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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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Inter RNAP–RNAP separation distributions along the DNA contour length. RNAP centre separation: (A) in the absence of all NTPs (n = 45), i.e. OPCs; (B) in the presence of ATP, GTP and UTP only (n = 131), i.e. SECs; (C) in the presence of ATP, GTP and CTP, followed by CTP after 20 min (n = 44); (D) formed by addition of all 4 NTPs together (n = 45) from the OPC state.
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fig5: Inter RNAP–RNAP separation distributions along the DNA contour length. RNAP centre separation: (A) in the absence of all NTPs (n = 45), i.e. OPCs; (B) in the presence of ATP, GTP and UTP only (n = 131), i.e. SECs; (C) in the presence of ATP, GTP and CTP, followed by CTP after 20 min (n = 44); (D) formed by addition of all 4 NTPs together (n = 45) from the OPC state.

Mentions: SECs were formed by the omission of CTP. The OPCs at each convergent promoter would then be expected to transcribe downstream (i.e. towards each other) until the first occurrence of a C base in the non-template strand. In our template, this C base occurred at positions +24 and +70 relative to the +1 sites of transcription initiation for each promoter respectively. The template was designed such that the sequence at each stall site was identical, hence the stalling and re-initiation behaviour of RNAP at either site should be identical. The appearance of the SECs by AFM was not greatly different to the OPC samples to the naked eye (cf. Figure 3A with 3B and 4A with 4B), but detailed DNA contour length analysis of the separation of the RNAPs along the template, reveals that the RNAP have converged consistent with the expected distance (see Figure 5). The number of DNA molecules on the mica surface with 1 or 2 SECs is reduced to about half of the OPC case, giving an overall yield of 15% of 1 SEC and 5% of 2 SECs with respect to the total number of DNA molecules observed.


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)

Inter RNAP–RNAP separation distributions along the DNA contour length. RNAP centre separation: (A) in the absence of all NTPs (n = 45), i.e. OPCs; (B) in the presence of ATP, GTP and UTP only (n = 131), i.e. SECs; (C) in the presence of ATP, GTP and CTP, followed by CTP after 20 min (n = 44); (D) formed by addition of all 4 NTPs together (n = 45) from the OPC state.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Inter RNAP–RNAP separation distributions along the DNA contour length. RNAP centre separation: (A) in the absence of all NTPs (n = 45), i.e. OPCs; (B) in the presence of ATP, GTP and UTP only (n = 131), i.e. SECs; (C) in the presence of ATP, GTP and CTP, followed by CTP after 20 min (n = 44); (D) formed by addition of all 4 NTPs together (n = 45) from the OPC state.
Mentions: SECs were formed by the omission of CTP. The OPCs at each convergent promoter would then be expected to transcribe downstream (i.e. towards each other) until the first occurrence of a C base in the non-template strand. In our template, this C base occurred at positions +24 and +70 relative to the +1 sites of transcription initiation for each promoter respectively. The template was designed such that the sequence at each stall site was identical, hence the stalling and re-initiation behaviour of RNAP at either site should be identical. The appearance of the SECs by AFM was not greatly different to the OPC samples to the naked eye (cf. Figure 3A with 3B and 4A with 4B), but detailed DNA contour length analysis of the separation of the RNAPs along the template, reveals that the RNAP have converged consistent with the expected distance (see Figure 5). The number of DNA molecules on the mica surface with 1 or 2 SECs is reduced to about half of the OPC case, giving an overall yield of 15% of 1 SEC and 5% of 2 SECs with respect to the total number of DNA molecules observed.

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