Adriana Salazar A01283039
SUMMARY
o Sources of DNA damage
o Types of mutations, their possible origin, and
how they may affect the encoded protein
o Lesion bypass
o Base excision repair mechanism and involved
molecules
o Mismatch repair mechanism and involved
molecules
o Nucleotide excision repair mechanism and
involved molecules
o Both types of double-strand damage
mechanisms and involved molecules
(Homology-directed repair and NHEJ)
o Transcription initiation, promoters,
polymerase and sigma factor, and
terminations (Rho dependent and
independent) in prokaryotes
o Riboswitches, the Lab Operon (including the
CAP- mediated regulation in the absence of
glucose), IPTG and X-gal, cis- and trans-
regulation of transcription, the Trp Operon
and Top Operon attenuation.
o Eukaryotic initiation of transcription
(molecules involved, transcription factors
and corresponding DNA sequence),
upstream regulatory elements (proximal
and distal), activators, proofreading.
o The 3 post-transcriptional events occurring
to the eukaryotic pre-mRNA
DNA Damage
Classes of DNA damage (Spontaneous and
Induced)
1. Single base changes: minor effect on overall
structure, it’s not likely to completely block
replication or transcription but may lead to
mutant RNA or protein product.
o Deamination: can occur because of
the action of water or included by a
chemical mutagen.
o Alkylation: caused by agents such as
nitrosamines. Guanine 🡪 O6-
methylguanine that mis pairs with
thymine.
o Oxidation: caused by ionizing
radiation and chemical agents.
Guanine 🡪 8-oxoguanine that mis
pairs with adenine.
2. Structural distortion:
o By UV light: cause the induction of
pyrimidine dimers between two
neighboring thymine bases. This
blocks replication and transcription.
o By intercalating agents: ethidium
bromide inserts between DNA
bases, binding and stacking with
them resulting in distortion of the
double helix. Can cause insertions or
deletions during replication.
o By base analogs: base analogs are
similar enough to normal bases that
they can be incorporated into DNA
during replication. They mispair
inappropriately. Thymine 🡪 5-
bromouracil mispairs with guanine.
3. DNA backbone damage:
o Abasic sites (depurination): loss of
the nitrogenous base from a
nucleotide by hydrolysis of the n-
glycosyl linkage (caused by water).
Leaves a hydroxyl (-OH) in its place
in the depurinated DNA.
o Double-strand breaks: can be
induced by x-rays, radioactive
materials, and chemical compounds;
attacking directly the deoxyribose
sugar and generating reactive
oxygen species.
Types of mutations
Mutations result from changes in the nucleotide
sequence (one time), deletions, and insertions
(throughout the DNA).
Point mutations: Nucleotide substitution (a
nucleotide pair in a DNA duplex is replaced with a
different nucleotide pair). This substitution
temporarily creates a mismatched base pair and
during the next round of replication, it becomes
permanently incorporated in the DNA sequence.
● Transition mutation (more common):
replace one pyrimidine base with another
or one purine base with another.
Tautomeric shift: “its easier to fit something
that fits”. Ex. Deamination.
● Transversion mutation: replace a pyrimidine
with a purine or vice versa.
Nucleotide substitutions may have a phenotypic
effect if they alter a critical nucleotide. Ex. In a gene
regulatory region (CDS), in the template for a
functional RNA molecule, in a protein-coding gene.
● Silent: altered codon codes for the same
amino acid. Happens in the promoter and
on CDS.
● Missense: altered codon codes for a
different amino acid. Happens only on the
CDS, the protein is often nonfunctional.
● Nonsense: altered codon is a termination
codon. Happens only in the CDS, protein
synthesis stops, and the protein is usually
nonfunctional.
Insertions or Deletions: the addition or deletion of
one or more base pairs results in a shift in the
reading frame of the resulting mRNA (frameshift
mutations). This leads to the production of a
nonfunctional protein. Happens on the CDS. If the
length if an insertion or deletion is not an exact
multiple of three nucleotides, the mutation shifts
the phase in which the ribosome reads the triplet
codons and alters all the amino acids downstream
the mutation.
DNA Repair
DNA mutations and damage pose a threat to
genomic integrity: they promote disease and cell
death. To cope with this problem cells have
developed: DNA repair enzymes and repair
polymerases. The cellular response to DNA damage
is either:
● To bypass the damage
● To remove the damaged section of DNA and
replace it with undamaged DNA
Lesion bypass
Normal replication uses high-fidelity DNA
polymerases that accurately copy nondamaged
template DNA but are unable to bypass DNA
lesions that cause structural distortion of the DNA
helix. Specialized low-fidelity “error prone” DNA
polymerases (DNA pol IV and V in E-coli & eta and
iota pol in humans) transiently replace the
replicative polymerases and copy past damaged
DNA in a process called translation synthesis (TLS).
This just allows cells to survive a fatal damage.
Removing damaged DNA
Repair systems that remove damaged DNA include:
i. Repair of single base changes/base excision
repair (BER): correction of a single base
changes that are due to conversion.
Initiated by DNA glycosylases.
1. Cleave the glycosidic bond
(deoxyribose sugar – damaged base)
2. Form an abasic site (Ex. human oxoG
repair enzyme (hOGG1), catalyzes
excision of oxoG).
3. The enzymes glycosylase-associated
β-lyase and APE1
