38.

Welches Protein muss stabilisiert werden, damit Signaltransduktion durch Wnt Liganden zu einer
transkriptionellen Antwort führt? Nach welchem Mechanismus erfolgt Stabilisierung? β-catenin needs to be stabilised
(i.e. not degraded) for Wnt signal to be transduced. The TCF-Smad4 (T-cell factor) complex is in nucleus & acts as a
transcr. repressor. When β-catenin binds to Smad4, complex acts as an activator. In order for β-catenin to be present in
nucleus, β-catenin GSK3 in the cytosol needs to be inactivated (it phosphorylates β-catenin which leads to ubiquitination
& then degradation in the proteasome). It is inactivated when a Wnt signal molecule is bound to the cell membrane
receptor Frizzled,leading to the activation of another complex, containing the protein Dishevelled, that inhibits the β-
catenin degradation complex
39. Isolieren eines Promoters aus einer Säugerzelle (!) und Analyse desselben (notwendige Schritte in Stichworten).
Promoter isolation: To know where the promoter is, need to know where transcr. starts bcs anything upstream of
transcription start (up to a certain point) is promoter. Take DNA containing gene of interest, including transcr. start & its
promoter. Nowadays we have entire genomes seq & can amplify gene by PCR, then find promoter. But PCR doesn’t work
well for large pieces of DNA, only for short. If you need a large piece, need f.ex. mouse DNA containing gene X from gene
bank. They send BAC & these can contain up to 300kb. They will then replicate in E. coli & can isolate them. BAC cloning
can’t be accomplished by classic genetic eng., use recombineering instead. this allows you to handle large pieces of DNA
→ technique based on homologous recombination systems, as opposed to the older/more common method of using
restriction enzymes & ligases to combine DNA seqs in a specified order.
Promoter bashing: You isolate DNA containing promoter, put it in plasmid, open up plasmid w/ restriction enzyme, add
exonuclease Bal31. Bal31 will digest from both ends, the longer you allow it to cleave the shorter the promoter
becomes. Then a 2nd restriction enzyme is used to cut away promoter seq. The promoter has a linker that allows it to be
cloned into another plasmid. Then get plasmids that have promoter fragments of different lengths (from short to long)
fused to a reporter gene. W/ current technology these steps would be substituted by amplification of promoter DNA
from annotated genomes via PCR.
40. Beschreiben Sie den thyroid hormone receptor: Struktur der DNA-Bindedomäne, Struktur von TR, Dimerisierung,
Mechanismus der Genaktivierung und -repression? TR is a heterodimeric receptor & binds to direct repeats in the DNA
together w/ the RXR family (TR/RXR heterodimer). The heterodimer sits in the nucleus, bound to DNA & acts as a
repressor as long as it doesn’t have the thyroid hormone bound. Binding of thyroid hormone leads to a conformational
change & the receptor then acts as an activator.
41. Welche spezifischen Fragestellungen beantworten sie mir folgender Methodik:
-) Nuclear run on assay Is there transcriptional control?
-) qPCR (= RT-PCR) How much of an expressed gene is in a cell?
-) Southern blot Detect specific DNA fragments in a genome.
-) Primer extension Determine the 5’ ends (transcription starts) of genes.
-) DNaseI footprint Determination of protein binding sites on DNA.
-) Y2H system Determine protein-protein interactions (e.g. of TFs w/ other proteins).
42. Welche Mechanismen zum Abschalten eines Gens auf Ebene der Transkription kennen Sie in pro- und
eukaryotischen Organismen?
-) Competition: Transcriptional repressors can repress genes by competing w/ activators.
-) Inhibition: Repressor interacts w/ activator, preventing other proteins from binding to it (e.g. coactivators).
-) Direct repression: Repressor interacts w/ an inhibiting mediator complex, e.g. to prohibit RNA pol promoter clearance
-) Indirect repression: Recruit deactivating histone modifying enzymes, such as HDACs. This is indirect, because repressor
doesn’t directly inhibit initiation but rather prevents initiation from happening as a whole.
43.U cemu se razlikuju 3 eukary. RNA pols?
All RNA pol r highly related to each other, they werent invented x times during evolution- rather they all go back to a
single ancestral enzyme. This is demonstrated by the subunit composition. All 3 eu- pol have 2 large subunits (like the β
subunits of prokaryotes), they are called L & L’. RNA pol II has a little tail called the C-terminal domain (CTD). The CTD
plays an important role in gene regulation. Eukaryotes have α-like subunits & then also a large number of extra subunits.
