1.
PROKARYOTIC TRANSCRIPTION
PRASANNA R KOVATH
ASSISTANT PROFESSOR
DEPARTMENT OF BIOTECHNOLOGY

2.
The region 5’ of the promoter sequence is called upstream sequence
The region 3’ of the terminator sequence is called downstream sequence

3.
TRANSCRIPTION PROCESS
•The transcription process involves three steps:
1.Initiation
2.Elongation
3.Termination

4.
TRANSCRIPTION INITIATION
• A particular DNA sequence called as promoter is required in
prokaryotes to find the right starting point of RNA polymerase enzyme
transcription .
• This section is located close to the start of transcription.
• Promoters are important part of the regulatory region of a gene.
• The synthesis of RNA transcript starts 5’ of the promoter and
continues towards 3’.
• Being in 5 prime of the gene, promoter region is also referred to
regulator 5 .

5.
Process : facilitated diffusion mediated by nonspecific binding.
It was proposed that sequences in the −35 region would affect
the initial binding of RNA polymerase to the promoter

6.
How does the enzyme move from a random
binding site on DNA to a promoter?
• There is considerable evidence that at least three different processes
contribute to the rate of promoter search by RNA polymerase .
• First, the enzyme may move in a one-dimensional random walk along
the DNA ("sliding" ) .
• Second, given the intricately folded nature of the chromosome in the
bacterial nucleoid, having bound to one sequence on the chromosome,
the enzyme is now closer to other sites, reducing the time needed for
dissociation and rebinding to another site ("intersegment transfer" or
"hopping").
• Third, while bound nonspecifically to one site, the enzyme may
exchange DNA sites until a promoter is found ("direct transfer" ).

7.
PROKARYOTIC PROMOTERS
• The promoters of prokaryotes are comprised of two short sequences
from the transcriptional start site which are located upstream at -10
and -35 bp (base pairs) position.
• Sequence at position -10 is called Pribnow box or the element of -10.
This sequence usually contains 6 nucleotide as TATAAT.
• Pribnow box is required for the initiation of transcription in
prokaryotes.
• The other sequence at position -35, is mostly composed of six
nucleotide comprising TTGACA.
• The presence of this -35 sequence position ensures the higher rate of
transcription

8.
• Binding of RNA-polymerase (RNAP) to the promoter region is
followed by a conformational change of the RNAP, and the first
nucleotide (almost always a purine) then associates with the initiation
site on the subunit of the enzyme.
• In the presence of the appropriate nucleotide, RNAP catalyzes the
formation of a phosphodiester bond, and the nascent chain is now
attached to the polymerization site on the subunit of RNAP.
• In both prokaryotes and eukaryotes, a purine ribonucleotide is usually
the first to be polymerized into the RNA molecule.
• After 10–20 nucleotides have been polymerized, RNAP undergoes a
second conformational change leading to promoter clearance.
• Once this transition occurs, RNAP physically moves away from the
promoter, transcribing down the transcription unit, leading to the next
phase of the process, elongation.

9.
Not all RNA polymerase complexes transcribe until the end of the
gene. Many transcription complexes dissociate from the template
after adding a couple of rNTPs, a process called abortive
transcription.

10.
STEPS OF INITIATION
• Sigma factor (σ) binds to promoter sequence (-10, -35 sequence).
• Core enzyme binds to the sigma factor (σ) and promoter but DNA is
still closed.
• This is called the closed promoter complex.
• Holoenzyme untwist the double strands of DNA.
• Untwisted promoter is called the open promoter complex.
• RNA polymerase binds to -10 sequence and placed in position to start
transcribing.
• Sigma factor (σ) is released so that core enzyme can go forward
transcribing.

11.
Structure of the σ Subunit RNA-DNA Hybrid Separation

12.
A ternary complex composed of RNA polymerase (RNAP), DNA template,
and RNA transcript is the central intermediate in the transcription cycle
responsible for the elongation of the RNA chain.