(apurinic/apyrimidinic
endonuclease) make nicks 3′ and 5′
to the abasic site in the DNA,
respectively.
4. DNA polymerase β replaces the
missing nucleotide
5. DNA ligase 3–XRCC1 complex seals
the gaps in the sugar–phosphate
backbone.
ii. Mismatch repair (MMR): mismatched base
pairs that result from DNA polymerase
errors during replication.
1. Recognition by MutS
2. MutS-L-H complex generates a nick
3. Exonuclease I (Exol) removes the
nicked fragment. (A large region of
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 DNA including the mismatch is
excised)
4. DNA pol delta replaces the strand
5. DNA ligase I seals.
iii. Nucleotide repair (NER): used to repair
structural distortion ex. Bulges from T-T
dimers.
1. XPA, XPC, RPA, TFIIH (XPD/XPB)
complex recognizes the error
2. TFIIH (Helicase) unwinds DNA
3. XPG (endonuclease) makes a nick at
3’ and XPF-ERCC1 (endonuclease) at
5’
4. The damaged strand is released
5. DNA polymerase δ or ε does the
repair synthesis
6. DNA ligase 1 puts it back together
iv. Double-stranded DNA damage:
● Homologous Recombination: most
important in prokaryotes and
unicellular eukaryotes. Retrieves
genetic information from an
undamaged homologous
chromosome.
1. Double strand break
2. Recognition of 3’-ends:
Rad52
3. Homologous strand invasion:
Rad51
4. Transitional joint molecule
5. Sequence information
restored: DNA synthesis
6. Interlinked molecules joint
by branch migration and
Holliday junction resolution
by resolvasomes.
7. DNA ligase.
● Nonhomologous End-Joining: most
important in mammals. Rejoins
double-strand breaks via direct
ligation of the DNA ends without any
requirement for sequence
homology. Frequently results in
indels at the break site.
1. Recognition of ends: Ku
complex
2. Ku recruits:
● DNA-PKCS (nuclease),
● DNA pol μ and λ and
● DNA Ligase IV
3. DNA-PKCS trims DNA at the
break site and keep ends
joint.
4. DNA pol μ and λ extends 3’-
and 5’
5. DNA ligase IV seals the ends.
Transcription in Prokaryotes
Transcription is the process by which the DNA
strand sequence of a gene is copied into its
complementary RNA strand. It’s the central event
in gene expression (process by which the gene
information is transformed into a protein):
Key enzyme: RNA polymerase
Minimum elements required for transcription:
promoter and RNA pol
Transcription and translation happen at the same
time (coupled), microRNA stops the transcription
process.
● Sense strand (coding or non template)
● Antisense strand (non coding or template)
● Promoter: binding site of the RNA
polymerase to DNA before transcription
begins. Consensus sequences:
o -10 sequence: TATAAT or TATA box
o -35 sequence: TTGACA
o *Uppercase are more conserved vs
lowercase*
● 5’ – and 3’ – UTR: untranslated regions,
regulatory sequences.
● CDS or ORF (Open Reading Frame):
sequence that encodes a protein by triplets
of nt’s (codons). AUG+Codons+Stop.
Translation begins at +1
● Terminator: sequence that indicates the
end of a gene
RNA polymerase
RNA polymerase is made up
by a holoenzyme
● Core-enzyme:
catalyzes the
polymerization of
RNA. 5 subunits
3
 compound (2 alpha, beta, beta prime, and
omega.
● Sigma factor: transcription factor,
recognizes the promoter (sequences -35
and -10). Contact takes place at -10
sequence and forms the transcription
bubble. Bacteria have several types of
sigma factors (i.e. σ70 - 70 kDa - more
abundant). Organisms adapted to a great
variety of environmental conditions have a
greater quantity of sigma factors.
INITIATION
1. RNA pol binds to the promoter (-25, -10)
2. Formation of a closed promoter complex
3. Formation of an open promoter complex
● 18-20 bp are opened (AT-rich), this
is irreversible.
4. NTP’s (nucleotides) are incorporated
5. Exit of the promoter
● 9-12 more NTP’s are added
● Sigma factor is released
● Elongation begins
ELONGATION (begins when sigma factor is
released)
1. RNA pol suffers a conformational change
and slides downstream the promoter
2. Synthesis 5’ 🡪 3’
3. RNA pol melts the DNA as it moves forward
4. Transcription bubble protects the 25-30 bp
against nucleases
Catalytic site:
● Subsite where the substrate (NTP’s)
bind.
● Subsite where the product binds (3’
end).
TERMINATION (RNA pol reaches a terminator)
● Rho dependent: Rho protein (hexamere)
gains access to mRNA at the end of the
gene and binds to a C-rich region: Rho
utilization site (Rut). It chases the RNA pol
and destabilizes the complex.
● Rho independent: inverted consensus
sequence that forms a stem loop followed
by 7-8 uracils in mRNA, the stem loop
destabilizes the RNA pol.
PROOFREADING
RNA pol has a reduced error rate (similar to DNA
pol).
● Short backtracking motion along the DNA
template through several (~5) base pairs.
● Nucleolytic cleavage by RNA pol and discard
the most recently added base(s).
Operons
In bacteria, genes are organized into operons. An
operon is a unit of bacterial gene expression and
regulation that includes structural genes and
control elements. Genes in an operon are
transcribed from a single promoter to produce a
single transcript. “Polycistronic mRNA” with several
genes (2-6)
Structure of an operon
● Regulator genes: codes for a repressor
protein
● Promoter: active site where RNA pol binds
to DNA
● Operator: located between the promoter
and the first structural gene, binding site for
repressor protein (ON or OFF switch)
● Structural genes: several genes that are
read by the same RNA pol producing a large
mRNA, translated into several polypeptides.
Lac Operon (positive inducible CAP)
