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Troels Linnet Nano

Davenport et al. (2000) Vs Adelman et. Al (2002) Possible states for Escherichia coli RNA polymerase. Troels Linnet Nano. Disposition. Metode afsnit Resume Resultater. Forsøgs opsætning. Davenport. Adelman. Stall. Lack of Cytosine Tri Phosphate. Constant force. Coverslip moves. Resume.

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Troels Linnet Nano

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  1. Navn på enhed (Indsæt --> Diasnummer) Davenport et al. (2000) Vs Adelman et. Al (2002)Possible states for Escherichia coli RNA polymerase Troels Linnet Nano

  2. Navn på enhed (Indsæt --> Diasnummer) Disposition Metode afsnit Resume Resultater

  3. Navn på enhed (Indsæt --> Diasnummer) Forsøgs opsætning Davenport Adelman Stall. Lack of Cytosine Tri Phosphate Constant force. Coverslip moves

  4. Navn på enhed (Indsæt --> Diasnummer) Resume Davenport Adelman Elongation kinetics are homogenous No fast/slow state(its statistic variation) • 1) Slow/fast transcription rate • 2) Diffent tendency to stop/pause • 3) Pause: Intermediate between elongation and stop

  5. Navn på enhed (Indsæt --> Diasnummer) DavenportFig 2SINGLE MOL Pause area Stop ved * Velong=2-10 bp/s 0,2 mM NTP (1mM NTPP 12 bp/s) Tension 8 pN Tension 2 pN

  6. Navn på enhed (Indsæt --> Diasnummer) 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

  7. Navn på enhed (Indsæt --> Diasnummer) DavenportFig 4 ENSEMBLE Pause 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

  8. Navn på enhed (Indsæt --> Diasnummer) 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

  9. Navn på enhed (Indsæt --> Diasnummer) Davenport. Table 1 Click

  10. Navn på enhed (Indsæt --> Diasnummer) Resume Davenport Adelman Elongation kinetics are homogenous No fast/slow state(its statistic variation) • 1) Slow/fast transcription rate • 2) Diffent tendency to stop/pause • 3) Pause: Intermediate between elongation and stop

  11. Navn på enhed (Indsæt --> Diasnummer) AdelmanFig 1SINGLE MOL Davenport: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 = 4,5 B8 Velong = 5,0 nt/s SD = 3,0 Descrive almost same elongation rate. With pauses

  12. Navn på enhed (Indsæt --> Diasnummer) 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%)

  13. Navn på enhed (Indsæt --> Diasnummer) 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

  14. Navn på enhed (Indsæt --> Diasnummer) 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 λ 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

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