These fall into 2 categories: 1) such that are common to all 3 pol; 2) those that are different for the 3 pol. Pol I has 5
additional subunits, pol II has 4 & III has 7
3 diff pol → they transcribe different groups of genes.
RNA Polymerase I is localised in the nucleolus and responsible for rRNA transcription; its core promoter is preceded by
an upstream regulatory sequence (URS) and followed by a downstream terminator sequence. RNA Polymerase II is
localised in the nucleoplasma where it transcribes pre-mRNAs and snRNAs (small nuclear). Protein coding genes possess
an upstream enhancer sequence, followed by URS and the TATA box motif, as well as a CAP binding site. RNA
Polymerase III is localised in the nucleoplasma as well, transcribing tRNAs, 5S rRNAs, snRNAs and ncRNAs (non-coding).
The promoter sequence of tRNAs consists of URS followed by the transcription start which is enclosed between URS and
two binding regions A and B; downstream sequences comprise poly-TA.
44. Wie führt man eine Chromatin Immunopräzipitation (ChIP) durch? Welche Frage versucht man mit einem ChIP
Experiment zu beantworten? We want to know if a TF only binds in vitro or if it would also bind in a cell? You can not
only determine wether or not a TF binds in the 1st place, but also how much of it binds. -) covalent crosslinking of
proteins w/ DNA, fragmentation of DNA by sonication -) purify protein-DNA complexes -) immunoprecipitation: use
antibody against TF coupled to beads, precipitate via centrifugation -) purify precipitated DNA & use proteases to
destroy TF → all obtained fragments contain binding sites for TF! -) quantitative determination of precipitated DNA by
qPCR
45. Skizzieren Sie den Mechanismus der Aktivierung des Enzyms Adenylatcyclase durch G-Protein-gekoppelte
Hormonrezeptoren. Welche weiteren Proteine und Mechanismen führen zur Änderung der Genexpression durch die
Aktivität der Adenylatcyclase? A group of large G proteins is associated w/ a receptor. When receptor binds its ligand,
GDP is exchanged for GTP & the G protein becomes activated, interacts w/ adenylate cyclase (AC) which also sits in the
plasma membrane & cyclates the phosphates on ATP → cAMP, our 2nd messenger. This leads to transcriptional
activation. (cAMP regulates protein kinase A which consists of 2 regulatory & 2 catalytic subunits. When the R subunits
bind cAMP, they dissociate from the C subunits, the C subunits then go to the nucleus & phosphorylate a TF called cyclic
AMP response element binding (CREB). It binds to a DNA seq that makes genes respond to cAMP. Unphosphorylated
CREB binds to DNA but it’s not active as a transcriptional activator. The phosphorylated form of CREB can function as a
transcriptional activator because it binds a co-activator: a histone acetyl transferase called CBP. CBP acetylates
surrounding histones & makes the gene more transcription-friendly) AC also becomes activated when there’s low levels
of ATP & high levels of AMP in the cell. It then takes AMP to produce cAMP, which again leads to transcriptional
activation, but through a different cascade than the 1 described in brackets above. Generally, AC responds to a low level
of energy, that’s what activates it.
46. Erklären Sie ein 2 Komponenten System. 2 component systems typically consist of a membrane-bound histidine
kinase that senses a specific environmental stimulus & a corresponding response regulator that mediates the cellular
response, mostly through differential expression of target genes (→ transcription factor). Binds to DNA in
phosphorylated state. Ex.: PhoR (phosphate regulator) sensor. 1 part sticks into the periplasm. When there’s enough
phosphate, the sensor is active. It has intrinsic enzymatic activity → histidine kinase. It attaches phosphates to histidines.
When there’s not enough phosphate in the periplasmic space, the ligand binding domain is not bound by PO34-. The
histidine kinase becomes active, phosphorylates a histidine on the kinase itself (histidine is part of the kinase). 2nd step:
PO34- is transferred from the histidine of the sensor to an aspartic acid residue on the response regulator. This transfer
of PO34- activates the response regulator.
47. Wie bzw. von welchem Co-Faktor wird CREB aktiviert? Von welchem pathway stammt das Signal? Cyclic AMP
response element binding (CREB) is activated by phosphorylation via the C subunits of protein kinase A.