Inducible Operon (normally OFF but transcription is
ON when allolactose is present). E.Coli uses glucose
as a source of C but when it uses up glucose it
starts digesting lactose.
● Lac I: makes repressor protein
● Lac Z: breaks down lactose into glucose and
b-galactose
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 ● Lac Y: helps in the assimilation
● Lac A: ?
● Cyclic AMP: binds to catabolic activator
protein (CAP) and promotes the digestion of
lactose when there is low glucose.
Mutants of the Lac Operon
Lactose is both and inducer and a substrate.
● Inducer only: IPTG
● Substrate only: X-gal
Operation protein mutant:*The mutation can
affect any chromosome, its diffusible/can move
around (trans)*
Trp Operon (negative repressible)
Repressible Operon (normally ON but transcription
is OFF when Trp is present in large amounts). Tpr is
a co-repressor.
Post-Transcriptional Control
Transcription attenuation of Trp operon
Differential folding of mRNA may stop transcription
and/or translation. During transcription of Trp
operon, the leading region can fold and form a
hairpin structure. The leader RNA contains a small
nucleotide coding region /trpL) which includes 2
Trp codons.
● If tRNA-Trp is abundant ribosomes will
tread the CDS continuously and TrpL will be
synthesized. A termination hairpin forms. It
interferes with RNA pol and transcription is
blocked.
● When tRNA-Trp is used up by the sell the
ribosome stall as a Trp codon in trpL which
forms an antiterminator hairpin. RNA pol
continues transcription and enzymes
responsible for Trp synthesis are expressed.
Riboswitches
Riboswitches are specialized domains within a
mRNA (5’UTR) that act as ON and OFF switches.
The bind metabolites arresting transcription and
preventing translation. They are formed by:
● Aptamer: RNA receptor that binds target
metabolite
● Expression platform: modulates folding
after metabolite-binding event
They are involved in metabolite sensing:
● When the metabolite is produced: it binds
the aptamer and forms terminator hairpin,
blocking translation
● When the metabolite is used up (low
concentrations): aptamer folds differently
forming an antitermination hairpin,
allowing translation
● When the metabolite is present at a
translational level: aptamer folds including
the ribosome biding site (RBS), blocking
ribosomes to initiate translation
Transcription in Eukaryotes
● Promoter: regulatory sequence required to
initiate transcription. Includes
o Core promoter: sequence
recognized by the RNA pol and
general transcription factors. TATA
box. (-31 to -26)
o Proximal promoter
elements/Upstream regulatory
elements: increase frequency of
initiation of transcription. (-200 to
-70)
o Long-range regulatory elements
(Distal): key regulators for cell type.
May contain enhancers or silencers,
bind TF`s, regulate promoter’s
activity. Can be found upstream,
downstream or within transcription
units.
● Transcriptional unit: total RNA strand
synthesized by RNA pol II
o Exons: CDS’s that will result in the
mature mRMA, UTS’s included.
o Introns: sequences of the transcript
that are removed to produce mature
mRNA, they do not encode a protein
but are involved in the regulation of
expression.
o Poly-A signal sequence: 3’- end
sequence used for maturation
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 o Termination region: indicates end of Without these modifications, the mRNA cannot be
transcription formed and can’t exit the nucleus.
Types of RNA pol
● RNA pol I: responsible for synthesis of large
rRNA LECTURE 1: DNA Damage
● RNA pol II: responsible for transcription of Forms of DNA damage unrelated to the process of
protein-encoding genes. No primer needed, replication occur relatively commonly.
has proofreading activity, it’s made up of 12 Exposure to a number of different types of agents, 
subunits and requires TF’s to start ● Oxygen free radicals
transcription. ● UV light
● RNA pol III: responsible for synthesis or ● Ionizing radiation
tRNA, 5s rRNA, and some snRNA. ● Various chemicals
Transcription factors  
Sequence-specific DNA-binding proteins (bind to DNA helix changes and prevents the double
promoters and distal regulatory elements). They melting of the replication. 
are responsible for recruiting and stabilizing RNA  
pol, meditate gene-specific transcriptional Spontaneous DNA damage
activation or repression. Depurination and Deamination
General TF’s: TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.  
Responsible for promoter recognition and
unwinding the promoter DNA.
Post-transcriptional modification of mRNA
1. 5’ – Guanosine Triphosphate Cap: in the
nucleus of the cell, the 5’ end of the pre-
mRNA is altered by the attachment of a
guanosine nucleotide via a 5’ – 5’
triphosphate linkage. The guanosine
nucleotide is quickly methylated at the 7
position to form the 7-methylguanosine.  
This cap protects the mRNA from  
degradation during protein synthesis, it also UV light causes pyrimidine dimers ?
stabilizes the mRNA and aids in
transportation across the nuclear
membrane.
2. Polyadenylation of 3’ end: the 3’ end of the
pre-mRNA is removed, and a series of
adenosine nucleotides are added. The 3’
end tail that contains the many adenines is
called the polyadenine tail. The ply-A tail
provides the mRNA with stability and keeps
the tail from degrading. About 200-250
adenine molecules are added to the tail.
3. Splicing of Exon: before the pre-mRNA
exists the nucleus, special proteins
(spliceosomes) will cut out the introns and  
splice together the exons
6

