Transcription

Flow of Genetic Information

RNA
Transcription
DNA Protein
Replication Translation
 Transcription

Transcription: production
of mRNA copy of the
DNA gene.

Eukaryote model
 Transcription

RNA
Not all RNA is translated into protein:

❧ Some RNA is structural - e.g. ribosomal RNA (rRNA)

❧ Some RNA is functional - e.g. transfer RNA (tRNA)
❧ Some RNA is chromosomal (some viruses)
 Transcription

From which DNA strand is RNA synthesized?

• Transcription usually takes place on only ONE

of the DNA strands
 Transcription

RNA growth always in the 5' → 3' direction

5'-GTCACCCATGGAGG-3' Nontemplate strand
3'-CAGTGGGTACCTCC-5' Template strand
5'-GUCACCCAUGGAGG-3' mRNA

3' mRNA 5'

5' 3' DNA
3' 5' DNA
5' mRNA 3' 5' mRNA 3' 5' mRNA 3'
 The classification of RNA molecules

• mRNA •Bring information from the DNA

•messenger RNA
•Transcript of protein-coding genes
hence
are translated into protein
•Not stable
2. rRNA •Ribosomal RNA
•80% of total RNA
•Component of ribosome
•Stable
•Involved in protein synthesis

3. tRNA •transfer RNA

•Has specific secondary and tertiery struc.
•Binds amino acid to mRNA
•Long lifespan
•Involved in protein synthesis
•There is a proofreading mechanism

4. snRNA •small nuclear RNA

•Involved in processing RNA
 Transcription
❧The process by which RNA molecules
are synthesized on a DNA template is
called transcription
❧Transcription results in the synthesis
of a single stranded RNA molecule
complementary to the DNA template
❧The ribonucleotide sequence written
in RNA is the genetic code which is then
capable of directing the process of
translation, which produces polypeptide
chains
Similarities and differences from
replication
SIMILARITIES DIFFERENCES

• 5’-3’ direction • RNAP not DNAP

• Many proteins • Proofreading
involved • Posttranscriptional
• Initiation, modification
elongation and • 1 strand copies not 2
termination • Not all transcribed
• Transcription
bubble
• Starts and stops at
specific sites
Prokaryotic transcription as in the
bacteria E. coli Requirements for Transcription

1. Single-stranded (ss) DNA

template
• Non-coding DNA strand acts as template

2. All 4 RNA triphosphate nucleotides

•ATP, GTP,UTP,CTP (NTP)
3. DNA dependent RNA Polymerase (a holoenzyme
consisting of subunits)

RNA polymerase/DNA dependent RNA polymerase

One in procaryote
Three in eucaryote (RNAPI-III)
 RNA Synthesis/ Transcription

Ingredients necessary for transcription

4. Bivalent ions
•Mg2+

5. Activators •Help in binding to DNA

•Increase the rate of transcription

6. Promoter
Determines when a gene is on/off
Has sequence to bind RNAP & σ
When you change this consensus seq- you alter rate of
transcription
Weak promoters have additional binding domains
 7. Transcription factor

Consensus sequences found in promoter

Protein Function Seq at –35

Seq at–10

Sigma 70 housekeeping TTGACA

TATAAT
Sigma 32 heat shock
TCTCNCCCTTGAA CCCCATNTA
Sigma 28 flagella synthesis CTAAA
CCGATAT
The consensus sequence share homology in
different genes of same organism or in one
gene or more genes of related organism

Cis acting element

Degree of RNAP binding to different

promoters varies and due to sequence
variation in the promoter which lead to
variable in gene expression
RNA POLYMERASE

•Transcribes all proc. DNA

•In euc.:

-RNA pol II* transcribes mRNA & snRNA

-RNA pol I * transcribes16S & 23S RNA (rRNA)

-RNA pol III * tRNA & 5S rRNA

-RNA pol IV * mitochondrial RNA

•Also has subunit α, β, β’ like the proc. RNAP

•Generally all RNAP are zinc metalloenzymes
•No proofreading: mistakes every 104-105 bases
RNA polymerase procaryote

4 polypeptide subunits 2α, 1 β,1 β’ & 1 σ (~500kDa)

