolle vorliegt?
Gro-Seq! Is an advancement of the Nuclear Run On Assay. It allo s the a al sis of a ge es →

even whole transcriptome analysis) at the same time:
The whole mRNA associated with chromatin (= this includes every pre-mRNA that is transcribed at
the poi t of isolatio is isolated → add labelled Nt. → h idize it ith hip → use bioinformatics for
evaluation = transcription rate of every gene transcribed under a certain condition. It is a true
t a s ipto e a a lsis, as it sepe ates RNA sta ilit , p o essi g,… .
Principle behind every gene:
Nuclear Run On Assay

Is used to analyse whether a certain gene (e.g. A) is transcribed (more or less) if a certain cell type is
put on a defined stimulus: Think that gene a is transcribed with a higher transcription rate ( the
transcription rate is defined as the number of RNA-Pol molecules, which are transcribing one gene at
the same time) in liver cells under lysine starvation (e.g.).
Afte isolatio , u lei a e put o i e → f eezi g stops RNA-pol, but they remain bound to DNA. Add
P32 labeled nucleotides a d put the app oa h u de a o ditio s → RNA-Pols finish
t a s iptio → i se t P la elled NT i g o i g RNA. RNA is isolated a d h idized to filte
containing to A complementary oligo- t → RNA of i ds to it → use a X a filte to detect the
sig al → the o e RNA of A is p ese t, the igge a d da e the spotted dot ill e.
B)Sie vermuten, dass der Transkriptionsfaktor Stat3 mit den identifizierten Genen wechselwirkt
bzw. diese stimuliert. Wie weisen Sie das nach?
I would make 2 co ditio s: : positi e o t ol → i this app o h WT “tat is p ese t a d k o k

do “tat o use a Muta t oe → o pa e the a ou t of RNA, ge e p odu ts G o-Seq or
Microarray analysis. In vivo and in virto possible e.g. by doxycycline induced knock down via shRNAs.
RNA Polymerase II - C-terminale Domäne. a) Wie ist diese beschaffen? b) Welche Rolle spielen
Phosphorylierungsereignisse im Transkriptionszyklus
See above 
04.04.2014
Welche Aussagen zur Transkriptionskontrolle sind richtig? (richtig = fett, falsch = normal)
1. Das Trp Operon in E. coli wird negativ kontrolliert und benötigt einen Korepressor
2. Verbiegen von DNA durch das CAP Protein erlaubt die Interaktion von Operonen des
lac Operons
3. Im bakteriellen Zweikomponentensystem fungiert ein Sensorprotein als
Transkriptionsfaktor
4. Das „rudder einer RNA Polymerase wird zur Trennung der DNA Stränge benötigt
5. Transkriptionsfaktor TFIIH ist eine RNA Polymerase II Kinase
6. „Backtracking der RNA Polymerase ist wichtig für reverse Transkription
7. Transkriptionsfaktoren können pausierende RNA Polymerase zur Elongation bewegen
8. Der Mediatorkomplex besteht aus generellen (basalen) Transkriptionsfaktoren
9. Histonchaperone halten Nukleosomen stabil, damit sie von der elongierenden RNA
Polymerase nicht zerstört werden
10. „Insulator Elemente trennen Exon und Intronsequenzen
2.a) Welche DNA Base kann in eukaryoten Organismen methyliert vorliegen? Skizzieren sie die
Struktur. Welche biologische Funktion hat die Methylierung der DNA?
C tosi e i CpG isla ds → e e postio in DNA where a C is followed a G.

2.b) Was ist der Unterschied zwischen „de novo“ und „maintenance“ Methylierung? Wie ist ein
DNA template für de novo Methylierung beschaffen und wie ein template für maintenance
Methylierung? (alle Antworten in Stichworte bzw. Skizzen)
See above 
Sketch
10.10.2014
GENAU DIE“ELEBEN FRAGEN ie . . ‼!
03.07.2015
10 ja/nein Fragen
 Z.B. In Eubakterien wird S5 der CTD phosphoryliert.

 DNase I footprinting wird zur Aufklärung der Mobilität von Promotoren verwendet.
DNase I wird zum Kartieren von hypersensitiven Bereichen verwendet. Was ist
Hypersensitivität/wie funktioniert das? Welche regulatorischen DNA elemente sind hypersensitiv?
Binding sites of CTCF are h pe se sit e to DNase digest → the efo e a e named HS something ^^
Binding sites for CTCF are mostly (in that case) calles HS something . That’s histo i , e ause these
egio s he e fi st a al sed Additio of DNase → egio s e essa fo t a s iptio e.g. to
regulate it) are less compacted → DNase a atta k su h egio s ette → uts the o e ofte
tha su h that o elate ith i a ti e egio s → o e o pa t → ot su h a essi le fo DNase .
Was kann man da noch erwähnen??

09.10.2015
10 Aussagen bei denen anzukreuzen war, was richtig ist. (wortwörtlich aus der Prüfungseinsicht mit
validierten korrekten Antworten):
 Sigma-Faktoren sind Teil der RNA Polymerase in der Elongationsphase (falsch)

