Chapter 11

Transcription

Central dogma
replication

transcription

DNA
Carrier of genetic
information in
chromosome

RNA

reverse
transcription

translation

protein
executor of genetic
information in
cytoplasm

Genetic Information Transfer

Transcription
The synthesis of RNA molecules using
single DNA strands as the templates
so that the genetic information can be
transferred from dsDNA to ssRNA.

Section 1
Template and Enzymes

Attention:The whole genome of DNA

needs to be replicated, but different
with replicationg only small portion of
genome(about 1%) is transcribed into
RNA.
DNA regions that can be transcribed
into RNA are called structural
genes.

1.1 Template
The template of transcription is single strand
DNA.(Only one strand is copied, but the other
strand is not)
The template strand The single strand DNA
being copied is called template strand or
Watson strand.
The coding strand The other strand that is not
copied is called coding strand or Crick strand.
It has the same sequence as the transcriptive
product RNA, except that the RNA contains U
instead of T.

5'

GCAGTACATGTC

3' coding

3'

CGTCATGTACAG

5'

strand
template
strand

transcription

5'

GCAGUACAUGUC

3'

RNA

Asymmetric transcription
The modle of transcription is asymmetric
transcription. It has two meanings:
First, in the double strand DNA, one
strand is copied, but the other strand is
not.
Second, the strand being copied is not
always in the same strand, or sometimes
one strand is copied, sometimes the
other.

5'

3'

3'

5'

1.2 RNA Polymerase

The enzyme responsible for the RNA
synthesis is DNA-dependent RNA
polymerase.
The prokaryotic RNA polymerase is a
multiple-subunit protein of ~480kD.
Eukaryotic systems have three kinds of
RNA polymerases, each of them is a
multiple-subunit protein and responsible
for transcription of different RNAs.

RNA-pol of Prokaryote

consists of 5 different subunits: 2 .

subunit

MW

36512

still discussing

150618

Catalyze polymerization

155613

Bind & open DNA template

70263

Recognize the promoter(start

region of gene)for synthesis
initiation

function

 Rifampicin, a therapeutic drug for

tuberculosis treatment, can bind
specifically to the subunit of RNApol, and inhibit the RNA synthesis.

RNA-pol of eukaryotes
RNA-pol

II

III

products

45S rRNA

hnRNA

5S rRNA
tRNA
snRNA

Sensitivity
to Amanitin

No

high

moderate

Amanitin is a specific inhibitor of RNA-pol.

rRNA
18S

28S

5.8S

45S-rRNA

transcription

splicing
18S-rRNA

5.8S and 28S-rRNA

1.3 Structure of transcription region

Only small portion of gene in the chromosome
can be transcribe into RNA. Each transcriptable
Unit is called operon.
One operon includes several structural genes
and upstream regulatory sequences (The
specific sequence on the DNA template that
regulate the transcription of structure gene).
The most important regulatory sequences is
promoter,which is the DNA sequence that RNApol can bind,and initiate the transcription
process. It is the key point for the transcription
control.

Operon
regulatory
sequences
5'
3'

RNA-pol

structural gene
3'
5'

promotor

promoter is the DNA sequence that RNA-pol can

bind,and initiate the transcription process. It is the
key point for the transcription control.

Prokaryotic promoter
3'

5'
3'

-50

-40

-30

-20

-35
region
TTGACA
AACTGT

-10

1
-10
region

10

5'

start

T A T A A T
A T A T T A
(Pribnow box)

Two important promoter consensus sequence are found

in prokaryote , one located about 10 nucleotides (-10
sequence)upstream of where transcription will begin
and one located about 35 nucleotides up stream(-35
sequence). The consensus sequence TATAAT located in
10 sequence is also called Pribnow box.

Consensus Sequence

Frequency in 45 samples

38 36 29
37 37 28

40 25 30
41 29 44

 The -35 region of TTGACA

sequence is the recognition site
(Recognize by subunit).
The -10 region of TATAAT is the
binding site of RNA-pol.