- is the holoenzyme

Subunit α & β form the core enzyme

β-provide the catalytic basis and active site for

transcription

Subunit σ70- needed for transcription i.e in initiation

of transcription
 The function of sigma factor
• the sigma subunit of RNA polymerase is an “initiation factor”

• there are several different sigma factors in E. coli that are

specific for different sets of genes

• sigma factor functions to ensure that RNA polymerase binds

stably to DNA only at promoters

• sigma destablizes nonspecific binding to non-promoter DNA

• sigma stabilizes specific binding to promoter DNA

• this accelerates the search for promoter DNA

 What is the function of sigma factor?

s factor is critical in promoter recognition, by

decreasing the affinity of the core enzyme for
non-specific DNA sites and increasing the
affinity for the corresponding promoter
s factor is released from the RNA pol after
initiation (RNA chain is 8-9 nt)

• Is involved in opening and closing of the DNA

double helix
• It helps to regulate the expression of a gene
set/family in different conditions
 • closed promoter complex (moderately stable)
• the sigma subunit binds to the –35/-10 region
s
RNA polymerase holoenzyme (+ s factor)

• open promoter complex (highly stable)

• the holoenzyme has very high affinity for
promoter regions because of sigma factor

s
• once initiation takes place, RNA polymerase does
not need very high affinity for the promoter
• sigma factor dissociates from the core polymerase
after a few elongation reactions

• sigma can re-bind

s other core enzymes • elongation takes place with

the core RNA polymerase

The sigma cycle

RNA polymerase function

• catalyzes the addition of 5’-ribonucleoside triP

at the 3’ end of growing polyribonucleotide

• Select the DNA template [3’-5’]

• Separates the two DNA strands

• Recoils the DNA after transcription

Procaryote Promoter

-10 site
•The start site for transcription
•The 5’-TATAAT-3’ is conserved in
E.coli and phage Pribnow Box
•Orientates RNAP to move from left to right

-35 site
•Is to the left of Pribnow Box
•Has a 6 base conserved sequence 5’-TTGACA-3’
•The RNAP binds to this site and initiates
transcription
Prokaryote Promoter Sequences
 Promoter

❧ conserved sequences required for specific

binding of RNA pol and transcription initiation
❧ RNA polymerase seeks out the consensus
sequences for proper orientation for binding to
initiate transcription.
❧ Note promoter sites have regions of similar
sequences at the -35 region and -10 region.
❧ Minus numbers represent bases upstream of
mRNA start point, +1 is the first base in the
RNA transcript.
❧ The -35 and -10 boxes contain consensus
sequences
 Promoter structure in prokaryotes

mRNA
5’ PuPuPuPuPuPuPuPu AUG

[ ]
-30 -10 +1

Promoter
transcription start site
mRNA
5’
-35 region -10 region
TTGACA TATAAT
AACTGT ATATTA
-36 -31 -12 -7 +1 +20
Pribnow box
T T G AC A T AT A AT
82 84 79 64 53 45% 79 95 44 59 51 96%
consensus sequences
Eucaryotic Promoter

•–25 site  consensus seq. 5’TATAAAA3’ 

TATA BOX
•Also known as the Hognes Box
•Mutation doesn’t effect transcription; effects
the start site of transcription
•CAAT Box  5’-GGCAATCT-3’ is on the left
(upstream) to the TATA Box
•Mutation of this site effects the rate of
transcription
•GC Box  5’-GGGCGG-3’ ; functions in binding
the RNAPII to the transcription site
Eucaryotic Promoter
TATA
(-25)

CAAT +1
(-75) Transcription start site

•No sigma factor 70

•There are about 6 subunits (inclusive 2α, 1 β,1 β’ )
•General transcription factors– 6 to 8 (e.g. TFIIA-J)

Enhancer
•Tissue specific expression at the right time
•Present upstream or down stream
•Binds with regulatory factors
 Transcription: organization of a gene

+1 site

Negative Positive
numbers numbers
Initiation of RNA chains:
• binding of RNAP holoenzyme to promoter region

