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Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy.

Crampton N, Bonass WA, Kirkham J, Rivetti C, Thomson NH - Nucleic Acids Res. (2006)

Bottom Line: Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC).After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template.Interestingly, the distances between the two RNAP show that they are not always at closest approach after 'collision' has caused their arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, University of Leeds, Leeds LS2 9LU, UK.

ABSTRACT
Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned lambda(pr) promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after 'collision' has caused their arrest.

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Related in: MedlinePlus

Summary of the observed RNAP collisions in this study. EC collisions are only observed in the two-step process. EC and SD collisions are observed in the one-step process. EC collisions occur between SEC that have failed to resume elongation and an active EC as well as between two active ECs. It is shown that SEC–EC collisions are more likely. Whether EC–EC collisions are observed is unclear and the collision of two active ECs may result in disengagement. Grey: OPCs; Orange: active ECs; Green: SECs. Short vertical arrows denote the stall sites.
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fig7: Summary of the observed RNAP collisions in this study. EC collisions are only observed in the two-step process. EC and SD collisions are observed in the one-step process. EC collisions occur between SEC that have failed to resume elongation and an active EC as well as between two active ECs. It is shown that SEC–EC collisions are more likely. Whether EC–EC collisions are observed is unclear and the collision of two active ECs may result in disengagement. Grey: OPCs; Orange: active ECs; Green: SECs. Short vertical arrows denote the stall sites.

Mentions: The largest proportion of collisions are occurring at or very close to the stall site and hence providing a block to re-initiation after a certain time-scale. SEC that fail to resume elongation were termed arrested complexes by Komissarova and Kashlev (11). The reason for such arrests is unclear, but it is known that they occur more frequently at some stall sequences. These arrested complexes lose their active site due to a small amount of spontaneous back-tracking (∼6 nt) whilst the complex retains a broadly similar structure. The force generated by two opposing active ECs may well be larger than that generated by a single EC being opposed by a stationary EC (23), which may result in RNAP disengagement after ‘active–active’ collisions. As mentioned previously, such disengagement will not be visualised in this experiment, since a proportion of naked DNA is present at all times. Relating these results to possible outcomes that were suggested in Figure 1, we are able to present the outcomes actually observed using our experimental setup in Figure 7.


Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy.

Crampton N, Bonass WA, Kirkham J, Rivetti C, Thomson NH - Nucleic Acids Res. (2006)

Summary of the observed RNAP collisions in this study. EC collisions are only observed in the two-step process. EC and SD collisions are observed in the one-step process. EC collisions occur between SEC that have failed to resume elongation and an active EC as well as between two active ECs. It is shown that SEC–EC collisions are more likely. Whether EC–EC collisions are observed is unclear and the collision of two active ECs may result in disengagement. Grey: OPCs; Orange: active ECs; Green: SECs. Short vertical arrows denote the stall sites.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636470&req=5

fig7: Summary of the observed RNAP collisions in this study. EC collisions are only observed in the two-step process. EC and SD collisions are observed in the one-step process. EC collisions occur between SEC that have failed to resume elongation and an active EC as well as between two active ECs. It is shown that SEC–EC collisions are more likely. Whether EC–EC collisions are observed is unclear and the collision of two active ECs may result in disengagement. Grey: OPCs; Orange: active ECs; Green: SECs. Short vertical arrows denote the stall sites.
Mentions: The largest proportion of collisions are occurring at or very close to the stall site and hence providing a block to re-initiation after a certain time-scale. SEC that fail to resume elongation were termed arrested complexes by Komissarova and Kashlev (11). The reason for such arrests is unclear, but it is known that they occur more frequently at some stall sequences. These arrested complexes lose their active site due to a small amount of spontaneous back-tracking (∼6 nt) whilst the complex retains a broadly similar structure. The force generated by two opposing active ECs may well be larger than that generated by a single EC being opposed by a stationary EC (23), which may result in RNAP disengagement after ‘active–active’ collisions. As mentioned previously, such disengagement will not be visualised in this experiment, since a proportion of naked DNA is present at all times. Relating these results to possible outcomes that were suggested in Figure 1, we are able to present the outcomes actually observed using our experimental setup in Figure 7.

Bottom Line: Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC).After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template.Interestingly, the distances between the two RNAP show that they are not always at closest approach after 'collision' has caused their arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, University of Leeds, Leeds LS2 9LU, UK.

ABSTRACT
Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned lambda(pr) promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after 'collision' has caused their arrest.

Show MeSH
Related in: MedlinePlus