Transcriptional Termination

1
Transcriptional Termination
2
Bacterial RNA Polymerase has two Modes of
Termination

• Transcription stops when RNA Polymerase
  encounters terminator sequence.
• RNA polymerase stops polymerization releases
  mRNA dissociates from DNA.
• Some terminators require a hairpin loop formed in
  the RNA.
• Two general types
• Factor independent termination
• Factor dependent termination

3
Characteristics of Factor Independent Terminator
Inverted repeats in GC rich region
5 GTCAAAAGCCTCCGGTCGGAGGCTTTTGACT 3 CAGTTTTCGGAG
GCCAGCCTCCGAAAACTGA
Run of As
When transcribed to RNA, hairpin loop formed at
GC rich repeats
4
When transcribed to RNA, hairpin loop formed at
GC rich repeats
5GTCAAAAGCCTCCGGTCGGAGGCTTTTGACT 3CAGTTTTCGGAGGC
CAGCCTCCGAAAACTGA
G U G - C C - G C - G U - A C - G C G C - G
Transcription product forms hairpin loop.
Note GC rich stem.
RNA
CUUUUOH3
5--GUCAAAG
5
5
polymerase
3
3
AGGCTTTT
G
ACT TGA
C
U
5
A
U
A
U
U
A
AG C C G C G U A C G C G G C
GU
A
A
TCCG
A
Fine structure of hairpin loop
6
5
3
3
AGGCTTTT
G
ACT TGA
C
U
5
A
U
A
U
U
A
AG C C G C G U A C G C G G C
GU
A
A
TCCG
A
UA rich region unstable
GC rich region very stable RNARNA hybrid very
stable. Tends to pull RNA from the bubble
20 less stable than rU-rA or rA-dT
Transcription bubble is destabilized
7
5
3
3
Combination of stable hairpin loop followed by
weak AU region destabilizes bubble.
5
5
3
RNA and polymerase fall off
8
Factor-Dependent Termination

• Requires proteins that interact with with
  polymerase or transcript to stop transcription
• E. coli has three termination factors
• Rho
• Tau
• NusA
• Rho (r) is best understood.

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Rho-Dependent Termination

• Works only if RNA not being translated (ribosomes
  not in the way)
• Bacteria can have simultaneous transcription/trans
  lation.
• Rho is RNA-dependent ATPase only cleaves ATP if
  RNA is present
• Rho is RNA-DNA helicase (hexamer). Will only
  unwind double helix if RNADNA hybrid.
• A pause site is also involved.

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Rho Utilization Site
Termination can not occur if rut is being
translated.
Protein
Rho binds to rut mRNA and slides along
mRNA, chasing polymerase
Ribosome
11
Rho sliding requires hydrolysis of ATP
RNA pauses at termination site Rho catches
polymerase
12
Helicase activity unwinds RNADNA hybrid and
burns ATP.
Disruption of RNA DNA hybrid causes RNA poly and
mRNA to fall off
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Summary of Termination

• Both factor independent and dependent mechanisms
  require polymerase and transcript to fall out of
  transcription bubble
• Factor independent uses a stable hairpin loop
  followed by an unstable AU region of transcript.
• Rho dependent relies on unwinding of RNADNA
  hybrid at end of gene.

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Bacterial mRNAs may be Polycistronic

• Single mRNA molecule can encode more than one
  polypeptide Polycistronic.
• cistron is the coding region for each
  polypeptide polycistronic means many coding
  regions.
• Include multiple translation initiation regions
  that signal ribosomes to begin translation.

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Genes
A B C
mRNA
B
A
Ribosomes
Bacterial genes are polycistronic. Coupled
translation-transcription
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Polarity

• Some mutations in one coding region of
  polycistronic mRNA can block transcription of
  next gene Polarity.
• Occurs if at least one Rho dependent termination
  site between the point where translation was
  blocked and the translation initiation region of
  the next.
• Rho will bind to mRNA and stop transcription of
  next gene.

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Normal condition
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If Translation of Gene A Blocked Rho catches up
with RNA polymerase
Transcription of Gene B halted prematurely