1) RNAP holoenzyme binds loosely to -35 region (dsDNA), then

tightly to the -10 region of dsDNA (closed promoter)
The transcription cycle is involves a series of
events between binding of RNAP to target
gene and dissociation of RNAP and the
completed RNA transcript from the DNA
The transcription cycle can be divided into 3
phases
c. Initiation
d. Elongation
e. termination
Trancriptin initiation can be divided into
3 steps
In the first step the RNAP bind to a region of DNA
call promoter. In bacteria this step involves the
initiation factor call sigma which recognized various
seq within the promoter
The RNAP with sigma attached bind to the promoter
in a defined orientation so the same stand always
transcribed from a given promoter
The RNAP and the promoter form a closed complex
In the second step, closed complex
undergo a transition to the open complex
conformation.
The pincer in front of the RNAP clamp
down tightly downstream of the DNA.
Sigma also changed conformation and the
DNA strand is seperated forming a bubble
of single stranded DNA
❧ In Step 3, once the open promoter
complex is formed, RNA Polymerase
catalyzes the insertion of the first 5’
ribonucleotide which is complementary to
the 1st nucleotide at the start site of the
DNA template.
❧ No primer is required!
❧ Subsequent complements are inserted and
linked together by phosphodiester bonds
❧ After several ribonucleotides have been
added, the sigma subunit dissociates and
elongation proceeds
E. coli RNA polymerase + σ subunit
RNAP synthesizes several short RNAs
before entering the elongation phase –
abortive initiation
Shot RNA molecules of less than ten
nucleotides in length
This is probably because the region of sigma
partially block the RNA exit channel.
Once this region has been ejected and
polymerase able to make an RNA longer than
ten bp, a stable ternary complex is formed,
consist of enzyme, DNA template and
growing RNA chain.
This is the beginning of elongation phase
2) Localized unwinding of the two strands of DNA provides
template strand for RNAP and exposes initiation site (+1)
4) phosphodiester bond
forms between the NTP’s of
RNA chain

3) unwinds dsDNA (≈ 17 bp) around

-10 region

5) RNAP chooses correct

strand to read (template)
template

6) initiates ≈ 8-9 bp then

σ factor is released
• Core has reduced affinity for promoter
When cores loses sigma
factor it moves away from promoter
• Transcription bubble ≈17 bp
• Core completes elongation
• RNAP unwinds dsDNA
• mRNA displaced from back
• RNA/DNA hybrid exists
 Elongation
● Core RNA polymerase adds nucleotides to form
complementary mRNA strand (transcript)
● Ribonucleotides enter the active site and are
added to the growing RNA chain under the
guidance of the template DNA strand
● Only eight to nine nucleotides of the growing
chain remain base-paired
● The remainder of the RNA chain is peeled off
and directed out of the enzyme
● In E. coli, 50 nucleotides/second at 37 degrees
C
During the elongation RNAP unwind the DNA
in front of the enzyme , synthesized RNA,
proofread RNA , dissociate RNA from the
DNA and re-anneal of DNA behind the
enzyme.

In contrast with DNAP, RNAP is able to do

this functions without the assistance with
other proteins
TERMINATION

Sequences called terminator trigger the

elongating polymerase to dissociate from the
DNA and release the RNA chain it has made
Two types of termination rho- independent
and rho-dependent

Rho-independent terminator also called

intrinsic terminator.

Consist of two sequence elements: a short

inverted repeat ~ 20 nucleotides followed by
a stretch of about eight A:T base pair
The RNA that result from the inverted
repeat is able to form a stem-loop
structure by base-pairing with itself
Is called a hairpin
The hairpin is believed to cause termination
by disrupting the elongation complex
The A:U base pairs are the weakest of all
base pairs, they are more easily disrupted
by the effect of stem loop on the
transcribing polymerase and allowing the
RNA to dissociate from DNA
 The steps in transcription: Termination

• rho-independent termination rho

Terminators
• complementary region (G:C-rich) which forms hairpin loop
in the ssRNA – RNAP pauses on UUU and falls off. Termination

C G G:C rich area

C G
complementarity causes hairpin
C G
mRNA C G
RNAP pauses on UUU region
3’
UUUU
5’ RNAP Falls off
Bound together by H bond

Terminators exist either:

