trancription in prokaryotes

1. TRANSCRIPTION IN
PROKARYOTES
MRS POONAM SINGH
ASSISTANT PROFESSOR
PG DEPARTMENT OF ZOOLOGY, MU

2. PROMOTER BINDING
The RNA polymerase core enzyme α2ᵝᵝ’ω has a general nonspecific affinity for DNA.
This is referred to as loose binding and it is fairly stable.
When σ factoris added to the core enzyme to form the holoenzyme, it markedly
reduces the affinity for nonspecific sites on DNA by 20 000-fold
In addition, σ factor enhances holoenzyme binding to correct promoter-binding sites
100 times.
Overall, this dramatically increases the specificity of the holoenzyme for correct
promoter-binding sites.

3. PROMOTER BINDING
The holoenzyme searches out and binds to promoters in the E. coli genome
extremely rapidly.
This process is too fast to be achieved by repeated binding and dissociation
from DNA, and is believed to occur by the polymerase sliding along the DNA
until it reaches the promoter sequence.
At the promoter, the polymerase recognizes the double-stranded –35 and –10
DNA sequences.
The initial complex of the polymerase with the base-paired promoter DNA is
referred to as a closed complex.

4. The σ subunit of prokaryotic RNApolymerase recognizes consensus sequences found in the promoter region upstream of
the transcription start sight. The σ subunit dissociates from the polymerase after transcription has been initiated.

5. DNA UNWINDING
In order for the antisense strand to become accessible for base pairing,
the DNA duplex must be unwound by the polymerase.
Negative supercoiling enhances the transcription of many genes, since
this facilitates unwinding by the polymerase.
However, some promoters are not activated by negative supercoiling,
implying that differences in the natural DNA topology may affect
transcription, perhaps due to differences in the steric relationship of the –
35 and –10 sequences in the double helix

6. DNA UNWINDING
For example, the promoters for the enzyme subunits of DNA gyrase are
inhibited by negative supercoiling.
DNA gyrase is responsible for negative supercoiling of the E. coli genome
(see Topic C4) and so this may serve as an elegant feedback loop for
DNA gyrase protein expression.
The initial unwinding of the DNA results in formation of an open complex
with the polymerase;
and this process is referred to as tight binding.

8. RNA CHAIN INITIATION
Almost all RNA start sites consist of a purine residue, with G being more
common than A.
Unlike DNA synthesis , RNA synthesis can occur without a primer (Fig. 1).
The chain is started with a GTP or ATP, from which synthesis of the rest
of the chain is initiated.
The polymerase initially incorporates the first two nucleotides and forms a
phosphodiester bond between them.

9. RNA CHAIN INITIATION
The first nine bases are added without enzyme movement along the DNA.
After each one of these first 9 nt is added to the chain, there is a significant probability
that the chain will be aborted.
This process of abortive initiation is important for the overall rate of transcription since
it has a major role in determining how long the polymerase takes to leave the
promoter and allow another polymerase to initiate a further round of transcription.
The minimum time for promoter clearance is 1–2 seconds, which is a long event
relative to other stages of transcription.

11. RNA CHAIN ELONGATION
When initiation succeeds, the enzyme releases the factor and forms a ternary
complex (three components) of polymerase–DNA–nascent (newly synthesized)
RNA, causing the polymerase to progress along the DNA (promoter
clearance)allowing re-initiation of transcription from the promoter by a further
RNA polymerase holoenzyme.
The region of unwound DNA, which is called the transcription bubble, appears
to move along the DNA with the polymerase.
The size of this region of unwound DNA remains constant at around 17 bp
(Fig.2), and the 5-end of the RNA forms a hybrid helix of about 12 bp with the
antisense DNA strand. This corresponds to just less than one turn of the RNA–
DNA helix

12. RNA CHAIN ELONGATION
The E. coli polymerase moves at an average rate of 40 nt per sec, but the
rate can vary depending on local DNA sequence.
Maintenance of the short region of unwound DNA indicates that the
polymerase unwinds DNA in front of the transcription bubble and
rewinds DNA at its rear.
The RNA–DNA helix must rotate each time a nucleotide is added to the
RNA.

14. RNA CHAIN TERMINATION
The RNA polymerase remains bound to the DNA and continues transcription until it
reaches a terminator sequence (stop signal) at the end of the transcription unit (Fig.
3).
The most common stop signal is an RNA hairpin in which the RNA transcript is self-
complementary.
As a result, the RNA can form a stable hairpin structure with a stem and a loop.
Commonly the stem structure is very GC-rich, favoring its base pairing stability due to
the additional stability of G–C base pairs over A–U base pairs.
The RNA hairpin is often followed by a sequence of four or more U residues.

15. RNA CHAIN TERMINATION
It seems that the polymerase pauses immediately after it has synthesized the
hairpin RNA.
The subsequent stretch of U residues in the RNA base-pairs only weakly with the
corresponding A residues in the antisense DNA strand.
This favors dissociation of the RNA from the complex with the template strand of
the DNA.
The RNA is therefore released from the transcription complex.
The non-base-paired antisense strand of the DNA then re-anneals with the sense
DNA strand and the core enzyme disassociates from the DNA.

16. RHO DEPENDENT TERMINATION
While the RNA polymerase can self-terminate at a hairpin structure followed by a
stretch of U residues, other known terminator sites may not form strong hairpins.
They use an accessory factor, the rho protein () to mediate transcription
termination.
Rho is a hexameric protein that hydrolyzes ATP in the presence of single-stranded
RNA.
The protein appears to bind to a stretch of 72 nucleotides in RNA, probably
through recognition of a specific structural feature rather than a consensus
sequence.

17. RHO DEPENDENT TERMINATION
Rho moves along the nascent RNA towards the transcription complex
There, it enables the RNA polymerase to terminate at rho-dependent
transcriptional terminators. Like rho-independent terminators, these
signals are recognized in the newly synthesized RNA rather than in the
template DNA.
Sometimes, the rho-dependent terminators are hairpin structures which
lack the subsequent stretch of U residues which are required for rho-
independent termination.