Unphosphorylated CREB binds to DNA but it’s not active as a transcriptional activator. Once phosphorylated, CREB can
bind a coactivator: histone acetyl transferase called CBP that acetylates surrounding histones & makes the gene more
transcription-friendly. Pathway: PKA has C & R subunits. When cAMP binds to the R subunits, the C subunits dissociate &
become active. cAMP is produced by adenylate cyclase (AC) which is activated by G protein coupled receptors.
48. Welche enzymatischen Aktivitäten von Zelloberflächenrezeptoren kennen Sie (Bsp.)? Wie sind die
Signaltransduktionswege beschaffen, die diese enzymatische Aktivität letztlich in veränderte Genexpression
übertragen (jeweils ein Bsp. in Form einer Skizze)?
Major enzymatic activities of membrane bound cell surface receptors are phosphorylation through kinase activities,
proteolysis and hydrolysis. Cytokine receptors can be coupled with JAK kinases which phosphorylate tyrosines of the TF
STAT, enabling its dimerisation and binding to target genes. RTK/Receptor tyrosine kinases are specific for growth factors
such as hormones or polypeptides, and binding results in induction of MAPK signalling pathways. RTK phosphorylates
itself (increasing own activity), leads to activation of various kinases including RAS, RAF, MEK and MAPK (in this order). G
protein-coupled receptors are receptors coupled to GTPases which by GTP hydrolysis stimulate cAMP production by
adenylate cyclases. Delta-Notch receptor (transmembrane protein binds transmembrane protein) interaction causes
cleavage of Notch receptor C-terminus that move to the nucleus and acts as a transcriptional regulator. Interaction
between Delta and Notch happens directly through cellcell contact. If Delta associates to Notch, NME complex
dissociates, and the C-terminal domain of Notch is cleaved off by a protease activity
49. Wie kann man die Teile des Promoters nachweisen, an die die RNA Pol bindet? DNAse I footprinting: DNAse I
cleaves DNA predominantly in the minor groove but in principle it can cleave after any nt. You add so little of DNAse I
that each of the DNA fragments is only cleaved once (on average). Now you get a collection of fragments where every nt
position conforms to 1 cleavage fragment. You can separate the fragments on a gel which gives you a DNA ladder. Then
you add RNA pol to the DNA & every cleavage site that is w/in the binding of RNA pol will be protected, no cleavage
occurs. → technique for determination of protein binding sites on DNA.
50. Erklären Sie, wie weit entfernt von der Initiationsstelle der Transkription bindende TF die Bindung der RNA Pol
beeinflussen können. TF partaking in transcriptional initiation either bind directly to the promoter seq or to distal
enhancers. DNA looping is an essential prerequisite for contacts between the IC and TF that bind to enhancers far
upstream of the transcription start. TF CTCF (involved in formation of chromatin hubs) → structures induced through
interaction of insulator seqs with each other or with CTCF → forming a loop which brings the LCR into close proximity
with the target genes → Interaction of LCR with promoter/enhancer seqs determines which genes within a
chromosomal domain are transcribed. To ensure communication between transcription factors and RNA pols, an
enhanceosome has to be formed. This structure requires association of high mobility proteins, bending the DNA so that
interaction of transcription factors is possible (IFN-ß enhanceosome mediates binding of histone modifying and
chromatin remodelling complexes to the IFN-ß promoter, as a prerequisite for the removal of the nucleosome at the
transcription start and initiation complex formation)
51. Worin unterscheiden sich Nukleosomen und Enhanceosomen? Welche funktionelle Bedeutung hat die Bildung
eines Enhanceosoms? Enhanceosome: Protein complex made up of activators, binds to enhancers & thus activates
transcription. Its structure is similar to that of a nucleosome. Ex.: IFN-β is made in response to viral infection to protect
the organism from it. When IFN-β is activated, an enhanceosome is formed. It causes histones to become acetylated,
meaning the chromatin becomes loosened, i.e. more transcription friendly, also causes recruiting of RNA pol II.
Nucleosome: Made up of histones instead of TFs, does not activate DNA transcription, DNA is wound around it, doesn’t
need specific seqs to bind to.
52. Was ist der Unterschied zwischen ‚de novo‘ und ‚maintenance‘ Methylierung? DNA methylation is facilitated by
DNA-methyl-transferases (DNMTs). There are 2 kinds: -) De novo methylases are responsible for establishing DNA
methylation. They methylate a DNA strand even if there’s no methylation on the opposite strand. -) Maintenance
methylases make sure that methylations are passed on to daughter cells. They profit from the fact that DNA replication
is semi-conservative: 1 strand is already methylated, they then use this template to methylate the newly formed strand.
