Sted og dato (Indsæt --> Diasnummer) Dias 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

Sted og dato (Indsæt --> Diasnummer) Dias 2 Disposition 1)Metode afsnit 2)Resume 3)Resultater Navn på enhed (Indsæt --> Diasnummer)

Sted og dato (Indsæt --> Diasnummer) Dias 3 Forsøgs opsætning DavenportAdelman Navn på enhed (Indsæt --> Diasnummer) Constant force. Coverslip movesStall. Lack of Cytosine Tri Phosphate

Sted og dato (Indsæt --> Diasnummer) Dias 4 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) Navn på enhed (Indsæt --> Diasnummer)

Sted og dato (Indsæt --> Diasnummer) Dias 5 Davenport Fig 2 SINGLE MOL Pause area Stop ved * V elong =2-10 bp/s 0,2 mM NTP (1mM NTPP 12 bp/s) Tension 8 pN Tension 2 pN Navn på enhed (Indsæt --> Diasnummer)

Sted og dato (Indsæt --> Diasnummer) Dias 6 Davenport Fig 3 ENSEMBLE Variation caused by load? No, V elong does not vary with force Average peak: 0,2 mM NTP V elong = 7, bp/s Average: 0,2 mM NTP V elong = 4, bp/s Average peak: 1 mM NTP V elong = 14, bp/s Average: 1 mM NTP V elong = 8, bp/s Stall force 15pN No template-dependant rate Rate MUST depend on RNAP slow/fast state Navn på enhed (Indsæt --> Diasnummer) Slow Fast To maximum value

Sted og dato (Indsæt --> Diasnummer) Dias 7 Davenport Fig 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 Navn på enhed (Indsæt --> Diasnummer)

Sted og dato (Indsæt --> Diasnummer) Dias 8 Davenport Fig 5 ENSEMBLE Is pause and stop state correlated? 1/V elong (small is big) Slow rate gives many pauses Many pauses Gives small distance between Arrest state NOTE: ONLY 0,2 mM NTP Navn på enhed (Indsæt --> Diasnummer)

Sted og dato (Indsæt --> Diasnummer) Dias 9 Davenport. Table 1 Navn på enhed (Indsæt --> Diasnummer) Click

Sted og dato (Indsæt --> Diasnummer) Dias 10 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) Navn på enhed (Indsæt --> Diasnummer)

Sted og dato (Indsæt --> Diasnummer) Dias 11 Adelman Fig 1 SINGLE 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 V elong = 14,27 nt/s SD = 4,5 B8 V elong = 5,0 nt/s SD = 3,0 Descrive almost same elongation rate. With pauses Navn på enhed (Indsæt --> Diasnummer)

Sted og dato (Indsæt --> Diasnummer) Dias 12 Adelman Fig 2 Skewed Gaussian shapes 128 WT pauses 421 B8 pauses V elong 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 Navn på enhed (Indsæt --> Diasnummer) Pause state. Small value arise from slight smearing of finite averaging. (7,8%)

Sted og dato (Indsæt --> Diasnummer) Dias 13 Adelman Fig 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. Navn på enhed (Indsæt --> Diasnummer) Active elongation Pause state

Sted og dato (Indsæt --> Diasnummer) Dias 14 Adelman Fig 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 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.differential equationpositive number Navn på enhed (Indsæt --> Diasnummer) 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.