 Der elektrophoretic mobility shift assay zeigt die Bindung von Transkriptionsfaktoren aus
Kernextrakten an regulatorischen DNA Sequenzen (richtig)
 Operons führen zur Erzeugung polycistronischer RNA (richtig)
 Öffnen einer Promoterstruktur führt zum Erliegen der Transkription (falsch)
 2-Komponentensysteme übertragen Phosphogruppen (richtig)
 Eukaryote Initiationskomplexe für alle RNA Polymerasen enthalten TBP (richtig)
 Der generelle TF TFIIB interagiert sowohl mit der RNA Polymerase als auch mit dem TF
TFIID (richtig)
 Der nuclear run-on assay misst die Initiation durch RNA Polymerase (falsch)
 Alle eukaryoten RNA Polymerasen werden in der C-terminalen Domäne phosphoryliert
(falsch)
 Elongation der Transkription erfordert vollständige Entfernung der Nukleosomen (falsch)
Wie können TFs Transkription beeinflussen? 3 Bsp
See above 
1) Binding to enhancer ele e t → interacting with mediator, which is used to enable crosstalk /
o u i atio et ee sig als o i g f o e ha e a d su h f o p o oto →
enhanceosome
2) Re uit e t of CRCs → aki g TF- binding sites for other TFs more accessible!
3) Recruitment of histone modifying o ple es → e.g. CREB he phospho lated e uits CPB
(=HAT)
4) Fo atio of a h o ati hu → e.g. hae oglo i ge es → eeds CTCF a d ohesi s
04.12.2015
10 Fragen:
1) TF stellt Kontakt zwischen Pol und TFIID her

2) TF-Monomere binden palindrome Sequenzen
3) prokaryotische TF führen alle zur negativen Regulation der Transkription
4) irgendwas mit beta-scaffold TF (?) binden direkt repeats
5) Zn-finger binden kleine Furche der DNA
6) Laktose ist ein Coaktivator des Lac-Repressors
7) Der TF TBP führt zur Biegung der DNA
8) Nuclear Hormone Receptors (beta-scaffold DBD) binden alle direct repeats
9) Der TF TFIIB stellt Kontakt zwischen Pol und TFIID her
10) ziemlich wörtlich: "TF binden die RNA-Polymerase" (kein Plan ob damit alle, manche, einer zu
interpretieren ist)
Aufbau eines Nukleosoms, Modifikationen: 3 Bsp + wie Transkription beeinflussen (Skizze)
See above 

Decker Genex-ErgänungFA

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Diese Fragenausarbeitung wurde von mir nach besten Wissen und

Gewissen verfasst. Trotzdem kann es natürlich sein, dass sich

1) Cross-li k h o ati so that it does ’t fall apa t oss-linker: e.g. formaldehyde)

There are different ways to read out a ChIP:

Note: STAT is the only TF that is activated by Tyrosinkinases

STAT = Signal Transducer and Activator of Transcription

If there comes no signal from extracellular (via a cytokine

If cytokine binds to receptor (cytokines are present because

In this case phosphorylation leads to dimerization, which is

I po ta t path a i i u e s ste → ithout NFkB path a oi u e espo se ould e

NFkB… Nu lea Fa to k B, o sisti g of su u its p a d p , hi h is ade as a p e u so =

IKK phosphorylates Inhibitor NFkB = IkB,

Phosphorylation leads to degradation of

Phospho latio of CBP i eases its affi it to NFkB → CBP i ds to NFkB

Fu the phospho latio of CBP de ease its affi it to p → phospho -

Phosphorylation of CBP leads to increased activity of NFkB → i eases t a s iptio al a ti it .

Developmental pathway, regulating cell growth.

Red coloured proteins prevent

Red proteins lead to

In this case phosphorylation

“TAT → hi h is o l i po ted to u leus, if it is a di e . See above.

MyoD: its activity is regulated by the formation of different heterodimers:

cIf a progenitor muscle cell should

If p oge ito should ’t diffe e tiate, M oD

Wnt-Beta-Cate i Path a → see a o e

Methods to detect such interactions:

Bait and Fish domain are located on 2 different plasmids.

2 ways of doing it:

A) If have already candidates identified → do as des ied a o e

Definiere epigenetische Genregulation. Nenne damit in Verbindung gebrachte Komplexe, welche

Epige etis he Ge egulatio edeutet das Ch o ati zustä de e e a si d → de )usta d, de

Dabei vererbten Chromatinstruktur werden im wesentlich durch (unterschiedliche) DNA-

Au h i diese Fall spiele T a sk iptio sfakto e ei e i htige Rolle → “ie kö e au h das

Aktivatorkomplexe wirken aktivierend, hingegen Silencing Komplexe Reprimierend

Histon 3.3 ist dem Histo ah e a dt → e de au h gege ei a de ausgetaus ht. H . ist

Nennen Sie 2 Chromatin-remodellierende Komplexe. Beschreiben Sie die biochemischen

Nennen Sie 2 Chromatin-remodellierende Komplexe. Beschreiben Sie die biochemischen

Chromatin Remodeling Complexes (CRCs) cause changes in the chromatin structure:

 Define Eu- and Heterochromatin

Examples for CRC families:

Biochemical activity of CRCs:

Nucleosome SLIDING (a):

In vivo and in vitro.

Nuclesome TRANSFERE (b):

Activity of CRCs is influenced by TFs!

 TFs cause modifications of histones, allowing CRCs to bind.

→ TFs a ause i se tio , epositio i g of u leoso es i o de to a ti ate o ep ess t a s iptio

1) Der Initiationskomplex der Transkription besteht aus Polymerase-associated factors (PAFs)

Ja/Nein Fragen zu den Themen:

Shows specific binding of an TF to DNA or specific

DNA fragment (restriction fragment,

Nuclear Run On Assay

Mit ChIP sind basenspezifische Kontakte nachweisbar

Mit ChIP-Seq ist spez. RNA-Spezies nachweisbar

ChIP: Chromatin Immuno-Preciptiation:

To determine, to which chromatin sequence a TF is binding:

Pimer extension and S1

Example: La Ope o → it is egative and positive regulated!

10 Ja/Nein Fragen, u.a:

1) H3.3 findet man in transkriptiv aktiven Regionen

 -35 region: TG rich

2 ways to determine, that -35 and -10 region define a promotor: