INFORMATION

READOUT:
TRANSCRIPTION
TRANSCRIPTION
 Mechanistically similar DNA replication
in terms of substrates and template-
directed growth
 2 major difference:
Only 1 DNA template strand is transcribed
for a particular gene
Only a small fraction of the entire genetic
potential of an organism is relaized in one
cell.
EXISTENCE OF mRNA
 Until about 1960 it was thought that
rRNA represented the set of templates
fro protein synthesis
 Jacob and Monod predicted the
existence of mRNA by analyzing E.coli
mutants that are altered in the control of
lactose metabolism
 Operon model
 T2 and T4 barcteriophage
CHARACTERISTICS OF
mRNA
 There is a high rate of mRNA synthesis
followed by rapid degradation
 Because of the rapid synthesis and
degradation, mRNA accumulates rapidly but
not to high steady state levels
 mRNA, being a copy of 2 or more
contiguous genes, is large and
heterogenous
 mRNA nucleotide sequence is identical to
one of the template strand of DNA
RNA POLYMERASE
 Polynucleotide phosphorylase was initially
thought to be the RNA-synthesizing enzyme
 Ultimately it turned out that it participates in
the degradation of bacterial mRNA
 In prokaryotes, a single RNA polymerase
catalyzes the synthesis of all 3 RNA classes
 In eukaryotes, 3 classes of polymerase
catalyze the different types of RNA
RNA POLYMERASE
 The 3 classes of RNA Polymerase differ
in their sensitivity to inhibition by α-
amanitin
 2 more inhibitors were used to confirm
other characteristics of transcription
Cordycepin
○ Transcription chain terminator
Actinomycin D
○ Intercalating agent
RNA POLYMERASE
KINETICS DNA POLYMERASE RNA POLYMERASE

Turnover number 500 -1000 50 nucleotides/ sec

nucleotides/sec

Number of enzyme/ 10 molecules 3,000 molecules

cell

Processivity High High

Accuracy 10-6 10-5

Error – correction 3’ –exonuclease GreA and Gre B

mechanism
RNA POLYMERASE STRUCTURE
SUBUNIT Mr # per Function
enzyme
molecule
α 36,500 2 Chain initiation , interaction with regulatory
proteins and upstream promoter elements

β 151,00 1 Chain initiation and elongation

0
β’ 155,00 1 DNA binding
0
σ 70,000 1 Promoter recognition

ω 11,000 1 unknown
RNA POLYMERASE STRUCTURE
INITIATION OF TRANSCRIPTION
ELONGATION: Incorporation of
Ribonucleotides
PROMOTER RECOGNITION
 The most frequently transcribed genes
initiate transcription about once every
10sec,whereas some genes are
transcribed as infrequently as once per
generation (30-60 min)
 Rate –limiting step
 Each gene transcribed in E.coli shared a
short adenine and thymine rich
sequence (CONSENSUS SEQUENCE)
CONSERVED SEQUENCES IN
PROMOTERS
TERMINATION
 In bacteria, 2 distinct types of
termination events exists:
Factor –Independent
Factor – Dependent
FACTOR-INDEPENDENT
 2 structural
features:
The symmetrical
GC-rich segments
that in the trasncript
have the potential
to form a stem-loop
structure
A downstream run
of four to eight A
residues
FACTOR DEPENDENT
TERMINATION
 Less frequent
 Rho protein is a
hexamer composed of
identical subunits
 It is an RNA –DNA
helicase which
contains nucleotide
triphosphatase acivity
that is activated by
binding to
polynucleotides
REGULATION OF TRANSCRIPTION

 Most genes are kept in a turned-off until

their products are needed
 Genes concerned primarily with
anabolism are kept turned-on unless the
product of the anabolic sequence is
present, then the genes are repressed
PROKARYOTIC REGULATION OF
TRANSCRIPTION
 The Lactose Operon
 Bacteriophage λ
 The SOS regulon
 Biosynthetic operons
THE LACTOSE OPERON
 Consists of 3 linked structural genes that
encode enzymes of lactose utilization
plus adjacent regulatory sites
 Structural genes z,y and a encode β-
galctosidase, β- galactoside permease
and thiogalactoside transacetylase
 In the presence of an inducer, all 3
enzymes accumulate simultaneously but
at different levels
REGULATION IN LAC
OPERON
 2 distinct mutant phenotypes:
Constitutive
Noninducible
 These 2 mutations mapped in 2 sited o
and i
 Transcription of the 3 structural genes is
initiated near an adjacent site called the
operator
 This will yield a polycystronic mRNA
REGULATION IN LAC
OPERON
ISOLATION AND PROPERTIES OF
REPRESSOR
 Isolated by W.
Gilbert and
B.Muller-Hill in 1966
 A tetramer, each
with 360 amino
acids
 i gene is expressed
at a very low rate
(10 molecules /cell)
but very efficient
THE REPRESSOR BINDING SITE
 The operator is composed of 35 bp, with
28 bp of symmetrical sequence
REGULATION BY
GLUCOSE
 Glucose repression or catabolite
repression
 Decrease in the levels of glucose would
increase the cAMP levels
 The increase would trigger activation of
the lac operon by its interaction with a
protein cAMP receptor protein (CRP)
BACTERIOPHAGE λ
BACTERIOPAHGE λ
 2 repressors: cI and Cro
 Each binds 2 different operators.
 The operators contain 3 repressor
binding sites and promoter sites
interspersed with the repressor binding
site
 Transcrition from the 2 promoter-
operator sites takes place in opposite
directions along the genome
Lambda cI REPRESSOR
 A dimeric protein with
a subunit molecular
weight of 27,000
 Transcription from
OLPL and ORPR is
controlled by cI and
Cro
GENES in LAMBDA PHAGE
 cI and cro – code for the repressors
 cII and cIII – stimulate cI synthesis
 rex – unknown function
 O and P – initiation of DNA replication
 N – product interacts with NusA to
prevent termination
 Q – product activates late gene
transcription
INTERACTION BETWEEN THE 2
REPRESSORS
 OLPL controls transcription of N through
interaction of its repressor binding site
with the cI protein
 However, most regulatory action occurs
at ORPR and it is here that the decision is
made between lysogenic and lytic
infection
Cro REPRESSOR
 Homodimer of 66-residue subunits
folded into 3 alpha-helical regions and 3
beta strands
 Helices 2 and 3 fit into the major groove
of the DNA
THE SOS REGULON
 REGULON – set of unlinked genes regulated by
a common mechanism
 Lytic infection is induced through DNA damaging
treatments (i.e. UV irradiation)
 lexA and recA gene products are the control
elements in E.coli SOS regulon
 RecA – stimulates strand pairing during
recombination and can stimulate proteolytic
cleavage of proteins when bound to ssDNA
 LexA – a repressor that binds at least 25 different
operators scattered in the genome
THE SOS REGULON
 In healthy cells, lexA and recA are expressed
at low levels
 The triger that activates the SOS system after
damage is ssDNA
 RecA binding within a gap activates LexA
proteolysis
 Decrease in conc of LexA removes LexA
barrier to recA transcription. RecA accumulates
 Simultaneously, cleavage of LexA protein
activates transcriptio of all genes under lexA
control (including cI repressor)
BIOSYNTHETIC OPERONS
 Since biosynthetic pathways utilize
energy, the regulatory goal is to repress
gene activity by turning off the synthesis
of the enzymes in the pathway when the
end product is available
 trp operon
POSTTRANSCRIPTIONAL
PROCESSING
 The major postranscritional event in
metabolism of prokaryotic mRNA is its
own degradation
 mRNA have half-lives of ~2-3 mins
 Degradation starts from the 5’ end
POSTTRANSCRIPTIONAL
PROCESSING
 rRNA processing
Contains 7 different operons for rRNA
species .
Each one encodes one copy of 16S, 23S
and 5S in a single transcript
It also includes sequences for 1-4 tRNA
molecules
Initial transcript form each operon is a 30S
RNA molecule
RNaseIII
POSTTRANSCRIPTIONAL
PROCESSING
 tRNA processing
tRNAs are
synthesized in
transcripts
containing 1-7
tRNAs each all
surrounded by
lengthy flanking
sequences
TRANSCRIPTION IN EUKARYOTIC
CELLS
 Transcription is precisely programmed
during development and tissue
differentiation
 Needs to deal with the complicated
levels of structure of eukaryotic
chromatin
 Presence of different polymerases which
requires transcription factors to bind to a
promoter and initiate transcription
RNA POLYMERASE I
 18S (small), 28S, 5.8S and 5S (large)
 28S, 18S and 5.8S all come from a large 45S pre-
mRNA transcript
 Pol I contains 13 sunbunits (600kDa) and requires at
least 2 TF
 The nucleolus is the site of ribosomal subunit
assembly
 About 6800 nucleotides will be discarded in the
process
 The processed rRNAs will combine with 5S from other
regions of the nucleus and are exported to the cytosol
RNA POLYMERASE III
 Largest and most complex
 Involves 14 subunits (700kDa), requires
at least 3 TF (TFIIIB,C and A)
 Catallyzes production of tRNAs and 5S
rRNA
RNA POLYMERASE II
FACTOR # of MW (kDa) FUNCTION
subunits

TFIID TBP -1 38 Core promoter recognition; TFIIB

(TBP+TAF) TAF -12 15-250 recruitment
Core promoter recognition; + and –
regulatory functions

TFIIA 3 12,19,35 Stabilization of TBP binding; stabilization of

TAF-DNA intxn
TFIIB 1 35 RNA polII-TFIIF recruitment ; start-site
selection by RNA pol II
TFIIF 2 30,74 Promoter targeting of polII; destabilization of
nonspecific polII-DNA intxn
RNA polII 12 10-220 Catalysis; recruitemnt of TFIIE

TFIIE 2 34,57 TFIIH recruitement, modulation of TFIIH

helicase, ATPase and kinase activities
TFIIH 9 35-89 Promoter melting using helicase activity,
MAJOR PROBLEMS IN
TRANSCRIPTION
 How can transcription factors and
initiation complex bind to DNA in the
presence of nucleosomes?
 How can the actively transcribing
polymerase pass through arrays of
nucleosomes?
PROBABLE ANSWERS
 Presence of hypersensitive sites
Susceptible to digestion by nucleases
Appear in the 5’ flanking regions of the
embryonic genes
These provide points at which TF and other
trans-acting proteins can gain access to
promoters and enhancers, allowing initiation
and stimulation of transcription
PROBABLE ANSWERS
 Chromatin Remodelling
Chromatin remodelling factors
SWI/SNF from yeast and NURF from Drosophila
Both require ATP hydrolysis
Exact mechanism is still unclear
Histone acetyltransferase and deacetylase
High acetylation of histone increases transcriptional
activity
Acetylation of histones in promoter nucleosomes
contributes to loosening of chromatin structures in
these regions
TERMINATION
 The eukaryotic pol II continues to transcribe
well past the end of the gene
 In doing so, it passes through 1 or more
AATAAA signals
 The pre-mRNA carrying this signal as
AAUAAA is cleaved by special
endonuclease
 After cutting at a site 11 to 30 residues 3’ to
it, a poly A tail is added by the non-template
directed polymerase
POST TRANSCRITIONAL
PROCESSING
 Addition of 5’ capping
 Splicing