De novo means the DNA template is entirely unmethylated. In all cells of our body we have a specific pattern of
methylated Cs, so when cells replicate the pattern of methylation also needs to be replicated, which is what
maintenance methylases, Dnmt1, do. This works because DNA replication if semi-conservative so 1 strand of the DNA
(the parental strand) is methylated & the other (daughter) isn’t.
53. Geben Sie eine Definition für genomic imprinting. Welche Modifikation ist damit verbunden und welches
Makromolekül wird modifiziert? DNA methylation controls genomic imprinting, an important epigenetic process. We
have 1 genome from our father w/ a paternal imprint, 1 from our mother w/a maternal imprint. Depending on which
parent the DNA is from, the expression can be different. This has a lot to do w/ DNA methylation. Genomic imprinting is
essential for the formation of an embryo. Androgenetic or gynogenetic embryos can’t survive. So genomic imprinting,
i.e. different genome expression coming from both parents is essential for viable offspring.
54. Unterscheiden Sie Steroid Rezeptoren aufgrund ihres funktionellen Aufbaus. TFs of the nuclear hormone receptor
family bind steroid or related lipophilic hormones in the nucleus. Lipophilic hormones are able to pass the plasma
membrane which means that their receptors can be inside the cell. Nuclear hormone receptors bind DNA either as
homodimers (for glucocorticoids, sex hormones) or as heterodimers (retinoic acid etc.) which are always formed w/ RXR.
There’s 2 different modes of action of the hormones: some hormone receptors reside in the cytoplasm & when f.ex.
glucocorticoids are bound they acquire the ability to go to the nucleus & do whatever they need to do there. The other
possibility is that nuclear hormone receptors 7 are permanently associated w/ DNA, never leave the nucleus. The
hormone then converts a transcriptional repressor into an activator. (The binding sites for nuclear hormone receptors
have some peculiarities. Receptors that bind DNA as homodimers all have their own palindromic binding seq. A
palindromic seq has rotational symmetry. That always means that homodimers bind, 1 monomer binds to each strand
basically. The spacing between the palindromes is always 3 nt. The receptors that bind to DNA as heterodimers w/ RXR
protein all have the same binding site. The specificity comes from the spacing between direct repeat seqs. The
heterodimer receptors bind direct rather than inverted seqs, specificity is given by the number of nt that sits between
the repeat seqs.)
55.Nennen Sie mindestens 2 Methoden zum Nachweis der physischen Interaktion zweier TF und eine Methode um zu
beweisen, dass ein TF wichtig ist für die Initiation der Transkription. Er hat glaub ich in der Vo nur eine Methode zum
Nachweis der Interaktion 2er TF besprochen. Proteomic approach to identification of protein interactions: -) purify
protein complexes -) separate into individual components (via gel electrophoresis) -) protease digestion → peptides of
individual proteins -) determination of peptide masses by mass spectrometry →characteristic fingerprint of peptide
masses → data base → identification of respective proteins Um nachzuweisen, dass ein TF wichtig ist für die Initiation
der Transkription: Reporter gene assay, try to see wether gene expression changes w/ and w/o adding the TF.
56. Welche Unterschiede bei Steroidrezeptoren gibt es hinsichtlich der Bindung an die DNA? Hinsichtlich der
Lokalisation in der Zelle? Wie kann ein Ligand so einen Rezeptor aktivieren (bzw. was passiert durch den Liganden)?
Some hormone receptors reside in the cytoplasm and when a hormone binds they acquire the ability to go to the
nucleus and do whatever they need to do there. The other possibility is that nuclear hormone receptors are
permanently associated w/ DNA, never leave the nucleus. The hormone then converts a transcriptional repressor into an
activator. The structure of NHRs has 3 important subdomains: 1) activation domain, that’s what every TF needs, that’s
how it talks to the machinery of transcriptional initiation and elongation 2) DNA binding domain 8 3) special for NHRs:
ligand binding domain. It is made in such a way that it undergoes quite a drastic (conformational) change upon the
binding of a ligand. As long as the structure is extended (before binding) it binds co-repressor proteins. When the ligand
is bound, the conformational change leads to dissociation of co-repressors and co-activator proteins can bind instead.
