Singlemolecule studies of RNAP

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Single-molecule studies of RNAP

• Overview of RNAP function and functional steps
• Brief overview of the variety of single-molecule
• experiments with RNAP
• Transcription initiation studies using smFRET
  (SW)
• Transcription initiation/elongation studies
  using
• magnetic tweezers

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Generalities on RNAP

• Transcribes DNA into RNA
• Monomeric enzyme in phages and mitochondria,
  multimeric in bacteria, archea and eukaryotes,
  but structurally similar with 3 channels for DNA,
  NTP and RNA
• Successive steps

RNAP
RNAP
RNAP
RNAP
RNAP
RNAP
Promoter
Gene
Terminator
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Single-molecule manipulation experimental
geometries
Block, Gelles, Bustamante, Wang, Heslot Groups,
etc
From Bai et al., ARBBS 35 (2006) 343 (PDF)
Non manipulation experiments fluorescence,
smFRET (diffusing or immobilized molecules)
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Closed complex
Open complex (RPo)
Initial transcribing complex (ITC)
Elongation complex (EC)
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Experimental system magnetic tweezers
2kb or 4 kb dsDNA containing the promoter
sequence, and a 100 or 400 bp elongation sequence
F 0.3 pN
1
-1
Revyakin et al., PNAS 101 (2004) 4776
(PDF) Revyakin et al., Science 314 (2006) 1139
(PDF)
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Promoter unwinding results in change in extension
?Lk ( 0) ?Tw ?Wr
Negatively supercoiled DNA Lk lt 0
Positively supercoiled DNA Lk gt 0
Promoter unwinds ?Tw -1
Promoter unwinds ?Tw -1
? nb of plectonemes increases
?Wr 1 ? nb of plectonemes decreases
DNA extension increases
DNA extension decreases
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Experimental results
Irreversible unwinding
Reversible unwinding
?lobs ?lunwinding ?lcompaction
?lunw 1.2 0.1 turn 13 1 bp ?lcomp 15
5 nm
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Twait (opening efficiency) depends on RNAP
and sgt0 Tunwound (open complex stability)
depends on sgt0
In the constant torque regime Twait/Tunw
cte Different regimes may be relevant in vivo
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Predictions of the different models of abortive
initiation
?Tw gt 0
?Tw 0
?Tw 0
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The initial transcribed sequence is
ATAAATTTG Adding (i) ATP, (ii) ATP and UTP plus
rifampicin (blocks transcription at N 2), and
(iii) ATP UTP, the abortion products are 1 bp
(A), 2 bp (AU) and 8 bp (AUAAAUUU) long
Notes no observable backtrack to the RPo state
ITC more stable
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scrunching (?unw gt 0) requires products
at least N 3 bp in length,and amounts to N
2 unwound bp
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Evolution of the unwound region during elongation
4 observed unwinding/rewinding transitions can be
assigned to 1 formation of open complex
(RPo) 2 formation of initial transcribing
complex (itc) (scrunching), 3 formation of
elongation complex (EC) (unscrunching) 4
termination
tsc
tsc
tsc
RPc
EC
RPo
RPitc
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Promoter escape scrunching occurs in most or all
transcription cycles
80 of transcription cycles have detectable
scrunching (gt 1 s)
percentage of transcription cycles with
scrunching (gt 1s AND lt 1s) 100
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(No Transcript)