Davenport et al. (2000) Vs Adelman et. Al (2002) Possible states for Escherichia coli RNA polymerase

1
Davenport et al. (2000) Vs Adelman et. Al
(2002)Possible states for Escherichia coli RNA
polymerase

• Troels Linnet
• Nano

2
Disposition

• Metode afsnit
• Resume
• Resultater

3
Forsøgs opsætning

• Davenport

• Adelman

Constant force. Coverslip moves
Stall. Lack of Cytosine Tri Phosphate
4
Resume

• Davenport

• Adelman

• 1) Slow/fast transcription rate
• 2) Diffent tendency to stop/pause
• 3) Pause Intermediate between elongation and
  stop

• Elongation kinetics are homogenous
• No fast/slow state(its statistic variation)

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DavenportFig 2SINGLE MOL

• Pause area
• Stop ved
• Velong2-10 bp/s 0,2 mM NTP (1mM NTPP
  12 bp/s)
• Tension 8 pN
• Tension 2 pN

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DavenportFig 3ENSEMBLE
Slow

• Variation caused by load?
• No, Velong does not vary with force
• Average peak 0,2 mM NTP
• Velong 7,3 - 2 bp/s
• Average 0,2 mM NTP
• Velong 4,3 - 2 bp/s
• Average peak 1 mM NTP
• Velong 14,5 - 4 bp/s
• Average 1 mM NTP
• Velong 8,0 - 3 bp/s
• Stall force 15pN
• No template-dependant rate
• Rate MUST depend on RNAP
• slow/fast state

Fast
To maximum value
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DavenportFig 4 ENSEMBLE

• Pause area
• Does pause depend on path?
• No, pause efflt100
• This support, that Pause is a state indepent of
  path.
• Pause and translocation are competitive states
• Does STOP depend on path?
• Apparently so
• Is pause and stop state correlated?
• NOTE
• ONLY 0,2 mM NTP

8
DavenportFig 5ENSEMBLE

• Is pause and stop state correlated?
• 1/Velong (small is big)
• Slow rate gives many pauses
• Many pauses
• Gives small distance between
• Arrest state
• NOTE
• ONLY 0,2 mM NTP

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Davenport. Table 1
Click
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Resume

• Davenport

• Adelman

• 1) Slow/fast transcription rate
• 2) Diffent tendency to stop/pause
• 3) Pause Intermediate between elongation and
  stop

• Elongation kinetics are homogenous
• No fast/slow state(its statistic variation)

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AdelmanFig 1SINGLE MOL

• DavenportReported elongation rate, significant
  slower than solution rates. 0,2 mM NTP
• (nucleoside triphosphate)
• Use 1 mM NTP. WT and HT-tag have same rate
  efficiency.
• Up to 5 mM NTP does not change rate.
• Force 4 pN.
• WT Velong 14,27 nt/s SD 4,5
• B8 Velong 5,0 nt/s SD 3,0
• Descrive almost same elongation rate. With pauses

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AdelmanFig 2

• Skewed Gaussian shapes
• 128 WT pauses
• 421 B8 pauses
• Velong for three RNAP
• No sites revealed, with large proportion of RNAP
  paused. (data not shown)
• Variation larger than SD (1,5 nt/s). Fluctuation
  as function of path.
• Slower-than-average RNAP more paused.
• Single-peak. Active elongation.
• Elongation is NOT a single-rate kinetic process

Pause state. Small value arise from slight
smearing of finite averaging. (7,8)
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AdelmanFig 3

• B8 mutation. Removed specific contact with mRNA.
  20 Å away from active site. Mutation (probably)
  does not change catalytic rate.
• Slower elongation
• Highly variable. Have WT burst.
• Slower rate (4.0) increased area for pause (33,3
  )
• Anomalously large velocities show that B8 retain
  ability to elongate at WT rates.

Active elongation Pause state
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AdelmanFig 4
Anomaly. B8 at WT burst.

• Fig 2 blue WT . Pause state
• Rate before and after pause. Conformational
  change must be shorter than res. limit 1s. No
  long-term change and memory of prev. states.
• C) WT 10s bin, B8 5s bin. Pause within time
  interval. Exponential decay. Stochatic
  uncorrelated event.
• D) WT 100nt bins, B8 50nt bins. Distance between
  pauses. Exponential decay
• E) WT 2s bins. Pause duration. Exponential decay
• F) B8 2s bins. Pause duration. Exponential decay
• C D E F
• A quantity is said to be subject to exponential
  decay if it decreases at a rate proportional to
  its value. Symbolically, this can be expressed as
  the following differential equation, where N is
  the quantity and ? is a positive number called
  the decay constant.

Anomaly. RNAP in altered configuration.
Anomaly. RNAP in altered configuration.
B8 pause more frequently, but pause duration is
not widely effected