Transcription in prokaryotes involves RNA polymerase binding to specific promoter sequences on DNA and synthesizing RNA. It occurs in three main steps - initiation at the promoter, elongation as the RNA chain grows, and termination. Key elements that regulate transcription include the -10, -35 promoter sequences, sigma factor, and rho-dependent or intrinsic terminator sequences.

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TRANSCRIPTION IN
PROKARYOTES

2. TRANSCRIPTION IN PROKARYOTES
Presented by: Christa Biju
19LS401011
MSc MB 2nd semester

3. Introduction:
The Central Dogma
In 1958, Crick proposed that information present in DNA is transferred to DNA (via Replication)
and RNA, and from RNA to proteins, this flow of genetic information is called as Central Dogma.
Transcription Unit
It is defined as the stretch of DNA, which is transcribed as a single RNA molecule. A typical
transcription unit has: A promoter at its beginning, a start point within the promoter, a
coding region that is transcribed and a terminator sequence at its end.

4. Prokaryotic Promoter
• A promoter is the sequence of DNA needed for RNA Polymerase to bind to the template and
accomplish the transcription initiation reaction.
• A generalised description of the promoter region in E. coli has 4 consensus sequences:
i. Startpoint : It is a region around the middle of a promoter region and is usually a single base,
usually a purine. Often it is the adenine residue of the triplet CAT.
ii. -10 sequence: It has a sequence TATAAT and is also called as the Pribnow box. It is responsible
for conversion of closed binary complex between DNA and RNA polymerase into the open binary
complex. It is the unwinding domain of the promoter.
iii. -35 sequence: Its sequence is TTGACA. It provides signal to RNA polymerase for the recognition
of the promoters and is therefore the recognition domain of the promoter.
iv. The distance separating -10 and -35 sequence: It is usually 16-18 bp in 90% of the cases and its
length is critical for appropriate geometry of RNA polymerase.
v. UP region: It is an A-T rich region located upstream of the -35 sequence and interacts with alpha
subunit of RNA Polymerase.

6. RNA Polymerase
The RNA polymerase is approx. 465kD and consists of:
i. Core enzyme- It helps in synthesising RNA after transcription has been initiated. It has
4 polypeptides, which are:
• α subunit- It is present in 2 copies and plays a role in promoter recognition and
interaction of RNA polymerase with some regulatory factors. It is encoded by rpo A.
• β and β’ subunit- The β and β’ subunits are encoded by rpoB and rpoC respectively
and are present in one copy each per enzyme molecule. These together form the
catalytic centre.
ii. The Sigma Factor
• It is a single polypeptide encoded by rpoD.
• It ensures that RNA polymerase binds stably only at the promoter.

7. Transcription
The steps in transcription are:
i. Template Recognition
• The sigma factor is required for recognition of promoter sequences and for initiation of transcription.
• Initially, the enzyme binds to the duplex DNA of the promoter region forming a closed binary complex.
• It then induces melting. The complex of melted DNA with the enzyme is known as open binary complex.
ii. Initiation
• It describes the synthesis of nucleotide bond in the RNA transcript.
• The nucleotides are incorporated by the enzyme at the beginning of the start point.
• When the first 2 nucleotides have been aligned, the enzyme catalyses the formation of a
phosphodiester linkage between them.
• The dinucleotide formed remains associated with the template and the enzyme forming
ternary complex.
• It is by the addition of new nucleotides and formation of phosphodiester bond that the RNA
chain keeps growing.

8. Transcription

9. ii. Elongation
• This step begins when the RNA polymerase leaves the promoter region and continues
synthesis of RNA.
• Initially, the polymerase loses its sigma factor and the core enzyme becomes tightly bound
to the DNA .
• The core enzyme- DNA- nascent RNA forms the elongation ternary complex.
• This step involves disruption of DNA structure so that single stranded regions are generated so
that RNA synthesis can continue.

10. iii. Termination
The prokaryotic termination are of 2 types:
a. Rho-independent Termination
• These terminators are also called as intrinsic terminators.
• The major feature is a typical hairpin having a stem of 7-20 bp and a loop is formed due to
base pairing. It has A-T rich region at the base of the stem.
• A run of approx. 6U residues are located 7-9 bp downstream of the hairpin.
• Termination usually occurs at any one of the several positions toward or at the end of the
U run.
• The RNA-DNA hybrid formed in the U run has an unusually weak base paired structure so
it requires least energy to dissociate.
• When the polymerase pauses, the RNA-DNA hybrid dissociates causing termination.

12. b. Rho- dependent termination
• This termination requires the rho factor, which is a hexamer of approx. 46kD and has
RNA binding site and ATPase activity.
• It binds to the RNA transcript at the recognition site located upstream of the termination site.
• The rho factor uses RNA dependent ATP hydrolysis for translocation along the RNA transcript
and for unwinding of the RNA transcript.
• The sequence in RNA transcript recognized by rho factor are rich in C and poor in G.

14. References:
• Singh B. D.; Genetics; Kalyani Publishers; 2nd edition; 2016
• Freifelder. David; Molecular Biology; Narosa Publishing House; 2nd edition
• Topp. Burton E; David Freifelder; Molecular Biology; Jones and Bartlett learning; 4th edition
• Cooper, Geoffrey M; The Cell: A molecular approach; 2nd edition