How chemical modifications of nucleotides
produce mutations
DNA Mutations
As a cell multiplies and divides,
● In most cases the genome is accurately
copied and
● Information is passed on to the next
generation with minimal error.
When DNA polymerases do make mistakes,
● Their proofreading activity generally corrects
the error
● Bu not always
 
Gene mutations affect their protein products in
different ways
Wild type--point mutation--truncation--deletion--
37C--25C
 
High fidelity vs.Low fidelity
● High fidelity DNA replication
● Beneficial for maintaining genetic information
over many generations
 
Low fidelity DNA replication
● Beneficial for the evolution of species, and
● For generating diversity leading to increased
survival
● When organisms are subjected to changing
environments
 
Relevance of Mutations
● Mutations are of fundamental importance in
molecular biology for several reasons.
 
1. Mutations are the major source of genetic
variation
-drives evolutionary change
1. Mutations may have consequences to an
organism or its descendants 
-deleterious or advantageous
1. Mutant organisms are important
tools for molecular biologists
-in characterizing the genes involved in cellular
processes.
 
DNA Repair 
● DNA mutations and damage pose a
continuous threat to genomic integrity.
● Promoting disease
● Cell death
-To cope with this problem cells have evolved
● DNA repair enzymes and 
● Repair polymerases
Origin of Mutations
● A spontaneous mutation
-occurs as a result of natural processes in cells, 
F Example, DNA replication errors.
● Induced mutation
-those that occur as a result of interaction of DNA with
an outside agent or mutagen
● That causes DNA damage Mutation
 
 
Types of Mutations
● Changes in the nucleotide sequence
● Deletions
● Insertions
 
 
Point Mutations
The simplest type of mutations
--A nucleotide substitution change
A nucleotide pair in the DNA duplex is replaced.
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 ●

 
1. Transitions mutations--occurs more  
Replace one pyrimidine base with. Another  Missense Mutations
● Altered codon codes for a different amino
One purine base with another one
1. Transversion mutations acids
Replace a pyrimidine with a purine   
 
● This substitution temporarily creates a 
●  mismatched base pair  
● During the next round of replication
● It becomes permanently incorporated in the
DNA sequence.
 
Types of Point Mutations  
   
Nucleotide substitutions may have a phenotypic ● The protein is often nonfunctional
effect if they alter a critical nucleotide:  
● In a gene regulatory region  
● In the template for a functional RNA molecule Nonsense Mutations
● Mutations in a protein-coding gene ● The new codon is a termination codon
-Silent ● Protein synthesis stops and the protein is
-Missense usually nonfunctional
-Nosense

Silent Mutations
● The altered codon codes for the same amino
acid
 
● Mutations changes in nucleotides that area
outside of coding regions (regulatory regions)
can also be silent. 
 
 
Insertions or Deletions
● The addition or deletion of one or more base
pair
-Results in a shift in the reading frame of the resulting
mRNA
-Leads to production of a nonfunctional protein  

8

Frameshift Mutations
● If the length of an insertion or deletion is not
an exact multiple of three nucleotides,
● The mutation shifts the phase in which the
ribosome reads the triplet codons and,
● Alters all of the amino acids downstream
from the site of the mutation.
Classes of DNA Damage
● A mutagen is any chemical agent that causes
an increase in the rate of mutation above the
spontaneous background.
● Mutagen can be present in our environment
or areas where we live, eat, drink, etc.
● Major impact on public health.
● Damage to DNA consists of any change
introducing a deviation from the usual
double-helical structure.
 
Three major classes of DNA damage
● Single base changes
● Structural distortion
● DNA backbone damage
 
Single Base Change
● A single base change or conversion affects the
DNA sequence.
● But has only a minor effect on overall
structure.
Single Base Change Deamination
● Replacement of the amino group of cytosine
with oxygen
-converts cytosine to uracil (U should only be in RNA)  bromide.
● Most frequent kind of damage, 
-Can occur spontaneously from the action of water, or 
-Induced by a chemical mutagen.
● Causes only a minor structural distortion in
the DNA double helix.
-Not likely to completely block replication or
transcription,
-May lead to a mutant RNA or protein product.
Single Base Change Alkylation
● Alkylating agents such as nitrosamines lead to
the formation of 06-methylguanine.
● This modified base often mis pairs with
thymine,
● Resulting in the change of a GC base pair into
an AT base pair.
 
