Transcription in Prokaryotes

Agustin Krisna Wardani

Gene Expression
The expression of a gene to form a polypeptide
occurs in two major steps:
1.

2.

Transcription copies the information of a DNA

sequence (the gene) into corresponding
information in an RNA sequence.
Translation converts this RNA sequence into
the amino acid sequence of a polypeptide.

The central dogma of molecular biology

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

DNA vs RNA

RNA much shorter than DNA

-DNA contains the code for making lots of different
proteins.
-Messenger RNA contains the information to make just
one protein
DNA has two strands arranged in a double helix. RNA
consists of a single strand.
DNA (deoxyribonucleic acid) has a backbone of alternating
deoxyribose and phosphate groups. In RNA (ribonucleic
acid), the sugar ribose replaces deoxyribose.
RNA uses the base uracil (U) rather than thymine (T):
A---U, G---C

RNA (ribonucleic acid) has the sugar ribose

replaces deoxyribose.

RNA (RiboNucleic Acid)

DNA (DeoxyriboNucleic Acid)

RNA uses the base uracil (U) rather than thymine (T):

The only difference between the two

molecules is the presence or absence of
the CH3 group.

Similarity between
replication and transcription

Both processes use DNA as the

template.

Phosphodiester bonds are formed in

both cases.

Both synthesis directions are from

5 to 3.

Differences between
replication and transcription
replication

transcription

template

double strands

single strand

primer

yes

no

Enzyme

DNA polymerase

RNA polymerase

product

dsDNA

ssRNA

base pair

A-T, G-C

A-U, T-A, G-C

The Amazing Gene Code (TAGC).

Transcriptional Control
DNA

RNA

Environmental change

Turn gene(s) on/off

protein
Proteins to deal with
new environment
Very important to:
1. express genes when needed
2. repress genes when not needed

The whole genome of DNA needs to be

replicated, but only small portion of genome
is transcribed in response to the
development requirement, physiological
need and environmental changes.
DNA regions that can be transcribed into

RNA are called structural genes.

Transcriptional Control
Many places for control

Transcription
DNA

RNA

protein

Initiation
Elongation
Termination

Processing
Capping
Splicing
Polyadenylation
Turnover

Translation
Protein processing

Prokaryotic Transcription

Operons
Groups of related genes transcribed
by the same promoter

Polycistronic RNA
(one mRNA code several genes)

Multiple genes transcribed

as ONE TRANSCRIPT

No nucleus, so transcription and

translation can occur simultaneously

Recognition of Origins

Each transcriptable region is called

operon.

One operon includes several structural

genes and upstream regulatory
sequences (or regulatory regions).

The promoter is the DNA sequence that

RNA-pol can bind. It is the key point
for the transcription control.

Nisin Operon

Template and Enzymes

Template
The template strand is the strand
from which the RNA is actually
transcribed. It is also termed as
antisense strand.

The coding strand is the strand

whose base sequence specifies the
amino acid sequence of the encoded
protein. Therefore, it is also called as
sense strand.

5'

GCAGTACATGTC

3' coding

3'

CGTCATGTACAG

5'

strand
template
strand

transcription

5'

GCAGUACAUGUC

3'

RNA

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 (I, II, III), each of which
is a multiple-subunit protein and
responsible for transcription of different
RNAs.

Types of RNA

Messenger RNA (mRNA) genes that encode

proteins

Ribosomal RNA (rRNA) form the core of

ribosomes

Transfer RNA (tRNA) adaptors that link

amino acids to mRNA during translation

Transcriptional Control
Transcription

Control of initiation
usually most
important.

Initiation
Elongation
Termination

Processing
Capping
Splicing
Polyadenylation
Turnover

Translation
Protein processing

General concepts

Three phases: initiation, elongation,

and termination.

The prokaryotic RNA-pol can bind to

the DNA template directly in the
transcription process.

The eukaryotic RNA-pol requires cofactors to bind to the DNA template

together in the transcription process.

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.

a. Initiation

RNA-pol recognizes the TTGACA

region, and slides to the TATAAT
region, then opens the DNA duplex.

The unwound region is about 171 bp.