• Intrinsic to RNA strand - intrastrand base pairing (rho-independent)
• Extrinsic - requires an accessory protein - rho to stop (rho-dependent)
 Termination

• Direct termination: The RNA hairpin loop of GC

(inverted repeats sequences and section of U
residues appear to serve as signal for RNA
polymerase release and termination of
transcription.
• ρ-independent Termination
• Inverted repeat downstream from stop
codon in DNA sequence will form “hairpin”
in mRNA transcript

❧ mRNA folds around center . When RNA

polymerase assembles hairpin, it pauses and
falls off
• .
• ρ-dependent Termination
• Less well-characterised
• ρ-dependent terminators have inverted
repeat in DNA sequence, but do not have
repeated A’s
• ρ- protein required for termination
• rho protein binds to specific sequences referred
to as rut. rho pulls RNA polymerase off RNA
Possible model for ρ-dependent termination
 Eukaryotic Transcription

❧ Transcription in Eukaryotes is much more complex than

the bacterial system.
❧ Three types of RNA Polymerases exist in Eukaryotes,
each responsible for transcribing certain types of genes.
❧ RNA Pol I, II and III (or A, B and C)
❧ Cis acting elements involved: CAAT (-70--80 from
start)
❧ and TATA (-25 from start)
❧ enhancers and transcription factors also involved
Eucaryotic Promoter
TATA
(-25)

CAAT +1
(-75) Transcription start site

Enhancer
•Tissue specific expression at the right time
•Present upstream or down stream
•Binds with regulatory factors
❧ transcription factors bind to the DNA to assist in
initiation: TFIID, TFIIB, TFIIA, and seven others
❧ In eukaryotes RNA Polymerase ll is the mRNA
producer
❧ hnrna: 5’ methylguanosine cap added, prior to transport
out of nucleus.
❧ poly a tail ( several to 250) added after cap; degrades
rapidly if tail missing
❧ Interviening sequences
● Introns = non coding regions
● ex. collagen has 50 introns
●
Histones and interferon have no introns
Transcription initiation in eucaryote

1. Binding to the promoters

• At TATA Box
• No sigma factor but has transcription
factors [TFIIA-J]
2. Start of transcription
•TFIIA-J will dissociate when transcription is initiated
•RNA polymerase open DNA helix
•RNA polymerase moves across template
•Transcript has similar sequence to non template strand
where urasil replaces tiamine
•RNAP will rewind the DNA that already been
transcribed
Formation of pre-initiation complex
Complete set of general transcription factors and
polymerase which bound together at the promoter and
ready for initiation, is called the pre-initiation complex
The formation of this complex begin at TATA element
TATA element is recognized by TFIID. TBP is a
component of TFIID which binds to TATA DNA sequence.
Once binding DNA, TBP extensively distorts the TATA
sequence.
The resulting TBP-DNA complex provides a platform to
recruit other general transcription factors and
polymerase
Formation of the pre-initiation complex then followed by
promoter melting, ATP is required and mediated by
TFIIF which has helicase like activity that can unwind
the promoter DNA
In eucaryote promoter escape involves phosphorylation
of the polymerase
The large subunit of Pol II has a C-terminal domain (CTD)
CTD contains a series of repeats of the heptapeptide
sequence: Tyr-Ser-Pro-Thr-Ser-Pro-Ser.
(Yeast 27 repeats in the yeast polII CTD, human 52)
Addition of phosphates group helps polymerase shed
most of the general transcription factors used for
initiation, and which the enzyme leaves behind as it
escape the promoter
Stepwise assembly of a transcription-initiation complex from
isolated RNA polymerase II (Pol II) and general transcription
factors
Once transcribed, eucaryotic RNA has to be processed
These processing include: capping of the 5’ end of the
RNA are, splicing and polyadenylation of the 3’ end
The first RNA processing event is capping
Critical for efficient translation of mRNA
and transport out of nucleus.

