This give detail mechanism and process of transcription Prokaryotes #ASMNAFISBIOLOGY

1. Transcription in Prokaryotes
Mechanism of: Initiation,Elongation and Termination
• By ASM NAFIS BIOLOGY
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2. Objectives
• Understand the basic mechanism of Transcription.
• Know the process of polymerase in Transcription
• Know the function of promoter elements and its
associating proteins.
• Know the proof reading mechanism
• Know the Transcription cycle(initiation,elongation
,termination).
• Understand the process of termination (by
independently or by involvement of rho protein).
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4. Bacterial promoters
There are several flavors of promoters
 and  recruit RNAP to promoter DNA
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5. RNA polymerases
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7. RNA Polymerase and its Promoter intraction
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8. Challenges in initiating transcription
1.Job of RNAP is to make transcripts—has to elongate
approximately without regard to sequence; and the
enzyme is specialized in this way; starting trx at defined
positions requires specific recognition; ortohogonal to
elongation—whole new apparatus to do this.
2.The initiating enzyme must not only recognize DNA but
open strands template strand is available to guide
transcription. Contours of that process just being
unraveled now—Discussion paper
3.How does the enzyme leave the promoter, when there
are all these DNA – protein interactions to bind it to the
promoter? Abortive initiation and its relationship to
initiation factors is key
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9. The Initiating Form of RNA Polymerase
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10. Is the -10 promoter element recognized as Duplex or SS DNA?
-10 logo-35 logo
Helix-turn-helix in Domain 4
Recognizes -35 as duplex DNA
Recognition of the prokaryotic promoter
Domain 2 recognizes -
10 as duplex DNA
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11. ‘holoenzyme’
'2
7
+ 7
‘core’
}
Can begin transcription
on promoters and can
elongate
}
Can elongate but cannot
begin transcription at
promoters
 factor is required for bacterial RNA polymerase to
initiate transcription on promoters
'2
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12. Initiating RNAP must open DNA to permit transcription:
Formation of the open complex
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13. is positioned for DNA recognition 14
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14. Undergoes a large conformational change upon binding
to RNA polymerase
Free  doesn’t bind DNA  in holoenzyme positioned
for DNA recognitionSorenson; 2006
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15. is positioned to affect key activities of RNA polymerase
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16. Fig-Recognition and melting of -10 element by sigma
region 2
Ref ch12 watson
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17. Remember that sigma linker 3-4 and the b reader of TFB are in
the RNA exit channel, providing a barrier to chain extension and
presumably blocking RNA exit ( not enough room for both sigma
linker and RNA)
It had been established that sigma domain 4 dissociates from
RNAP first.
That brings us to this very curious process carried out by all RNA
–abortive iniitation—many short RNA chains made prior to
extension.
Tell you about recent work of ebright to understand the
mechanistic basis of this process, and the insights it provided into
the role of abortive initiation in promoter excape
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18. Steps in transcription initiation 19
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19. Promoter escape and Abortive Initiation
during abortive initiation, RNAP synthesizes many
short transcripts, but reinitiates rapidly. How can
the active site of RNAP move forward along the DNA
while maintaining promoter contact?
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20. Three models for Abortive initiation
#1
Predicts expansion and contraction of RNAP
Predicts expansion and contraction of DNA
Predicts movement of both the RNAP leading and trailing edge relative to DNA
#2
#3
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21. • One model postulates that the RNAP molecule makes transient downstream
excursions on the template, briefly breaking its bonds with the promoter, until the short
RNA is released, and then the enzyme diffuses back to the promoter Such a model is
not easily reconciled with bulk footprinting data, which suggest that the abortive
initiation process results from an inability of RNAP to break its promoter contacts.
These observations led Straney & Crothers (to propose that the energy required to
break free of the promoter might be somehow stored in a “stressed intermediate” and
that abortive initiation was a consequence of this energy not being used productively.
