Characterization of a set of T7 RNA polymerase active site mutants

Gary Bonner, Eileen M. Lafer, Rui Sousa

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Abstract

We have evaluated the elongation rates, processivities, and abortive transcription characteristics of a set of T7 RNA polymerase mutants that map to the polymerase active site. The effects of these mutations on transcription are complex: they cause decreases in activity and processivity during both the processive and abortive phases of transcription and exhibit disproportionate decreases in activity and processivity on poly(dA)·poly(dT) or poly(dT) versus poly(dG)·poly(dC) templates. They also exhibit an increase in the proportion of slippage dependent poly(G) transcript synthesis during the initial stages of transcription. It is shown that these multiple, distinct effects on transcription can be attributed to decreases in the mutant enzymes' phosphodiester bond formation rates. Estimates of the decreases in these rates are derived from the measured transcript elongation rates and processivities of the mutant enzymes.

Original languageEnglish (US)
Pages (from-to)25120-25128
Number of pages9
JournalJournal of Biological Chemistry
Volume269
Issue number40
StatePublished - Oct 7 1994

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Bonner, G., Lafer, E. M., & Sousa, R. (1994). Characterization of a set of T7 RNA polymerase active site mutants. Journal of Biological Chemistry, 269(40), 25120-25128.