Promoter of eukaryote
cis-acting element
structural gene
GCGC

CAAT

TATA

exon

intron

exon

start
TATA box

enhancer

CAAT box

(Hogness box)

-25 region

GC box

The promoter consensus sequence of eukaryote is TATA which

locate on the -25 region, also called Hogness box or TATA box.
There are other promoters upstream the transcriptive start site,
such as Inr box(YYCARR) CAAT box and GC box .

Section 2
Transcription Process

2.1 Transcription of Prokaryotes

Initiation phase: RNA-pol recognizes the
promoter and starts the transcription.
Elongation phase: the RNA strand is
continuously growing.
Termination phase: the RNA-pol stops
synthesis and the nascent RNA is
separated from the DNA template.

Transcription in bacteria
RNA polymerase loosely bound to
DNA moves, and bind tightly to the
promoter region.
RNA polymerase opens up DNA helix,
and begin to synthesize RNA from
one strand of DNA (sense strand).
Sigma factor is released after
synthesizing about 10 nucleotides of
RNA.
RNA synthesis is being continued,
and reaches to the termination site.

RNA polymerase is being released

and join the sigma factor to the next
transcription.

Fig. 7-9

1. RNA is made at the speed of 1,500 bases/50 seconds.

2. Many RNA copies are made from the same gene in a relatively
short time, and the synthesis of the next RNA usually being
started before the first RNA is completed.
3. If 15 RNA polymerases work on a gene, they can make 1,000
transcripts made in an hour.

Fig. 7-8

c. Termination
The RNA-pol stops moving on the DNA
template. The RNA falls off from the
transcription complex.
The termination of prokaryote using two
different manners :
1.Rho -dependent manner.
2.Rho -independent manner.

The termination function of Rho factor

The Rho factor, a hexamer, is a helicase. It

can combine with PolyC residues specially.

Rho-independent termination
The termination signal is a stretch of 3040 nucleotides on the RNA transcript,
consisting of many GC followed by a
series of U.
The sequence specificity of this nascent
RNA transcript will self-complementary
to form particular stem-loop structures
to terminate the transcription.

DNA

rplL protein

5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 3
5TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3

RNA

UUUU...

Stem-loop

Stem-loop disruption
The stem-loop structure alters the
conformation of RNA-pol, leading to
the pause of the RNA-pol moving.
Then the competition of the RNA-RNA
hybrid and the DNA-DNA hybrid
reduces the DNA-RNA hybrid stability,
and causes the transcription complex
dissociated.

2.2 Transcription of Eukaryotes

a. Initiation
Promoter will be recoganizd and binded
by RNA-pol to start transcription just like
the initiation of prokaryote.

Promoter of eukaryote
cis-acting element
structural gene
GCGC

CAAT

TATA

exon

intron

exon

start
TATA box

enhancer

CAAT box

(Hogness box)

-25 region

GC box

The promoter consensus sequence of eukaryote is TATA which

locate on the -25 region, also called Hogness box or TATA box.
There are other promoters upstream the transcriptive start site,
such as CAAT box and GC box .

Transcription factors(TF)
Unlike prokaryote, the RNA-pol of eukaryote
cant bind the promoter directly. Some kind of
protein which can recognize and bind directly
or indirectly on promoter ,and regulate its
activity is necessary.Such of these protein
are called as transcription factor (TF).
RNA-pol II associates with six transcription
factors, TFII A.B.D.E.F.H.

Pre-initiation complex (PIC)

RNA pol II
TF II F
TF II
A

TBP TAF

TATA

TF II
B

TF II E

TF II H

DNA

Phosphorylation of RNA-pol
TF II H is of protein kinase activity to
phosphorylate CTD of RNA-pol. (CTD
is the C-terminal domain of RNA-pol)
Only the p-RNA-pol can move toward
the downstream, starting the
elongation phase.
Most of the TFs fall off from PIC
during the elongation phase.

b. Elongation
The elongation is similar to that
of prokaryotes.

c. Termination

The termination signal of eukaryote is

AAUAAA sequence followed by GT
repeats.

