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Davenport et al. (2000) Vs Adelman et. Al (2002)

Possible states for Escherichia coli RNA polymerase

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Disposition

1)Metode afsnit2)Resume3)Resultater



Forsøgs opsætning

Davenport Adelman


Constant force. Coverslip movesStall. Lack of Cytosine Tri Phosphate


Resume

Davenport

1) Slow/fast transcription rate

2) Diffent tendency to stop/pause

3) Pause: Intermediate between elongation and stop

Adelman

1)Elongation kinetics are homogenous

2)No fast/slow state(its statistic variation)



DavenportFig 2SINGLE MOLPause areaStop ved *Velong=2-10 bp/s 0,2 mM NTP (1mM NTPP 12 bp/s)Tension 8 pN

Tension 2 pN



DavenportFig 3ENSEMBLE

Variation caused by load?No, Velong does not vary with force

Average peak: 0,2 mM NTPVelong = 7,3 +- 2 bp/sAverage: 0,2 mM NTPVelong = 4,3 +- 2 bp/s

Average peak: 1 mM NTPVelong = 14,5 +- 4 bp/sAverage: 1 mM NTPVelong = 8,0 +- 3 bp/s

Stall force 15pN

No template-dependant rate

Rate MUST depend on RNAPslow/fast state


Slow

Fast

To maximum value


DavenportFig 4 ENSEMBLEPause area

Does pause depend on path?No, pause eff<100 %

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



DavenportFig 5ENSEMBLE Is pause and stop state correlated?

1/Velong (small is big)Slow rate gives many pauses

Many pausesGives small distance betweenArrest state

NOTE:ONLY 0,2 mM NTP



Davenport. Table 1


Click


Resume

Davenport

1) Slow/fast transcription rate

2) Diffent tendency to stop/pause

3) Pause: Intermediate between elongation and stop

Adelman

1)Elongation kinetics are homogenous

2)No fast/slow state(its statistic variation)



AdelmanFig 1SINGLE MOLDavenport:Reported 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 = 4,5B8 Velong = 5,0 nt/s SD = 3,0 Descrive almost same elongation rate. With pauses



AdelmanFig 2Skewed Gaussian shapes

128 WT pauses421 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%)


AdelmanFig 3

B8 mutation. Removed specific contact with mRNA. 20 Å away from active site. Mutation (probably) does not change catalytic rate.

Slower elongationHighly 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


AdelmanFig 4

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 FA 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 λ is a positive number called the decay constant.


Anomaly. B8 at WT burst.

Anomaly. RNAP in altered configuration.

B8 pause more frequently, but pause duration is not widely effected

Anomaly. RNAP in altered configuration.

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