57. Wie bewirkt die Phosphorylierung die Aktivierung eines TF im NFκB, Wnt und STAT pathway?
-) NFκB pathway: IκB is the inhibitory subunit of NFκB → as long as NFκB is associated w/ IκB, it’s inactive. IκB sits on the
nuclear localisation signal of NFκB, so when they’re associated NFκB can’t go to the nucleus. IκB kinase phosphorylates
IκB, which stimulates ubiquitination and degradation of IκB. Finally, NFκB dissociates from IκB, its NLS is no longer
masked, it can go to the nucleus and activate genes.
-) Wnt pathway: β-catenin interacts w/ a protein called TCF in the nucleus which interacts w/ Smad4. The complex
formed by TCF and Smad4 is a repressor. If TCF-Smad4 binds to β-catenin, it’s converted to a transcriptional activator. So
wether or not the genes that bind TCF are repressed or activated depends on wether β-catenin is present or not. β-
catenin degradation happens when there’s no signal from the Wnt pathway → the protein kinase GSK3 phosphorylates
β-catenin which causes it to be ubiquitinated and degraded. No β-catenin in the nucleus, no transcription of target
genes. When Wnt binds to its receptor, another complex becomes active which inhibits the protein complex that
degrades β-catenin.
-) JAK-STAT pathway: STATs are a group of TF that are different from most others because they’re activated by tyrosine
phosphorylation. Biologically, Tyr phosphorylation vs. f.ex. Ser phosphorylation means very different things. There’s a
plasma membrane cytokine receptor (cytokines are important proteins for the immune system, allow communication of
cells that interact in an immune response → hormones of the immune system) that has associated Janus (protein
tyrosine) kinases (JAKs). JAKs phosphorylate STATs on a Tyr, which allows the STATs to form dimers because each STAT
also contains an SH2 domain (these domains can recognise and interact w/ phosphorylated Tyr). The STAT dimers go to
the nucleus and are able to contact the binding sites of their target genes. STATs have an unconventional bipartite
nuclear location seq (NLS), that means as long as they’re not dimerised their NLS isn’t formed, it’s only formed through
the process of dimerisation.
58. bHLH Proteine können als inaktive und aktive Dimere wirken. Erklären Sie die strukturellen und mechanistischen
Hintergründe und nennen Sie ein Bsp. HLH proteins have a basic region for DNA binding, they can bind only as dimers,
so they need sth. that makes them dimerise which is an amphipathic helix → 1 hydrophilic and 1 hydrophobic side, the
hydrophobic side makes the 2 proteins interact. If 2 complete (i.e. containing dimerisation and binding domains) TFs
associate, the dimer will be transcriptionally active. But there’s also members of this family that only have the HLH motif
for dimerisation but not a DNA binding region. The resulting heterodimer is transcriptionally inactive because the dimers
need 2 basic regions to bind to DNA, otherwise there’s insufficient affinity. An ex. for this are MyoD and Id: MyoD has
both domains, Id only has a dimerisation domain. Basic region → a lot of aa are present that are positively charged at
physiological pH. DNA backbone is negatively charged so that makes the interaction possible. MyoD is full of pos.
charged Arg, Id has only 1. But both proteins have the ‚relevant‘ aa for the HLH motif.
59.Welche Strukturen benötigen TF für die Bindung an DNA? Nennen Sie Charakteristika und je ein Bsp. Nennen Sie in
vitro/vivo Methoden um TF-Bindungssequenzen zu verifizieren. TFs have a DNA-binding domain also called basic
region.It contains a number of (under physiological conditions) positively charged aa. It also contains aa that make base-
specific contacts to DNA. Ex. for basic domains: -) Leucine zippers - coiled-coil structures formed by amphipathic helices,
combined w/ an Nterminal basic region that binds DNA. E.g.: CREB -) Helix loop helix - Also coiled-coil structure but w/
an aa loop between. Also coupled w/ Nterminal basic region. E.g.: MyoD -) Helix turn helix - 2 α-helices w/ a turn
between them, 1 of them, called recognition helix, binds DNA. -) Zn fingers - compact array of β-sheets and α-helix
through coordination of ZN2+. -) β scaffold - made up of β sheets To verify TF binding sites in vitro: EMSA And in vivo:
ChIP-Seq, reporter gene assay

60. Sie haben mittels DNA-microarray nachgewiesen, dass die Zugabe von einem Wachstumsfaktor die Expression von
120 Genen erhöht. Wie können Sie nachweisen, dass tatsächlich Transkriptionskontrolle vorliegt? Sie vermuten, dass
der TF STAT3 mit den identifizierten Genen wechselwirkt bzw. diese stimuliert. Wie weisen Sie das nach? Transcr.