• One particular instance of this concept, the “inchworming” model, postulates that
flexible elements inside RNAP might allow the active center to move forward
transiently with respect to the upstream face during synthesis, storing up energy like a
stretched spring that retracts upon aborted synthesis. In a third model, the flexible
element that stores the energy ultimately used for promoter escape lies not in RNAP
but in the single-stranded DNA of the transcription bubble and its interactions with the
enzyme.
• In this scrunching model, RNAP functions more or less as a rigid body. The
downstream DNA is pulled progressively into the enzyme with each nucleotide
addition cycle, producing a scrunched form within the enzyme footprint .Abortive RNA
transcripts lead to the release of the scrunched DNA, which is then extruded out the
downstream face of RNAP (1, 56–58), only to be reeled in again upon further RNA
synthesis.
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24. 3.Termination
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27. 3
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28. summery
1. Multisubunit RNA polymerases are conserved among all organism and
cannot initiate transcription on their own. In bacteria 70 is required to
initiate transcription at most promoters. Among other functions, it
recognizes the key features of most bacterial promoters, the -10 and -35
sequences.
2. E. coli RNA polymerase holoenzyme, (core + ) finds promoter sequences
by sliding along DNA and by transfer from one DNA segment to another.
This behavior greatly speeds up the search for specific DNA sequences in
the cell and probably applies to all sequence-specific DNA-binding proteins.
3. Transcription initiation proceeds through a series of structural changes in
RNA polymerase, 70 and DNA.
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29. • A key intermediate in E. coli transcription initiation is the open complex, in
which the RNA polymerase holoenzyme is bound at the promoter and ~12
bp of DNA are unwound at the transcription startpoint. Open complex
formation does not require nucleoside triphosphates. Its presence can be
monitored by a variety of biochemical and structural techniques.
• Recognition of the -10 element of the promoter DNA is coupled with strand
separation
• When the open complex is given NTPs, it begins the ‘abortive initiation’
phase, in which RNA chains of
5-10 nucleotides are continually synthesized and released.
• Through a “DNA scrunching” mechanism the energy captured, during
synthesis of one of these short transcripts eventually breaks the enzyme
loose from its tight connection to the promoter DNA, and it begins the
elongation phase.
• Aspects of the mechanism of initiation are likely to be conserved . .
in eukaryotic RNA polymerase
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30. Refrences
• Young BA, Gruber TM, Gross CA. (2004) Minimal machinery of RNA
polymerase holoenzyme sufficient for promoter melting. Science. 303:1382-
1384
• *Kapanidis, AN, Margeat, E, Ho, SO,.Ebright, RH. (2006) Initial transcription
by RNA polymerase proceeds through a DNA-scrunching mechanism.
Science. 314:1144-1147.
• Revyakin A, Liu C, Ebright RH, Strick TR (2006) Abortive initiation and
productive initiation by RNA polymerase involve DNA scrunching. Science.
314: 1139-43.
• Murakami KS, Masuda S, Campbell EA, Muzzin O, Darst SA (2002).
Structural basis of transcription initiation: an RNA polymerase holoenzyme-
DNA complex. Science. 296:1285-90.
• Chpter 12 of mol bio 6th edition (2008) by Watson and bekar.
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31. Discussion Paper
**Feklistov A and Darst, SA (2011) Structural basis for Promoter -10 Element
recognition by the Bacterial RNA Polymerase  Subunit. Cell 147: 1257 –
1269
Accompanying preview: Liu X, Bushnell DA and Kornberg RD ( 2011) Lock
and Key to Transcription:
 –DNA Interaction. Cell: 147: 1218-1219
***Paul BJ, Barker MM, Ross W, Schneider DA, Webb C, Foster JW,
Gourse RL. (2004) DksA: a critical component of the transcription initiation
machinery that potentiates the regulation of rRNA promoters by ppGpp and
the initiating NTP.
Cell. 6:311-2
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32. Thanks