Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability

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

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

We have constructed a series of plasmid templates that allow T7 RNA polymerase (RNAP) to be halted at defined intervals downstream from its promoter in a preserved sequence context. While transcription complexes halted at +3 to +6 are highly unstable, complexes halted at +10 to +14 dissociate very slowly and gradually lose their capacity to extend transcripts. Complexes halted at +18 and beyond dissociate more readily, but the stability of the these complexes is enhanced significantly in the presence of the next incoming nucleotide. Unexpectedly, the stability of complexes halted at +14 and beyond was found to be lower on supercoiled templates than on linear templates. To explore this further, we used synthetic DNA templates in which the nature of the non-template (NT) strand was varied. Whereas initiation complexes are less stable in the presence of a complementary NT strand, elongation complexes are more stable in the presence of a complementary NT strand, and the presence of a non-complementary NT strand (a mismatched bubble) results in even greater stability. The results suggest that the NT strand plays an important role in displacing the nascent RNA, allowing its interaction with an RNA product binding site in the RNAP. The NT strand may also contribute to stabilization by interacting directly with the enzyme. A mutant RNAP that has a deletion in the flexible 'thumb' domain responds to changes in template topology in a manner that is similar to that of the wild-type (WT) enzyme, but halted complexes formed by the mutant enzyme are particularly dependent upon the presence of the NT strand for stability. In contrast, an N-terminal RNAP mutant that has a decreased capacity to bind single-stranded RNA forms halted complexes with much lower levels of stability than the WT enzyme, and these complexes are not stabilized by the presence of the NT strand. The distinct responses of the mutant RNAPs to changes in template structure indicate that the N-terminal and thumb domains have quite different functions in stabilizing the transcription complex. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)1049-1062
Number of pages14
JournalJournal of Molecular Biology
Volume302
Issue number5
DOIs
StatePublished - Oct 6 2000

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DNA-Directed RNA Polymerases
Thumb
RNA
Enzymes
Enzyme Stability
Plasmids
Nucleotides
Binding Sites
DNA
bacteriophage T7 RNA polymerase

Keywords

  • Elongation complex
  • Initiation complex
  • Isomerization
  • RNA:DNA hybrid
  • Termination

ASJC Scopus subject areas

  • Virology

Cite this

Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability. / Mentesana, Pamela E.; Chin-Bow, Stephen T.; Sousa, Rui J; McAllister, William T.

In: Journal of Molecular Biology, Vol. 302, No. 5, 06.10.2000, p. 1049-1062.

Research output: Contribution to journalArticle

Mentesana, Pamela E. ; Chin-Bow, Stephen T. ; Sousa, Rui J ; McAllister, William T. / Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability. In: Journal of Molecular Biology. 2000 ; Vol. 302, No. 5. pp. 1049-1062.
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abstract = "We have constructed a series of plasmid templates that allow T7 RNA polymerase (RNAP) to be halted at defined intervals downstream from its promoter in a preserved sequence context. While transcription complexes halted at +3 to +6 are highly unstable, complexes halted at +10 to +14 dissociate very slowly and gradually lose their capacity to extend transcripts. Complexes halted at +18 and beyond dissociate more readily, but the stability of the these complexes is enhanced significantly in the presence of the next incoming nucleotide. Unexpectedly, the stability of complexes halted at +14 and beyond was found to be lower on supercoiled templates than on linear templates. To explore this further, we used synthetic DNA templates in which the nature of the non-template (NT) strand was varied. Whereas initiation complexes are less stable in the presence of a complementary NT strand, elongation complexes are more stable in the presence of a complementary NT strand, and the presence of a non-complementary NT strand (a mismatched bubble) results in even greater stability. The results suggest that the NT strand plays an important role in displacing the nascent RNA, allowing its interaction with an RNA product binding site in the RNAP. The NT strand may also contribute to stabilization by interacting directly with the enzyme. A mutant RNAP that has a deletion in the flexible 'thumb' domain responds to changes in template topology in a manner that is similar to that of the wild-type (WT) enzyme, but halted complexes formed by the mutant enzyme are particularly dependent upon the presence of the NT strand for stability. In contrast, an N-terminal RNAP mutant that has a decreased capacity to bind single-stranded RNA forms halted complexes with much lower levels of stability than the WT enzyme, and these complexes are not stabilized by the presence of the NT strand. The distinct responses of the mutant RNAPs to changes in template structure indicate that the N-terminal and thumb domains have quite different functions in stabilizing the transcription complex. (C) 2000 Academic Press.",
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AB - We have constructed a series of plasmid templates that allow T7 RNA polymerase (RNAP) to be halted at defined intervals downstream from its promoter in a preserved sequence context. While transcription complexes halted at +3 to +6 are highly unstable, complexes halted at +10 to +14 dissociate very slowly and gradually lose their capacity to extend transcripts. Complexes halted at +18 and beyond dissociate more readily, but the stability of the these complexes is enhanced significantly in the presence of the next incoming nucleotide. Unexpectedly, the stability of complexes halted at +14 and beyond was found to be lower on supercoiled templates than on linear templates. To explore this further, we used synthetic DNA templates in which the nature of the non-template (NT) strand was varied. Whereas initiation complexes are less stable in the presence of a complementary NT strand, elongation complexes are more stable in the presence of a complementary NT strand, and the presence of a non-complementary NT strand (a mismatched bubble) results in even greater stability. The results suggest that the NT strand plays an important role in displacing the nascent RNA, allowing its interaction with an RNA product binding site in the RNAP. The NT strand may also contribute to stabilization by interacting directly with the enzyme. A mutant RNAP that has a deletion in the flexible 'thumb' domain responds to changes in template topology in a manner that is similar to that of the wild-type (WT) enzyme, but halted complexes formed by the mutant enzyme are particularly dependent upon the presence of the NT strand for stability. In contrast, an N-terminal RNAP mutant that has a decreased capacity to bind single-stranded RNA forms halted complexes with much lower levels of stability than the WT enzyme, and these complexes are not stabilized by the presence of the NT strand. The distinct responses of the mutant RNAPs to changes in template structure indicate that the N-terminal and thumb domains have quite different functions in stabilizing the transcription complex. (C) 2000 Academic Press.

KW - Elongation complex

KW - Initiation complex

KW - Isomerization

KW - RNA:DNA hybrid

KW - Termination

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