Section 3
Post-Transcriptional
Modification

 The nascent RNA, also known as

primary transcript, needs to be modified
to become functional tRNAs, rRNAs,
and mRNAs.
The modification is critical to eukaryotic
systems.

3.1 Modification of hnRNA

Primary transcripts of mRNA are
called as heteronuclear RNA
(hnRNA).
hnRNA are larger than matured
mRNA by many folds.
Modification includes
Capping at the 5 - end
Tailing at the 3 - end
mRNA splicing

a. Capping at the 5 - end

OH

OH

O
N
H2N

O
H2C

5'

HN

O
O

P
O

O
O

P O

NH

5'

CH2

NH2

N
O

CH3

Pi

OH

O
O

AAAAA-OH 3'

m7GpppGp---Immediately after synthesis , the 5 end of the primary

transcript is modified by the addition of a methylated
guanine cap.

 Function of 5- cap :
It can protect primary RNA that
cant be broken up by exonuclease.
The primary RNA which havent 5cap structure will be broken up by
exonuclease immediately.

b. Poly-A tailing at 3 - end

The endonuclease cleaves the RNA transcript
at a site of a AAUAAA sequence that is called
a polyadenylation signal. Poly(A) polymerase
then adds 100-200 A residues to the new 3
end using ATP as precursor.
Attention:There is no poly(dT) sequence on
the DNA template. The tailing process does
not depend on the template.

 Function of PolyA tailing:

It contribute to the stability and
integrity of mRNA. The length of PolyA
tailing is accord with the survival time
of mRNA in vivo.

c. mRNA splicing
mRNA

primary RNA

The matured mRNAs are much shorter than

the primary RNA.

Split gene
The structural genes of eukaryote are
discontinuous ,and are composed of
coding and non-coding regions that are
alternatively separated.
L

B C D

7 700 bp

A~G no-coding region 1~7 coding region

DNA: prokaryote vs. eukaryote

Prokaryote DNA

Eukaryote DNA

Fig. 7-13

Exon and intron

Exons are the coding sequences that
appear on split genes and primary
transcripts, and will be expressed to
matured mRNA.
Introns are the non-coding sequences
that are transcripted into primary
mRNAs, and will be cleaved out in the
later splicing process.

mRNA splicing

RNA splicing is the precise cutting out of

intron sequences and joining the ends of
neighboring exons to produce a functional mRNA
molecule.

Signal sequences of intron removal

1. Three sequences are required to remove the introns.

2. The sequences are recognized by snRNP.

Fig. 7-15

RNA splicing
mechanism
1. Splicing is done by enzyme complex,
called small nuclear
ribonucleoprotein particles
(snRNP), pronounced snurps.
2. snRNPs are composed of protein
and RNA.
3. A specific sequence A in the intron
attacks 5-end of the intron, and cut
the RNA.
4. The cut 5-end of the intron becomes
covalently joined to the A, forming
a loop called lariat.
5. The free 3-end of the exon then cuts
and joins with the second exon.
6. The lariat is released and digested.

Fig. 7-16

3.2 Modification of tRNA

Cleavage

RNAase P
endonuclease

ligase

Addition of CCA-OH

tRNA nucleotidyl
transferase
ATP

ADP

Base modification

1
1

1. Methylation
AmA, GmG
2. Reduction
UDHU
3. Transversion
U

3
4

4. Deamination
AI

3.3 Modification of rRNA

45S transcript in nucleus is the
precursor of 3 kinds of rRNAs.
The matured rRNA will be assembled
with ribosomal proteins to form
ribosomes that are exported to
cytosolic space.

rRNA
18S

28S

5.8S

45S-rRNA

transcription

splicing
18S-rRNA

5.8S and 28S-rRNA

(ribozyme)
RNA

 rRNA

rRNA
5-

 rRNA tRNA mRNA

(hammerhead structure)
60