control can be determined by Gro-Seq (genomic run on sequencing) which is an advancement of the nuclear run on
assay. This method allows simultaneous analysis of transcription of various genes and even transcriptome analysis (i.e.
genome wide analysis of nascent transcripts). Bioinfo analysis of results enables evaluation of data, providing info on
transcript freq. The more a certain part of a gene has been transcribed, the higher the sequence reads of this particular
gene and its transcription. To determine whether or not Stat3 influences expression of a certain gene under a certain
condition, a reporter gene assay can be implemented. (A cell is co-transfected with two plasmids, the first carrying the
gene encoding the transcription factor Stat3, the second a reporter gene and a Stat3 binding site. If Stat3 stimulates
transcription upon environmental stimulation, more reporter gene transcripts will be synthesised as Stat3 is more
active.)
61. DNase I wird zum Kartieren von hypersensitiven Bereichen verwendet. Was ist Hypersensitivität, wie funktioniert
sie? Welche regulatorischen DNA-Elemente sind hypersensitiv?
Accessible/open regions of chromatin can be mapped by their DNase I hypersensitivity. If chromatin is exposed to low
concentrations of DNase I, the enzyme will cleave sequences specifically where nucleosome have either been removed
or altered (DNA is no longer tightly wound around the nucleosome). Furthermore, promotors, enhancers and other
transcriptional control regions are hypersensitive to DNase I; the more loosely the DNA is packed, the more accessible it
is to a nuclease such as DNase I. Specific band patterns indicate hypersensitive regions: undigested chromatin forms
thick bands at the top of the blot whereas lightly digested chromatin forms bands nearer the middle of the blot.
However, heavily digested and therefore hypersensitive chromatin forms no fragment at all
62. Welche Rolle spielen polycomb und trithorax Proteine bei epigenetischer Regulation? Polycomb complexes
convert open chromatin into heterochromatin. They contain enzymes that modify histones so that the chromatin
becomes repressive. Most important mark: trimethylation of H3K27 (Lys 27 in H3) and methylation (mono-, di- or tri-) of
H3K9. These marks lead to a conformation that is repressive towards gene expression. Trithorax proteins are the
antagonists to polycomb complexes, they are involved in the formation of open chromatin. They contain methylases and
the methylations they introduce correspond to active chromatin.
63. Welche Konsequenzen hat das DNA supercoiling für die DNA-Struktur? Transcription causes a positive supercoil in
the DNA. That means that per 10 nt there’s more than 1 rotation of the 2 strands around each other. The winding
number is too high. In front of the RNA pol we have positive supercoiling, behind it negative supercoiling. If the supercoil
becomes too positive (DNA becomes too tightly wound), the energy needed for RNA pol to advance would be too high
and it could not continue transcription.
64.Erklären Sie die proofreading-Aktivität der Pol. Proofreading by RNA pol prevents the accumulation of mistakes. It
comprises 2 things: The enzyme knows when a mistake has occurred and it can correct that mistake. Pyrophosphorolytic
editing: the last nt of the growing RNA is replaced by pyrophosphate. Hydrolytic Editing: RNA Pol moves backwards for a
few nt and removes a piece of mismatched RNA. Hydrolytic editing is probably more important and occurs during
‚backtracking‘ → the RNA pol moves backwards a little bit on the DNA. When a mistake occurs, the nt that was added
cannot form H-bonds to the template strands, the strands are apart. When this non-H-bound-nt is sensed, the enzyme
goes back until the wrong nt is in the funnel region. An elongation factor TFIIS binds in the enzyme and displaces the
backtracked RNA from binding to the enzyme and, in the end, cleaves it away. Then, the RNA pol can start to elongate
again.
65. Beschreiben Sie die Termination bei Bakterien. Termination of transcription in bacteria can be rho-dependent or -
independent. -) Rho-independent: The end of the transcript contains a poly(A) run and a GC rich inverted repeat. An
inverted repeat is a self-complementary seq of DNA/RNA. That means that one part of the repeat can bind to the other
and the 2 parts have the ability to form a structure called a hairpin. GC bp are more stable than AT/AU bp. So you now
have a stretch of RNA-DNA hybrid in the poly(A) region that is relatively unstable and can be separated easily. The
folding of the GC hairpin delivers the energy needed to separate the poly(U) run of the RNA from the As of the DNA. The
hairpin is formed spontaneously because it is an energetically favourable process. -) Rho-dependent: Requires energy.
