Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability 1 1Edited by M. Gottesman

Mentesana, Pamela E.; Chin-Bow, Stephen T.; Sousa, Rui; McAllister, William T.

doi:10.1006/jmbi.2000.4114

Cited by 55 publications

(78 citation statements)

References 50 publications

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“…Strikingly, the presence of a noncomplementary NT strand in the region of the RNA-DNA hybrid stabilizes these complexes and allows the assembly of functional, salt-resistant scaffolds having duplex DNA upstream (scaffolds 15 and 20). Similar stabilizing effects of a noncollapsing transcription bubble on the stability of halted ECs were observed previously (11).…”

Section: Fig 6 Organization Of Rnap-scaffold Complexessupporting

confidence: 85%

“…Unlike binary promoter-RNAP complexes and initiation complexes, which are rapidly inactivated by exposure to 200 mM NaCl, T7 RNAP elongation complexes are resistant to salt challenge (1,2,9,11,30). As shown in Fig.…”

Section: Assembly Of Functional Scaffoldmentioning

confidence: 99%

“…The RNA-DNA Hybrid May Only Be Extended to 12-13 bpAlthough the length of the RNA DNA hybrid is 8 bp in halted elongation complexes (11), recent studies have shown that during the transition to a stable EC the length of the hybrid may reach 10 bp before the transcription bubble collapses to 8 bp (13). When scaffold 2 (in which the primer-template is 8 bp) was incubated in the presence of all four NTPs, the primer could be efficiently extended to 12-13 nt but not beyond (Fig.…”

Section: Fig 6 Organization Of Rnap-scaffold Complexesmentioning

confidence: 99%

“…13 and see below). The final phase (between 10 and 14 nt) appears to involve displacement of the 5Ј end of the RNA from the upstream end of the hybrid and its association with an RNA product-binding site (11,15). Crystal structures have now been solved for free RNAP, for RNAP complexed with a specific inhibitor of transcription (T7 lysozyme), for a binary promoter-RNAP complex, and for an initiation complex that has transcribed the first three bases in the template strand (16 -19).…”

mentioning

confidence: 99%

“…The RNA-DNA hybrid is ϳ8 bp (14,15,22) and is enclosed in a transcription bubble of ϳ9 bp. The association of the displaced nontemplate (NT) strand of the transcription bubble with the RNAP may help to stabilize the complex (11). Fluorescence studies and KMnO 4 sensitivity indicate that the upstream edge of the transcription bubble in the EC is very close (within 1 bp) to the point at which the RNA is displaced from the template and that downstream border is very close (within 1 bp) to the 3Ј end of the RNA (in the active site) (14,22).…”

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confidence: 99%

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Characterization of T7 RNA Polymerase Transcription Complexes Assembled on Nucleic Acid Scaffolds

Temiakov

Anikin

McAllister

2002

Journal of Biological Chemistry

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We have used synthetic oligomers of DNA and RNA to assemble nucleic acid scaffolds that, when mixed with T7 RNA polymerase, allow the formation of functional transcription complexes. Manipulation of the scaffold structure allows the contribution of each element in the scaffold to transcription activity to be independently determined. The minimal scaffold that allows efficient extension after challenge with 200 mM NaCl consists of an 8-nt RNA primer hybridized to a DNA template (T strand) that extends 5-10 nt downstream. Constructs in which the RNA-DNA hybrid is less than or greater than 8 bp are less salt-resistant, and the hybrid cannot be extended beyond 12-13 bp. Although the presence of a complementary nontemplate strand downstream of the primer does not affect salt resistance, the presence of DNA upstream decreases resistance. The addition of a 4-nt unpaired "tail" to the 5 end of the primer increases salt resistance, as does the presence of an unpaired nontemplate strand in the region that contains the 8-bp hybrid (thereby generating an artificial transcription "bubble"). Scaffold complexes having these features remain active for over 1 week in the absence of salt and exhibit many of the properties of halted elongation complexes, including resistance to salt challenge, a similar trypsin cleavage pattern, and a similar pattern of RNA-RNA polymerase cross-linking.During the early stages of transcription, T7 RNAP 1 (like all known RNAPs) forms an unstable initiation complex (IC) that synthesizes and releases short abortive initiation products before clearing the promoter and forming a stable elongation complex (EC) (1-3). The transition is accompanied by release of upstream promoter contacts, changes in the size of the footprint of the polymerase on the DNA, increased resistance to challenge with agents such as salt and heparin (which disrupt and inactivate the IC), and changes in accessibility to cleavage by a variety of proteases (4 -7). Taken together, these changes suggest that significant alterations in the organization of the complex occur during the transition.A variety of lines of evidence demonstrate that the transition is complex and may involve multiple steps (6, 8 -13). Recent fluorescence probing and nuclease sensitivity experiments indicate that promoter clearance and collapse of the upstream edge of the transcription bubble occur when the RNA-DNA hybrid achieves a length of 8 -9 bp (13, 14) but that the length of the hybrid increases to 10 bp before the transcription bubble collapses to yield a hybrid length of 8 bp, as is observed during elongation (Ref. 13 and see below). The final phase (between 10 and 14 nt) appears to involve displacement of the 5Ј end of the RNA from the upstream end of the hybrid and its association with an RNA product-binding site (11, 15). Crystal structures have now been solved for free RNAP, for RNAP complexed with a specific inhibitor of transcription (T7 lysozyme), for a binary promoter-RNAP complex, and for an initiation complex that has transcribed the first thre...

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Section: Fig 6 Organization Of Rnap-scaffold Complexessupporting

confidence: 85%

Section: Assembly Of Functional Scaffoldmentioning

confidence: 99%