Single Base Change Oxidation
 
● Potent oxidizing agents (ROS) are generated
by ionizing radiation and by chemical agents.
● Can generate 8-oxoguanine (oxoG)
-a guanine containing an extra oxygen atom.
● OxoG is a highly mutagenic because it can
mismatch with A GC--TA
 
Structural Distortion by UV light
● Generated by UV light
● Bases absorb UV light with a wavelength of
about 260 nm.
● The most frequent lesions are the induction
of pyrimidine dimers between two
neighboring thymine bases.
● Block replication and transcription.
 
Structural Distortion by Intercalating Agents
● Intercalating agents such as ethidium
9
 ● Insert between the DNA bases, binding and
stacking with the DNA bases.
- Resulting in distortion of the double helix,
-can cause insertions or deletions during DNA
replication.
- 5-bromouracil (analog of Thyme) can mispair with
guanine
- TA-CG
 
DNA Backbone Damage Abasic Sites 
● Loss of the nitrogenous base from a
nucleotide
● By hydrolysis of the N-glycosyl linkage by the
action of water.
● Leaves a hydroxyl (-OH) in its place in the
depurinated DNA.
 
DNA Backbone Damage Double-Strand Breaks
● DSB´s can be induced by:
● X-rays, radioactive materiales
-Attacking directly the deoxyribose sugar
-Generating reactive oxygen species
● Wide range of chemical compounds
LECTURE 2: DNA Repair
● Radioactividad como método de vacuna
contra enfermedades. Agua con
isótopos radioactivos. En la época de
Madame Curie se empezó a añadir
Radio al Agua.
 
Cellular Response to DNA Damage
● There are a variety of complex responses to
different types of DNA damage in both
prokaryotes and eukaryotes.
● The responses fall into these main categories:
● those that bypass the damage;
● those that remove the damaged section
of DNA and replace it in with
undamaged DNA.
 
Lesion Bypass
● The normal replication machinery uses high-
fidelity DNA polymerases.
● These high-fidelity polymerases accurately
copy non damaged template DNA.
● But are unable to bypass DNA lesions that
cause structural distortion of the DNA helix.
● Specialized low-fidelity, “error-prone” DNA
polymerases
● DNA pol IV and V in E. coli.
● Several DNA (eta, iota..) pol in humans.
● Transiently replace the replicative
polymerases (might generate mutations)
● copy past damaged DNA in a process called
translesion synthesis (TLS).
Just allows cells to survive a fatal damage
 
Removing Damaged DNA
 
● Repair systems that remove damaged DNA
include:
● Repair of single base changes
● Mismatch repair
● Nucleotide repair
● Double-stranded DNA damage
 
Repair of Single Base Change Base Excision
Repair
● Base excision repair
● correction of single base changes that
are due to conversion.
● Initiated by DNA glycosylases.
● Cleave the glycosidic bond (deoxyribose
sugar- damaged base).
● Forming an abasic site.
● For example, human oxoG repair
enzyme (hOGG1), catalyzes excision of
oxoG.
10
● The enzymes glycosylase-associated β-lyase
and APE1 (apurinic/apyrimidinic
endonuclease) make nicks 3′ and 5′ to the
abasic site in the DNA, respectively.
● DNA polymerase β replaces the missing
nucleotide
● DNA ligase 3–XRCC1 complex seals the gaps
in the sugar–phosphate backbone.
Structural Distortion Mismatch Repair
 
● Mismatch repair (MMR).
● Mismatched base pairs that result from
DNA polymerase errors during
replication.
1. Recognition by MutS
2. MutS-L-H complex generates a nick
3. Exonuclease I (ExoI) removes the nicked
fragment.
1. A large region of DNA including the
mismatch is excised.
2. DNA pol delta replaces the strand
3. DNA ligase I seals.
Nucleotide Excision Repair
 
● NER is used to repair structural distortion,
● E.g. bulges from T-T dimers.
● Recognition:
● XPA, XPC, RPA, TFIIH (XPD/XPB)
complex.
● Unwinding:
● TFIIH (Helicase).
● 3’-nick:
● XPG (endonuclease)
● 5’-nick:
● XPF-ERCC1 (endonuclease)
● The damaged strand is released.
● Repair synthesis:
● DNA polymeras
● e δ or ε
● Ligation:
● DNA ligase 1.
 
Double Strand Break Repair
● Homologous Recombination:
● Most important in Prokaryotes and
unicellular eukaryotes.
● Retrieves genetic information from an
undamaged homologous chromosome.
● Nonhomologous End-Joining
● Most important in mammals.
● Rejoins double-strand breaks via direct
ligation of the DNA ends without any
requirement for sequence homology.
● Frequently results in indels at the break
site.
Homologous Recombination:
● Most important in Prokaryotes and
unicellular eukaryotes.
● Retrieves genetic information from an
undamaged homologous chromosome.
● Recombinación
Nonhomologous End-joining:
● Ligación directa de ambas secuencias,
pega las terminales.
 