You need a rho-factor that basically binds to a seq in the transcript called rho utilisation (rut) seq. That is a part of the
RNA that can be recognised by the rho factor. The factor binds to the 3’ end of the RNA and moves towards the pol, then
pulls the RNA out of the RNA pol. Movement of the rho factor requires ATP. Poly(U) run and inverted repeat leads to rho
independent termination, rut seq leads to rho dependent termination → the RNA seq determines the termination.
66. Interpretieren Sie: Zellen werden mit Cytokin behandelt. Im nuclear run on assay findet man vermehrt Aktivität,
im Northern blot hingegen sieht man keine Steigerung der RNA-Menge. Nuclear run on assay zeigt uns, dass die
Transkription durch Zugabe des Cytokins (positiv) beeinflusst wurde, weil mehr mRNA produziert wird. Wenn sie beim
Northern blot nicht mehr nachweisbar ist, könnte das daran liegen, dass sie sofort wieder abgebaut wurde (instabil ist).
67. Erklären Sie die Bestimmung des Transkriptionsstarts. -) S1 mapping: Hybridise mRNA to a S1 probe that is
radioactively labelled. Then add S1, which digests ssRNA and ssDNA. The parts where to mRNA and the probe hybridised
will not be digested. Then you analyse the length of the remaining radioactively labelled S1 probe w/ gel
electrophoresis. -) Primer extension: Label primer at 5’ end, it hybridises to mRNA. Then you extend it w/ reverse
transcriptase and the fragment will end at the 5’ end of the mRNA. Again, all you have to do is measure the length of the
radioactive fragment. 12 S1 is an endonuclease that digests ss- but not ds nucleic acids. It can also digest partially
mismatched ds molecules with such sensitivity that even a single bp mismatch can be cut and hence detected. In
practice, a probe of end-labeled dsDNA is denatured and hybridised to complementary RNA. S1 is used to recognise and
cut mismatches or unannealed regions and the products are analysed on a denaturing polyacrylamide gel. A number of
different uses of the S1 nuclease have been developed to analyse mRNA taking advantage of this property. Both
qualitative and quantitative information can be obtained in the same experiment. -) CAGE = cap analysis of gene
expression. mRNAs are capped at the 5’ end. Take random primers (short, random seqs). All RNAs we have will be
randomly primed. Then primer extension, you get a cDNA whose 3’ end corresponds to a 5’ end of mRNA. To isolate the
new cDNAs you label the cap of the RNA w/ biotin. You then purify (centrifugation w/ beads covered in streptavidin).
Then you get ss cDNA and then you make a 2nd strand using linkers (little DNA fragments) that bind to the 5’ end of
cDNA. The linker contains a binding site for a restriction enzyme called MmeI. MmeI cleaves not where it binds but
upstream. The cleavage by MmeI gives us a fragment of 20 bp + the linker. This you isolate, then seq and you get the 5’
ends of all the RNAs you used in the beginning.
68. Wie bekommt man Plasmide in Säugetierzellen? -) CaPO4-co-precipitation → not used much anymore today. You
mix DNA w/ a salt that contains Ca, then slowly add another salt that contains PO43-. When you add the 2nd salt,
insoluble CaPO4 crystals form that contain the DNA. When you add that onto cells they will endocytose the crystals and
part of the DNA then gets into the cell and gets expressed. -) Electroporation → zapping cells w/ high current in a salt
buffer. Cell membranes become leaky, DNA can penetrate into cells and gets expressed. -) Lipofection → you make
complexes of a lipid that is positively charged and that interacts w/ the negative charges on the cell membrane. The lipid
is complexed w/ the DNA we want to get into the cell. Through mechanisms that aren’t well understood, the cells take
up the lipid and some of the DNA is then expressed in the cell. -) Insert construct into lentiviral vectors, form lentiviral
particles and infect cells w/ these (attenuated) viruses. -) Microinjection → microscope that contains a microinjector
which is a needle that has a little bit of DNA in it, when you poke the cell nucleus and inject nl of DNA into the cells.
69. Beschreiben Sie die Aktivierung von SREBP durch Proteolyse. SREBP = Sterol regulatory element binding protein, is
a TF that allows genes to respond to cholesterol, which is a sterol. It is made as a larger precursor protein which sticks in
the membrane of the ER where it’s immobilised. In the ER; there’s a protease called SCAP which has a cholesterol
binding site → it senses how much cholesterol is in a cell. As long as there’s enough, SCAP is inactive. When cholesterol
levels drop there’s no C bound to SCAP, it becomes active and cleaves the N terminal of SREBP away. The N-terminal
domain is the active TF, it can go to the nucleus, bind to promoter elements and stimulate transcription of genes that
are important for cholesterol uptake and synthesis.