13.
• RNA polymerase (core enzyme) moves along to transcribe the DNA
sequence into a single strand RNA of the coding gene.
• When transcribing, the RNA polymerase interact with DNA sequence
forming transcription bubble.
• DNA double helix is reformed as the RNA polymerase moves
forward.
• Few RNA nucleotides (newly synthesized) form an RNA/DNA hybrid
within RNA polymerase.
• As transcription proceed, single strand RNA gets out of the RNA
polymerase.
TRANSCRIPTION ELONGATION

14.
A transcription bubble is a molecular structure formed during DNA transcription when
a limited portion of the DNA double strand is unwound. The size of a transcription
bubble ranges from 12-14 base pairs. A transcription bubble is formed when the RNA
polymerase enzyme binds to a promoter and causes two DNA strands to detach. It
presents a region of unpaired DNA, where a short stretch of nucleotides are exposed on
each strand of the double helix.

15.
TRANSCRIPTION TERMINATION
• Once a gene is transcribed, the prokaryotic polymerase needs to
be instructed to dissociate from the DNA template and liberate
the newly made mRNA.
• Depending on the gene being transcribed, there are two kinds of
termination signals. One is protein-based and the other is RNA-
based.
• There are specific signals for the termination of transcription
(stop). Terminators are:
1. Rho-independent terminator (type 1 terminator)/
Intrinsic termination
2. Rho-dependent terminator (type 2 terminator)

16.
• Rho-dependent terminators were first discovered
in bacteriophage genomes.
• Rho's key function is its helicase activity, for which energy is provided
by an RNA-dependent ATP hydrolysis.
• The initial binding site for Rho is an extended (~70 nucleotides,
sometimes 80–100 nucleotides) single-stranded region, rich
in cytosine and poor in guanine, called the rho utilisation site (rut).
• Rho binds to RNA and then uses its ATPase activity to provide the
energy to translocate along the RNA until it reaches the RNA–DNA
helical region, where it unwinds the hybrid duplex structure. small
mutations in the sequence disrupts its function.
• In short, Rho factor acts as an ATP-dependent unwinding enzyme,
moving along the newly forming RNA molecule towards its 3′
end and unwinding it from the DNA template as it proceeds.

17.
Rho-dependent termination
• Rho-dependent termination is controlled by the rho protein, which tracks
along behind the polymerase on the growing mRNA chain.
• Near the end of the gene, the polymerase encounters a run of G
nucleotides on the DNA template and it stalls.
• As a result, the rho protein collides with the polymerase. The interaction
with rho releases the mRNA from the transcription bubble.

18.
Rho-independent terminator (type 1 terminator)/
Intrinsic termination
• Intrinsic, or rho-independent termination, is a process
in prokaryotes to signal the end of transcription and release the newly
constructed RNA molecule.
• intrinsic termination does not require a special protein to signal for
termination and is controlled by the specific sequences of RNA.
• As the polymerase nears the end of the gene being transcribed, it
encounters a region rich in C–G nucleotides.
• The mRNA folds back on itself, and the complementary C–G
nucleotides bind together. The result in secondary structure a
stable hairpin also known as a Stem-loop.

19.
• This RNA hairpin is followed by multiple uracil nucleotides.
• The bonds between uracil and adenine are very weak.
• A protein bound to RNA polymerase (nusA) binds to the stem-loop
structure tightly enough to cause the polymerase to temporarily stall.
This pausing of the polymerase coincides with transcription of the
poly-uracil sequence.
• The weak adenine-uracil bonds lower the energy of destabilization for
the RNA-DNA duplex, allowing it to unwind and dissociate from the
RNA polymerase and liberate the new mRNA transcript.

20.
• The purpose function of intrinsic termination is to signal for the
dissociation of the ternary elongation complex (TEC), signaling the
end of a transcript in prokaryotes.