Recombinación Homologa, existe cierto
reconocimiento. 18 a 25 pares de bases
1. Double strand break
2. End processing and recognition
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 3. Strand invasion and DNA synthesis
4. Branch migration
5. Holliday junction Resolution and ligation
● Recognition of 3’-ends: Rad52
● Homologous strand invasion: Rad51
● Transitional joint molecule.
● Sequence information restored: DNA
synthesis.
● Interlinked molecules joint by branch
migration and holliday junction resolution by
resolvasomes.
● DNA ligase.
Nonhomologous Joining
1. DNA-PK o KU80-KU70
Reclutan a la enzyma-endonucleasa
2. Ligasa para unir
● Recognition of ends: Ku complex
● Ku recruits:
● DNA-PKCS (nuclease),
● DNA pol μ and λ and
● DNA Ligase IV
● DNA-PKCS trims DNA at the break site and
keep ends joint.
● DNA pol μ and λ extends 3’- and 5’
● DNA ligase IV seals the ends
Homologous Recombination PASOS
1. Recognition of 3 ends: Rad52
2. Homologous strand invasion: Rad51
3. Transitional joint molecule
4. Sequence information restored: DNA
synthesis
5. Interlocked molecules joined by branch
migration and holliday junction
resolution by resolvasomes. resolvases
6. DNA ligase
Homologous Recombination
1. Recognition of ends: Ku complex
2. Ku recruits:
● DNA PKcs nuclease
● DNA pol u and gamma and
● DNA Ligase IV
3. DNA-PKcs trims DNA at at the break site
and keep joins
LECTURE 3: Transcription in Prokaryotes
 
T. Transcription
 
● Process by which the DNA strand
sequence of a gene is copied into its
complementary RNA strand.
● Central event in gene expression.
● Gene expression: process by which the
gene information is transformed into a
protein.
● Key enzyme:
● -DNA-dependent-RNA polymerase or
● -RNA polymerase
Example:
1. 5´-3´Sense strand, coding or non
template
2. 3´-5´Antisense strand, non coding or
template
3. mRNA messenger RNA
4. Protein
microRNA: attaches to the RNA and cannot express
T. Transcription in Prokaryotes
● Transcription and Translation coupled,
efficiency because it has no nucleus
● Single RNA can create multiple proteins
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 Characteristic DNA Pol RNA Pol
 
P. Promoter: Synthesizes DNA RNA
● Binding site of RNA polymerase to DNA
Synthesis 5’🡪3’ 5’🡪3’
before transcription begins.
Direction
 
CDS or ORF (Open Reading Frame): Template DNA (both Antisense DNA
● Sequence that encodes a protein by strands) strand
triplets of nt´s codons. AUG plus Codons
plus Stop Template 3’🡪5’ 3’🡪5’
  reading
T. Terminator: Substrate dATP, dGTP, ATP, GTP, CTP,
● Sequence that indicates the end of a
dCTP, dTTP UTP
gene
  RNA Primer Required None
5´and 3´UTR:  
● Untranslated Regions regulatory
 
sequences.  Holoenzyme:
  ● Core, enzyme
Minimum elements required for transcription: ● Sigma factor
   
● Promoter
C. Core-enzyme
● RNA polymerase
● Catalyzes the polymerase of RNA 
  ● 5 subunits compound ➙ alpha (2α),
P. Promoter beta (β), beta prime (β') and omega (ω).
 
● Consensus sequences:
● -10 sequences TATAAT or TATA
● -35 sequence TTGACA
 
ORF

● Sigma factor (σ) (Transcription Factor)

● Recognizes the promoter (sequences
-35 and -10); 
● -10 sequence: contact takes place and
forms the transcription bubble
● Bacteria have several types of sigma factors
RNA Polymerase (i.e. σ70 - 70 kDa - more abundant).
 

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 ● organisms adapted to a great variety of  
environmental conditions have a
greater quantity of sigma factors.
Initiation
● RNA pol binds the promoter (-35,-10).
1. Formation of a closed promoter complex.
2. Formation of an open promoter complex.
● 18-20 bp are open up (AT-rich)
Termination
● Irreversible
2. NTP’s are incorporated.
1. Exit of the promoter
● After 9-12 NTPs are added
● Sigma factor is released
● Elongation begins
 
 
 
Enlongation
● Begins when sigma factor is released.
● RNA pol suffers a conformational change and
slides downstream the promoter.
● Synthesis 5’ 🡪 3’
● RNA pol melts the DNA as it moves fwd.
● Transcription bubble protects the 25-30 bp
against nucleases.
● Catalytic site
● Subsite where the substrate (NTPs)
binds.
● Subsite where the product binds (3´-
end).
● Rho dependent:
● Rho protein (hexamere) gains access to
mRNA at the end of the gene.
● Binds to a C-rich region: Rho utilization
site (Rut)
● Chases the RNA pol and destabilizes the
complex.
● RNA pol reaches a terminator
● Rho independent:
● Inverted consensus sequence that forms
a - stem loop - followed by 7-8 Uracils in
mRNA
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 ● The stem loop destabilizes the RNA pol  
LacZ breaks lactose units
 
LacY use by bacteria to
use the units
LacA if not the
assimilation of Lactose is not good
Proofreading  CapSite - Promoter - Operator -
● RNA pol has a reduced error rate (similar to ORF> Open Reading Frame
DNA pol).  
Regulatory
● Short backtracking motion along the
CapSite: Catabolic Activator Protein
DNA template through several (~5) base
Catabolism> breaking down
pairs.
 