Involves addition of a methylated guanine base to the 5’

end of the RNA by unusual 5’-5’ linkage
8. Removal of phosphate group from 5’ end of the
transcript
9. Addition of GTP
10. Modified by addition of methyl group
 The Ends of Eukaryotic mRNAs: 5’- Capping

Features:
• Triphosphate bridge
• 5’ to 5’ linkage of guanine (reverse orientation)
• Guanine is methylated 7’ position
• First 2 NT’s of RNA can be methylated
• NO cap coded for in DNA
• Essential for ribosome to bind to 5’-end
 Polyadenylation

•The final processing event and intimately linked with

termination of the transcription

• Once polymerase has reached the end of a gene it

encounters specific sequences after being transcribed
into RNA, trigger the transfer of the polyadenylation
enzyme to that RNA

•Leading to three events: cleavage of the message,

addition of many adenine residues to its 3’ end and
termination of transcription by polymerase

•Thought to stabilize mRNA from degradation

• Aids in efficiency of translation
Embedded in transcript is a poly A site

Cuts 11-30 NT downstream of

poly A site

Uses ATP
 RNA splicing
• Group 1
• Introns of primary transcript in rRNA
• Intron has autolytic Ribozyme
• Will self splice with guanosine cofactor
• Group 2
• mRNA and tRNA, mitochondrial and chloroplast RNA
• also self splicing but no cofactor necessary
• Spliceosome formation
 In Eukaryotes :
• RNA splicing – removal of intron sequences
R-loops which correspond to areas missing in mature RNA
Pre-RNA or DNA
Mature RNA
Exons – amino acid coding regions
“Expressed sequences” found in both a gene’s
DNA and in the mature mRNA

Introns – non-amino acid coding regions

“Intervening sequences” found in a gene’s DNA but
not in the mature mRNA (removed from the primary
transcript)
Splicing

Gene is 2.5 million base pairs in length

Exons ≈ 50-2,000 Bp
Introns ≈ 50-100,000 Bp
Introns very common in
eukaryote genes
l
 How are Introns Removed?
• RNA primary transcript has all introns and exons
RNA splicing involves:
• removal of introns
• stitching together of exons to form contiguous RNA
• very precise mechanism to protect integrity of
reading frame!
Must have mechanism in place to distinguish between
intron and exon junction
Must have enzymes to splice out the introns

Splicing of introns is usually carried out by a complex of enzymes

known as a spliceosome
 Short sequences dictate the sites of splicing

• Introns begin with 5’-GU and end with 3’-AG

• Also intron includes a branch-point sequence upstream of 3’ splice site
• Key base is the Adenine of the branch site

• Process is usually carried out by a complex of proteins

• known as the spliceosome
• made of snRNPs (small nuclear ribonuclear proteins)
 Splice Acceptor site

Splice Donor site

Regulated Process:
snRNPs
• cut at junction of
exon 1/ intron

• loop intron and join

to branch-point A

• cut at junction of
intron/exon 2

• Exons joined
• lariat degraded
 In Prokaryotes :

5’ end of transcript has a triphosphate,

rather than a methylated cap

No tail at 3’ end

No introns
 Prokaryotic vs. Eukaryotic Transcription
❧ Prokaryote
❧ All promoters upstream of functional gene
❧ Main promoter consensus sequences TATAAT (-10) and TTGACA (-35)
❧ One RNA polymerase with σ subunit makes mRNA, tRNA, rRNA
❧ No enhancers
❧ mRNA is primary transcript – “ready to go” – short lifetime (just a few
minutes)

❧ Eukaryote
❧ Promoter positions differ for each polymerase- not all upstream
❧ Main consensus sequence TATA box (-25) and CAAT box (-60 to -120)
Plants have AGGA instead of CAAT
❧ RNA POL I – rRNA
❧ RNA POL II – mRNA
❧ RNA POL III – ss rRNA, tRNA
❧ DNA enhancer regions work with some promoters to increase transcription
❧ Initial product of transcription is not usable mRNA. Primary transcript must
be processed to form mRNA. Longer lifetime (hours/days)
Review Questions
❧ What are the enzymes involved in replication?
❧ What are the characteristics of replication?
❧ Describe the entire process of replication
noting correctly the sites, proteins and
enzymes involved
❧ What is the difference if any between the
procaryotic and eucaryotic replication
process?
❧ What is the function of methylation in the
regulation of replication?