A Slide based on Transcription in Prokaryotes

1. Transcription in Prokaryotes
Puja katwal
Msc 1st sem,
Microbiology
Department

2. INTRODUCTION
The process of synthesis of RNA by copying the template strand of
DNA is called transcription.
During replication entire genome is copied but in transcription only
the selected portion of genome is copied.
The enzyme involved in transcription is RNA polymerase. Unlike
DNA polymerase it can initiate transcription by itself, it does not
require primase. Transcription is an essential step in using the
information from genes in our DNA to make proteins

4. RNA polymerase
In prokaryotes only a single enzyme, RNA polymerase
holoenzyme, (core + σ), governs the synthesis of all cellular RNAs.
Specifically, RNA polymerase builds an RNA strand in the 5' to 3'
direction, adding each new nucleotide to the 3' end of the strand.
The holoenzyme is a complete RNA polymerase consisting of core
enzyme and a sigma factor(σ), hence called complex enzyme. The
shape of holo enzyme has been described as a crab-claw
appearance. The core enzyme consists of 5 subunits: two α
subunits, one β, one β’and one ω. The holoenzyme binds DNA at
specific sites call promoter and transcribes specific length of RNA.
And σ factor helps to recognize this promoter site.

5. The functions of 4 subunits of core enzyme (~400 kDa):
• α : the two α subunits assemble the enzyme and also
recognizes regulatory factors.
• β: has the polymerase activity (catalyzes the synthesis of
RNA).
• β': binds to DNA .
• ω: function not known clearly. However it has been observed
to offer a protective/chaperone function to the β' subunit and is
supposed to take part in unwinding.
RNA polymerase synthesizes RNA at the rate of 40
nucleotide per minute at 37 degree Celsius.

7. The steps of transcription
Transcription is an enzymatic process. The mechanism of
transcription completes in three major steps:
• Initiation
• Elongation
• Termination.

8. Initiation
To proceed the initiation process the components such as template,
RNA polymerase, template activated precursors and divalent metal ions
(Mg ++ or Mn++) are required. Initiation process can be further
divided as:
1. Promoter Recognition
RNA polymerase binds to a sequence of DNA called the promoter,
found near the beginning of a gene. Promoters are the specific base
sequences ( 20-200 bases long). Each gene has its own promoter. The
RNA polymerase plays a key role in recognition and binding of
initiation site. The holoenzyme transcribes only one of the two DNA
strands.

9. The σ factor of holoenzyme recognizes the promoter region of the
DNA. The two conserved nucleotide sequence (promoter region)
found in most prokaryotes are:
a. Pribnow box (5ˈ-TATAAT-3ˈ): It is found as a part of all
prokaryotic promoters. It is a stretch of 6 nucleotides situated at
8-10 nucleotides upstream of the transcription start site.
b. 35-sequence (5ˈ-TTGACA-3ˈ): It is a second conserved
sequence found about 35 nucleotides upstream of the transcription
start site. This site is also known as recognition sites.

10. 2. Binding of RNA polymerase to promoter:
Binding occurs with the help of β’ after recognition of promoter
by σ . The strength of binding of RNA polymerase to different
promoter varies. There are promoters that have binding sites for
proteins rather than RNA polymerase. Therefore, for these
promoters the site must be occupied by that protein for RNA
polymerase to bind correctly. One of the most common binding site
for this type is one that binds a complex of cyclic AMP receptor
protein (CRP). The concentration of AMP governs activity of these
promoters.

11. 3. Unwinding of DNA
The super helical nature of The chromosome play a role in
promoter function as the torsional stress imposed by super coiling
makes the certain area of DNA easier to separate by RNA
polymerase. Binding of ω factor of RNA polymerase results in
unwinding of a DNA helix. After binding to the DNA, the RNA
polymerase switches from a closed complex to an open complex.
This change involves the separation of the DNA strands to form an
unwound section of DNA of approximately 13 bp, referred to as
the “transcription bubble”.

12. 4. Synthesis of First Base of RNA Chain:
The first base of RNA synthesized is always in the form of
purines i.e either triphosphate guanine or triphosphate adenine.
Most of the chains in E. coli is started with pppG. Initiation ends
after the formation of first inter nucleotide bond.

13. Elongation
Elongation is the stage when the RNA strand gets longer after
synthesis of RNA more than 10 bp long. Sigma factor releases
from the holoenzyme and elongation occurs by the core enzyme
that moves along the DNA template. The released sigma factor
can combine with any of the core enzyme and thus is reused for
the initiation of a new chain. Only one of the two strand serve as a
template DNA which is copied in 5’ to 3’ direction.
The region of the template that has been transcribed regain its
double helical from behind the bubble and the next region of DNA
which is to be transcribed unwinds.

14. The RNA transcript does not elongate uniformly along the template.
This is due to the presence of pausing sites at certain regions in the
template. It has been found that generally the pausing sequence
contains GC rich regions about 16-20bp upstream of 3ˈ-OH end of
paused transcript and dyed symmetry also causes pausing. However
the mechanism is not clear.
It is believed that after release of sigma factor the NusA and NusG
proteins become associated with the core enzyme and modulate the
rate of elongation.
The RNA transcript is nearly identical to the non-template,
or coding, strand of DNA. However, RNA strands have the base
uracil (U) in place of thymine (T), as well as a slightly different sugar
in the nucleotide.

15. During this phase the growing end of RNA forms hybrid duplex
i.e short temporary base paring with DNA to form RNA:DNA
double helix which is of 8 bp long. Supercoiling is solved by
topoisomerase enzyme as in DNA replication.

16. Proof Reading
There are two known proof reading mechanisms,
pyrophosphorolytic editing, where the incorrect base pair is
immediately removed and hydrolytic editing is where RNA
polymerase has to backtrack to fix an incorrect pairing. The two
proteins GreA and GreB enables the RNA polymerase to back up
and cleave 2-3 nucleotides from 3ˈ end of nascent transcript. This
is similar to 3ˈ-5ˈ exonuclease activity of DNA polymerase.

19. Termination
Termination is the end of transcription, when an RNA strand is fully
formed. As seen in the image to the right, both the newly
formed RNA and the RNA polymerase complex dissociate.
Transcription occurs in two ways:
a. Rho dependent transcription termination
b. Rho independent transcription termination
a. Rho dependent transcription termination: it is dependent on a
protein factor called rho factor. This protein encoded by rho gene.
The Rho factor is hexamer. This factor binds to C rich region near 3’
end of RNA and migrates toward RNA polymerase enzyme in 5’ to
3’ direction.

20. This protein moves faster and catches the polymerase enzyme.
The movement is facilitated by hydrolysis of ATP that provides
energy. The contact between Rho protein and RNA polymerase is
facilitated by NusA or NusG proteins. The wrapping goes
continuously and disrupts the covalent bonding that holds the
newly formed RNA to DNA and RNA polymerase.
Rho pulls the RNA transcript and the template DNA strand apart,
releasing the RNA molecule and ending transcription.

22. b. Rho-independent termination: It depends on specific sequences in
the DNA template strand. As the RNA polymerase approaches the end
of the gene being transcribed, it hits a region rich in C and G
nucleotides. The RNA transcribed from this region folds back on itself,
and the complementary C and G nucleotides bind together. The result
is a stable hairpin that causes the polymerase to stall. The hairpin is
followed by a stretch of U nucleotides in the RNA, which match up
with A nucleotides in the template DNA. The complementary U-A
region of the RNA transcript forms only a weak interaction with the
template DNA. This, coupled with the stalled polymerase, produces
enough instability for the enzyme to fall off and liberate the new RNA
transcript.

24. THANK YOU