● Nucleolytic cleavage by RNA pol and With Glucose in the Operator there is a
discard the most recently added base(s). barrier and the info can't be transcribe.
 
With Lactose there is in the repressive protein
a little part where allolactose can binds with
the repressor protein.
 
Polycistronic
ATP makes cAMP in the Cap there is cAMP,
small metbolite that binds with the capsite.
 
TRANSCRIPTION
● Process by which the DNA strand sequence of
a gene is copied into its complementary RNA
strand. 
● Central event in gene expression.
● Gene expression: 
     Process by which the gene 
     information is transformed
     into a protein.
 
 
▪ OPERON units of the genome that are
composed of regulatory elements. Regulation of Gene Expression
  ● Other genes get expressed (regulated) at
▪ Transcription of several genes at once. different times.
Because all of those are necessary for
● Depending on the metabolic requirements of
the same function.
  the cell.
▪ The LAC OPERON o Inducible genes: 
  ● Turn ON when they are required
Glucose. Lactose. Allolactose

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 ● Catabolic pathways (substrate ●When needed, changing their
usage) “machinery” quickly to adjust to
o Repressible genes: changing conditions
The genes required for a specific reaxion or
● Always active, but turn OFF when metabolic pathway are usually grouped in a
they are not required any more complex called Operon
● Anabolic pathways (production of  
metabolites)
Why is Regulation Necessary?
 
Operon
● Other genes get expressed (regulated) at ● In bacteria, genes are organized into operons
different times.
● Operon:
● Depending on the metabolic requirements of
● unit of bacterial gene expression and
the cell.
regulation,
o Inducible genes: 
o including structural genes and
● Turn ON when they are required
control elements
o
● Catabolic pathways (substrate ● Genes in an operon are transcribed from
usage)
● a single promoter
o Repressible genes:
● to produce a single transcript
● Always active, but turn OFF when “polycistronic mRNA.”
they are not required any more
o With several genes (2-6)
●Anabolic pathways (production of
metabolites)  
● Allows cells to take advantage of variable or
different environmental conditions or
● Quickly adjust to changing conditions
● Usually several proteins are involved in a
metabolic pathway (synthesis or catabolic
Rxns)…
● They save energy (more efficient) when  
not required proteins are not
expressed ! Structure of an Operon
● Promoter
● Allow efficient expression of other
● Active site where the RNA polymerase
required proteins.
binds to the DNA
●
Highly orchestrated !
● Structural genes
● Evolution has forced prokaryotes to grow and
reproduce as fast as possible ● Several genes that are read by the same
● Being highly efficient on taking RNA pol producing a large mRNA
advantage of a limited but present ● Translated into several polypeptides
nutrient ● Operator

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 ● Located between the promoter
and the first structural gene
● Binding site for another protein
● ON or OFF switch
● Regulator gene(s)
● Codes for a repressor (protein)
● Repressor protein recognizes a
specific operator
● They determine if the structural
gene(s) get expressed or get silent.
 
Types of Control
 
● Transcriptional level
● Most common type of control
● Factors that regulate transcription
● Interfere with RNA pol
● Most efficient
● Post-transcriptional level
● Factors that regulate expression after
transcription
● Less frequent*
● Influence over mRNA
● Inducible Operon
● An inducer compound (metabolite)
binds the repressor
o Repressor “falls” off the Operator
o Transcription can take place
● Transcription is ON when a nutrient is
present
o Catabolic pathways
▪ Break of a nutrient into
smaller compounds
 
● Inducible Operon
● E. coli uses glucose as source of C
● When it uses up glucose but lactose is
present
o It starts using lactose
 
● Lactose binds repressor protein
● Repressor Protein releases the Operator
● RNA pol can transcribe the operon
●
Break down lactose
● When lactose is used up
● The repressor binds the operator again
● Lac Operon is blocked
● Enzymes to use lactose are not needed
anymore !
 