70. Steroidhormonrezeptoren: Welche 2 Typen gibt es? Wo befinden sie sich? Wo und wie binden sie DNA?
Some steroid hormone receptors may reside in the cytoplasm and enter the nucleus upon ligand binding (glucocorticoid
receptor), while other (nuclear) receptors such as RXR family members reside inside the nucleus, constantly bound to
DNA. Homodimeric hormone receptors such as glucocorticoid and oestrogen receptors bind inverted repeats
(palindromes) of DNA. Heterodimeric hormone receptors such as TR/RXR and RAR/RXR (retinoid X receptor) recognise
the same direct repeats.
71. Erklären Sie den TGFβ (Smad) pathway. The Smad pathway involves Ser phosphorylation. Receptors bind proteins
from the TGFβ family (which are sort of hormones/cytokines). The receptor itself is a Ser kinase and phosphorylates R-
Smad. Phosphorylation allows R-Smad to dimerise w/ a common Smad (which isn’t modified in any way), e.g. Smad 4.
The R-Smad/Smad4 dimer can then go to the nucleus where it binds to DNA and functions as a transcription factor.
72. Wie wird der TF NFκB reguliert? NFκB is activated when a receptor binds a cytokine and activates the IκB kinase. IκB
is the inhibitory subunit of NFκB → as long as NFκB is associated w/ IκB, it’s inactive. IκB sits on the nuclear localisation
signal of NFκB, so when they’re associated NFκB can’t go to the nucleus. IκB kinase phosphorylates IκB, which stimulates
ubiquitination and degradation of IκB. Finally, NFκB dissociates from IκB, its NLS is no longer masked, it can go to the
nucleus and activate genes. How does the system go back into the homeostatic state? There’s a very simple negative
feedback loop for NFκB: 1 of the target genes of NFκB is the gene that encodes its inhibitory subunit, IκB. When there’s
enough IκB in the cell, it binds to NFκB and inhibits it. → Classic ex. of neg. feedback loop. NFκB is also regulated by a
2nd post-transcriptional modification: acetylation. Acetylation allows proteins to interact w/ histones. It’s very similar to
NFκB. When the pathway is active, CBP (histone acetyl transferase) acetylates p65 which allows it to be activated.
73. Erklären Sie wie TF die Modifikation und Struktur von Nukleosomen beeinflussen können am Bsp. von Gal1. Gal1
is required by yeast to metabolise Galactose. In the absence of galactose, the transcription start is bound by Gal80 which
keeps Gal4 from being transcriptionally active. If galactose is present, it abrogates the interaction between Gal4 and 80,
4 can exert its transcriptional activity. It does that by recruiting SAGA (histone acetylation) to acetylate histones by the
transcription start, and it recruits a chromatin remodelling complex that removes the nucleosomes that occlude the
transcription start.
74. Was können TF? -) Impact the initiation of transcription: contacts w/ TAFs or mediator, sometimes through
coactivators -) Impact elongation (pausing pol II): pTEFb recruitment (through mediator or other mechanisms) -) Impact
DNA structure: bending (e.g. TBP), DNA loops, chromatin hubs (CTCF) 14 -) Impact chromatin modification and structure:
recruitment of histone-modifying enzyme complexes (HAT, HDAC, methyltransferases, demethylases etc.), association
w/ remodelling complexes (e.g. SWI/SNF), association w/ activator complexes (e.g. Trx), association w/ silencer
complexes (e.g. PCR2)
75. Erklären Sie, wie Transkription durch Pol I und III funktioniert. RNA pol I sits in the nucleolus and transcribes the
rDNA. We have a core promoter and an upstream control element UCE, that has 2 binding sites for the upstream binding
factor UBF. Binding of UBF allows formation of initiation complex SL1 that includes TBP (also present in transcriptional
initiation of RNA pol II!). For RNA pol III, initiation occurs from w/in the gene. This was discovered by deleting regions of
the DNA and then they found that at some point deletion w/in the gene fucked shit up. The region contains 2 binding
sites for TFIIIC. During initiation, TFIIIC associates w/ BoxA and B seqs, then TFIIIB (that has TBP in it) can bind. TBP is part
of the initiation complexes for each pol. Its function is not necessarily to find a TAATA box but probably the change of
DNA structure.