Transcription in

Prokaryotes
M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

Gene expression begins with
transcription

RNA copy of a gene made by an RNA
polymerase

Prokaryotic RNA polymerases are assemblies of
several different proteins
Bacterial genomes have simple gene structure

- Promoter
-35 sequence (T82T84G78A65C54A45) 15-20 bp
-10 sequence (T80A95T45A60A50T96) 5-9 bp (Pribnow Box)
- Start of transcription : initiation start: Purine90 (sometimes
its the A in CAT)
- Translation binding site (Shine-Dalgarno) 10 bp upstream of
AUG (AGGAGG)
- One or more Open Reading Frame
start-codon (unless sequence is partial)
until next in-frame stop codon on that strand ..
Separated by intercistronic sequences.
- Termination
Bacterial Gene: Structure of signals
Gene 1
Gene 2
RNA polymerase must know where the
start of a gene is in order to copy it

RNA polymerase has weak interactions
with the DNA unless it encounters a
promoter
A promoter is a specific sequence of
nucleotides that indicate the start site for RNA
synthesis
General Steps of Transcription
Initiation:
Binding of RNA polymerase to double stranded DNA
Development of closed promoter complex
Development of open promoter complex
Start of transcription by adding the first two
ribonucleotides.
Elongation:
- Formation of transcription bubble or Transcription
elongation complex.
Progression of the complex gradually in the
3 direction to elongate the initiated RNA chain.
Rapid process: up to 40 nucleotides per second.
On the same gene there are several RNA strands being
transcribed in a staggered fashion.
Termination:
Terminator sequences signal stop of transcription.
Sigma
dissociates
Initiation
Irreversible form of Open Complex Formation
Generation of Abortive Initiation
Products
Transcription Bubble
RNA Elongation
Reads template 3 to 5
Adds nucleotides 5 to 3
(5 phosphate to 3
hydroxyl)
Synthesis is the same as
the leading strand of DNA
Polymerization is polar: enzyme works by adding
to a free 3 hydroxyl in growing mRNA chain.
RNA Synthesis

RNA pol moves
nt by nt,
unwinds the
DNA as it goes
Will stop when
it encounters a
STOP.
RNA pol
leaves,
releasing the
RNA strand
Termination of Transcription
Factor-independent termination

Factor-dependent termination
3 factors
Rho (), Tau () and NusA
Rho best studied
Termination of transcription
RNA: single stranded nucleic acid
can form secondary structures

Rho-dependent termination: protein signal
Rho binds to RNA; able to cause RNA &
RNA polymerase to leave DNA
termination

Rho-independent signal: hairpin or stem-
loop RNA structure forms, followed by
several uracils
termination
Terminator Sequences
In prokaryotes there are two types:

1. Intrinsic: Rho () independent terminator

Contains a G-C rich region followed by six or more
A-T sequences.
Causes the formation of a double stranded RNA
called a hairpin loop.
Retards the movement of the RNA polymerase
along the DNA molecule, and causes
termination at the A-T rich region.



2. Extrinsic: Rho-dependent terminator
Requires a protein factor called Rho ().
Rho protein trails the RNA polymerase until
it reaches a GC rich region, when Rho
catches up with the polymerase.

Rho protein pulls off RNA from transcription
bubble.


1. Rho-independent terminator site
RNA transcript at the terminating site is
self-complementary
The bases can pair to form a hairpin
structure with a stem and loop, a structure
favored by its high G-C content
The stable hairpin is followed by a
sequence of 4 or more U residues
The RNA transcript ends within or just
after them
Intrinsic termination site
String of Us
Intrastrand
complementary
bases
Mechanism of Rho-independent
Termination
RNA polymerase pauses when it encounters
such a hairpin formed at the terminator site
The RNA-DNA hybrid helix produced after
the hairpin is unstable because of its content
of rU-dA base pairs, the weakest of the four
kinds of pairs
Nascent RNA is pulled off from the DNA
template and then from the enzyme
DNA template strand now joins its partner to
form the DNA duplex

Control of trp operon by attenuation:
stalled translation allows region
2 to interact with region 3

3 & 4 cannot interact

regions 3 & 4 interact;
termination results
Global control systems in E. coli:
In global control systems: many genes, pathways regulated
simultaneously in response to a specific environmental signal
e.g., regulon: collection of genes and/or operons controlled
by common regulatory protein

Sporulation in Bacillus: another global control system:
2. Rho-dependent terminator
Rho () Protein
Rho is an RNA-dependent ATPase
Also an RNA-DNA helicase
It is an hexamer, with a mass of 275 kDa (each
subunit is of 46 kDa)
It binds to ssRNA at Rut site a stretch of 72 nt
is bound, 12 per subunit
It is brought into action by sequence located in
the nascent RNA
ATPase activity enables it to move
unidirectionally along the nascent RNA
Effect of rho protein on the size of
RNA transcripts
Rho factor: factor
mediated termination
In an ATP-mediated
reaction, a rho protein
complex binds to the
mRNA and unwinds
RNA from the DNA
template
Recognition sites
may not have hairpins
or U tracts; tend to be
C-rich
?
Rho-dependent:
One Transcriptional unit
Two contiguous genes
RNA is released so we can make many
copies of the gene, usually before the first
one is done
Can have multiple RNA polymerase molecules on
a gene at a time
Initiation
site
Termination
site
RNA fibrils
Summary
Types of mRNA
In Bacteria:
Monocistronic mRNA
Polycistronic mRNA

In Eukaryotes:
Monocistronic mRNA
Polycistronic mRNA
Many prokaryotic mRNAs are polycistronic
Contain sequences specifying the synthesis of
several proteins

A polycistronic mRNA molecule possesses a
series of start and stop codons
In case it codes for three proteins:
Start, Protein1, Stop Start, Protein2, Stop
Start, Protein3, Stop

Abou 5-20 bases may be present between one
stop codon and the next start codon. These are
called Spacers.

The segment of RNA corresponding to a DNA
cistron is called a Reading frame
Polycistronic vs Monocistronic mRNA
spacers
Processing of pre-rRNA transcripts
in E. coli