Transcriptional Control
 
● Repressible Operon
● Transcription always ON but
● When an inducer (co-repressor) is
present it makes the repressor block
transcription
o Anabolic pathways
▪ The enzymes have
produced enough of
their product,
▪ the excess of product
acts as a co-repressor
 
 
Trp Operon
● Repressible Operon
● When Trp is present in large amounts
● It acts as a co-repressor
● binding the repressor protein and
ACTIVATES it
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 ● The activated repressor protein binds
the Operator
o Transcription is blocked
● Trp Operon is in charge of expressing
enzymes involved in Trp biosynthesis.
● When the cell has produced enough
amounts of Trp
● Trp binds the Repressor protein
o To stop the expression of the
enzymes responsible for its
production
▪ No enzymes 🡪 Stop
production of Trp !
● When Trp is used up
● The free repressor protein (without Trp
bound to it) cannot bind the Operator
o Transcription of Trp Operon takes
place.
▪ More Trp is poduced.
Types of Control
● Transcriptional level
● Most common type of control
● Factors that regulate transcription
● Interfere with RNA pol
● Most efficient
● Post-transcriptional level
● Factors that regulate expression after
transcription
● Less frequent*
● Influence over mRNA
 
Post-Transcriptional Control
 
● Expression is regulated at the mRNA level.
● Differential folding of mRNA may stop
transcription and/or translation.
 
Transcription attenuation of Trp operon
 
● Transcription attenuation of Trp operon
● During transcription of Trp operon, the
leading region of the transcript can fold
and form a hairpin structure.
o Terminator
o Antiterminator
● The leader RNA contains a small nucleotide
coding region (trpL)
● Includes 2 Trp codons
● If tRNA-Trp is abundant
● Ribosome will read the CDS
continuously
● TrpL is synthesized
o Terminator hairpin forms
● Interfering with RNA pol
●
Transcription is blocked
● When tRNA-Trp is used up by the cell
● Ribosome stalls at a Trp codon in trpL
● Stalling makes mRNA to form an
antiterminator hairpin
● RNA pol continues transcription
● Enzymes responsible for Trp synthesis
are expressed.
 
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 Post-Transcriptional Control ●
 
● Riboswitches
● Specialized domains within a mRNA (5’-
UTR)
● Act as switches ON or OFF
● Binding metabolites
● arresting transcription
● preventing translation
 
 

● Involved in:
● Metabolite sensing
  o When metabolite is produced
Riboswitches o It binds the aptamer
  o Forms a terminator hairpin
● Structure:
o Blocks transcription
● Aptamer:
o RNA receptor
o Binds target metabolite
● Expression platform
o Modulates folding after
metabolite-binding event

●
● Involved in:
● Metabolite sensing
o When metabolite is used up (low
concentrations)
o Aptamer folds differently
o Forms a antitermination hairpin

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 Allowing transcription
o o
● Involved in:
● Metabolite sensing at translational
level
o When metabolite is present
o Aptamer folds including the
Ribosome binding site (RBS)
Blocking ribosomes to initiate translation
LECTURE 4: Transcription in Eukaryotes
● Transcription and Maturation Steps, Has
several Checkpoints
 
Gene Structure
● Promoter:
● Regulatory sequence required to initiate
transcription.
o Core promoter and
Proximal (and distal) promoter
elements.
● Transcriptional Unit:
● Total RNA strand synthesized by RNA
pol
 
● Core Promoter:
● Sequence recognized by the RNA pol
and
● General Transcription Factors
● Contains the TATA box
● Set of sequences required to start
transcription
● Proximal Promoter Elements
● Also called “upstream promoter
elements” or “Upstream regulatory
elements”
● Located 5´ of the Core Promoter (-70 to
-200)
● Recognized by TF´s
● Increase frequency of initiation of
transcription
 
● Long-Range Regulatory Elements (Distal)
● Key regulators for cell type
● 1 kb or more from core promoter
● May contain enhancers or silencers
o About 500 pb length
o Bind TF´s
o Up- or downregulate Promoter´s
activity
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 o May be found upstream, ● Responsible for synthesis of tRNA, 5S
downstream or within rRNA, and some snRNAs.
transcription units.  
 
  RNA POL ll
Transcription Unit ● RNA pol II
 
● No primer needed
● Transcription Unit:
● Proofreading activity
● Total RNA strand synthesized by RNA
● 12 subunits
pol II
● Requires TF´s to start transcription
● 5´- and 3´-UTR´s:
 
● Regulatory sequences that flank the Transcription Factors
transcript.
● Sequence-specific DNA-binding proteins
 
● Bind to gene promoters and
● Exon:
● CDS´s that will result in the mature ● Long-range regulatory elements
mRNA ● Responsible for recruiting and stabilizing RNA
● UTR´s included pol.
● Intron ● Mediate gene-specific transcriptional
activation or repression.
● Sequences of transcript that are
 
removed to produce a mature mRNA.
● Do not encode a protein
● Involved in regulation of expression
● Poly-A signal sequence
● 3´-end sequence used for maturation  
● Termination Region  
● General TF´s
● Indicates end of transcription
  ● “transcription factor for RNA
  polymerase II,”
RNA POL
● TFIIB, TFIID, TFIIE, TFIIF, and TFIIH
● RNA pol II
● Responsible for promoter recognition and
● Responsible for transcription of protein-
● Unwinding the promoter DNA.
encoding genes
 
● RNA polymerase I
Transcription Mechanism
● Responsible for synthesis of the large
rRNA. ● First transcript = Transcriptional Unit (DNA)

● RNA polymerase III ● Transcript must be processed before

translation

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● Maturation increases stability
● Eliminates non-coding sequences (introns)
● Matured mRNA leaves the nucleus to be
translated

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