Rho dependent termination;Rho independent termination

1. Prokaryotic Transcription
Presented ,

4. Prokaryotic Transcription
• Reaction can be divided into three stages
1. Initiation
2. Elongation
3. Termination
Is binding site of RNA polymerase Is dissociation site of RNA Polymerase

5. • Transcription start when RNA Polymerase binds to DNA Sequence called as
promoter.
• RNA Polymerase move along the template strand , extending its growing RNA
Chain in the 5’3’ direction by the stepwise addition of Ribonucleotides until
it reach a termination signal ,, at which point the newly synthesizing RNA chain
and RNA Polymerase release from DNA.
• 6bp consensus sequence or motif = -10bp and -35bp.
• -10bp sequence or box also known as pribnow box..5’TATAAT3’
• -35bp sequence 5’TTGACA3’ ..
• -10bp and -35bp sequence distance between two sequence is =16-18 bp sigma
factor binding site of this sequence.

6. Promoter
• Strong promoter having AT rich sequence.
• Strong promoter sequence located in -40 to -60 nucleotide of upstream
region … this is called UP elements (Upstream promoter element)..
• UP elements recognized by α-subunit CTD (Carboxyl Terminal Domain)of
RNA polymerase.
• Alternative sigma factors recognize different consensus sequence with the
promoter.

7. Strong promoter
 Promoter closer to consensus sequence
 This sequence found upstream region
UP-Elements (40-60nt) at upstream region
 Recognized by α-subunit CTD (Carboxyl
Terminal Domain)of RNA polymerase.
 AT Rich sequence
weak promoter
consensus sequence absent

8. Holoenzyme = RNAP + σ factors
• In a Bacteria single RNA Polymerase which are interact with different
σ- factors - activation of different gene ..
Initiation factor σ factors ..
Nif gene

9. Terminator Sequence
Start site
5’
3’
3’
5’
-10bpSequence(6bp)=TATAAT
-35bpSequence(6bp)=TTGACG
Spacer Sequence- Are not conserved
Spacer sq
Conserved sequence exact similar
Upstream Downstream
Upstream – In upstream sequence - Promoter , Operator (Regulatory sequence)
Downstream-- In Downstream sequence - Coding sequence, Terminator Sequence

10. Initiation
• RNA Polymerase as a holoenzyme binds to promoter = Closed Binary Complex. Means DNA remain duplex
•
• Sigma factor recognize consensus sequence(more similar).
• Sigma factor change DNA binding properties , with sigma factor RNA Polymerase more affinity with DNA.
• RNA Polymerase holoenzyme cover the 70-80 bp of DNA.
• Close complex-- Open complex(melting short region of DNA)  transcription bubble.
• First two ribonucleotide make phosphodiester bond between them.- Ternary complex (RNA,DNA,Polymerase).
• After synthesis of 8-9 ribonucleotides sigma factor release from Holoenzyme- Core RNA Polymerase 
Synthesis of RNA.
• RNA polymerase doesn’t require a primer.
• First nucleotide with purine Nitrogen base (A,G) present in the transcript at 5’ End (Nucleoside Triphosphate—
pppG or pppA)

12. Elongation
• Nucleotides are covalently added to 3’ end of growing RNA chain and RNA DNA hybrid forms (8-
10 bp)
• During each nucleotide addition the β and ϒ phosphates are removed from the incoming
nucleotides and Hydroxyl group is removed from 3’ carbon
• RNA pol speed= 40 nt /sec at temp 37 celcious
• when RNA pol transcribe DNA , Unwinding and rewinding occurs.
• RNA pol move forward along the doble helix it generate positive supercoiling ahead the and
leave negative supercoiling behind.
• Negative supercoiling helps in Transcription.
• Gyrase and Topoisomerase I removes +ve supercoiling and introduce negative supercoiling.

13. Elongation

14. Termination
• No further addition of nucleotides.
• Termination sequence  Transcription termination .
• In bacteria Termination occurs by Two Ways
1)Intrinsic Termination (Rho Independent)
2)Rho dependant Termination

15. 1)Intrinsic Termination(Rho Independent)
• formation of Hairpin structure in growing transcript because of GC rich
inverted repeat sequence followed by U rich region in RNA at 3’ site .
• Due to formation of Hairpin structure in RNA, the conformational change
occur in RNA pol. And Termination occur.

16. 2)Rho dependant Termination
• Rho protein mw= 275kDa – Hexameric protein
• It bind RNA at rut site (Rho utilizing site)
• rut site rich in C Residues and poor in G Residues
• Direction 5’3’ along RNA
• It having ATPase activity (ATP dependant RNA stimulate Helicase which
disturb RNA-DNA complex.