Initiation

RNA polymerase
Promoter DNA
RNAP binding sites

RNA Polymerase in prokaryote

RNA-pol of E. Coli
subunit

MW

function

36512

Determine the DNA to be

transcribed

150618

Catalyze polymerization

155613

Bind & open DNA template

70263

Recognize the promoter

for synthesis initiation

Initiation

RNA polymerase
4 core subunits
Sigma factor ()
determines promoter
specificity
Core + = holoenzyme
Binds promoter sequence
Catalyzes open complex and
transcription of DNA to RNA

RNA Polymerase II in eukaryote

Promoter

The first thing that the enzyme has to do is to find the

start of the gene on the coding strand of the DNA.
These base sequences are known as promoter
sequences.

Promoter

regulatory
sequences
5'
3'

promotor
RNA-pol

structural gene
3'
5'

Prokaryotic promoter
3'

5'
-50
3'

-40

-30

-20

-35
region
TTGACA
AACTGT

-10

-10
region
TATAAT
ATATTA
(Pribnow box)

Consensus sequence

10

start

5'

Consensus Sequence

The -35 region of TTGACA sequence

is the recognition site and the
binding site of RNA-pol.

The -10 region of TATAAT is the

region at which a stable complex of
DNA and RNA-pol is formed.

RNAP binds specific promoter

sequences
Sigma factors recognize consensus
-10 and -35 sequences

RNA polymerase promoters

TTGACA

TATAAT

Deviation from consensus -10 , -35 sequence leads to

weaker gene expression

Bacterial sigma factors

Sigma factors are transcription factors

Different sigma factors bind RNAP and recognize
specific -10 ,-35 sequences
Helps melt DNA to expose transcriptional start site
Promote broad changes in gene expression
E. coli 7 sigma factors
B. subtilis 18 sigma factors
Generally, bacteria that live in more varied
environments have more sigma factors

Sigma factors
Sigma subunit

Type of gene controlled

70 RpoD

Growth/housekeeping

54 RpoN

N2; stress response

~15

RpoS

Stationary phase, virulence

~100

RpoH

Heat shock

~40

RpoF

Flagella-chemotaxis

~40

Extreme ?heat shock, unfolded proteins

~5

Ferric citrate transport

~5

32 RpoE
FecI

# of genes controlled
~1000

E. coli can choose between 7 sigma factors and about 350

transcription factors to fine tune its transcriptional output
An Rev Micro Vol. 57: 441-466 T. M. Gruber

elongation
RNA-pol, DNA form a complex called
the transcription bubble.
The 3 segment of the nascent RNA
hybridizes with the DNA template, and
its 5 end extends out the
transcription bubble as the synthesis
is processing.

Once the enzyme has attached to the DNA, it

unwinds the double helix over a short length, and
splits the two strands apart. This gives a "bubble" in
which the coding strand and template strand are
separated over the length of about 10 bases.

Transcription bubble

the enzyme moves along the DNA, zipping it up

again behind it. Essentially it moves the bubble
along the chain, adding new nucleotides all the
time. The growing RNA tail becomes detached from
the template strand as the enzyme moves along.

RNA synthesis

Transcriptional Control
Transcription
Initiation
Elongation
Termination

Processing
Capping
Splicing
Polyadenylation
Turnover

Translation
Protein processing

How does the enzyme know where to stop after it

reaches the end of the gene?

there will be a termination sequence of bases.

Once the enzyme gets to those, it stops adding new
nucleotides to the chain and detaches the RNA
molecule completely from the template chain.

Transcriptional Termination

Bacteria need to end transcription at the

end of the gene
2 principle mechanisms of termination in
bacteria:

Rho-independent (more common)

Rho-dependent

Rho-independent termination

Termination sequence has 2 features:

Series of U residues
GC-rich self-complimenting region
GC-rich sequences bind forming stem-loop
Stem-loop causes RNAP to pause
U residues unstable, permit release of RNA chain

Rho-dependent termination

Rho is protein (50kDa) that terminate

transcription

Rho attach rut (rho utilization site),

Binds the 70-80 base segment of RNA

Rho moves along RNA and catch up to

RNAP, then unwinds DNA/RNA hybrid

Rho has ATPase activity which can

induce release of the polymerase from
DNA.

Mechanism of action of rho (p) factor in termination of transcription, involving

its travel along RNA till it catches RNA polymerase pausing at a RNA hairpin

RNA hairpins followed by a run of U residues in rho independent

terminator, but not in rho dependent terminator of transcription

Quorum Sensing

Bacteria produce and secrete chemical signal

molecules (autoinducers)
Concentration of molecules increases with
increasing bacterial density
When critical threshold concentration of
molecule is reached, bacteria alter gene
expression
Way for communities of bacteria to talk to
each other