The low processivity of T7 RNA polymerase over the initially transcribed sequence can limit productive initiation in vivo

Journal of Molecular Biology
P J LopezM Dreyfus

Abstract

In vitro, after binding to the promoter to form a catalytically active complex, RNA polymerases abortively cycle over the first transcribed nucleotides (initial transcribed sequence or ITS) before leaving the promoter. With the bacteriophage T7 enzyme, the extent of abortive transcription varies with the nature of the ITS and with the elongation speed of the polymerase. Here, we compare in vitro and in vivo the yield of long transcripts from T7 promoters, with two different ITSs, the T7 gene10 and the lactose operon ITSs, and two different T7 RNA polymerases, the wild-type and a 2.7-fold slower mutant (G645A). The use of non-cognate ITS and/or slow polymerase decreases the yield of long transcripts in vitro and in vivo in a parallel fashion, with low polymerase speed and non-cognate ITS acting synergistically. In vitro, this decrease is mirrored by an increase in the average number of abortive cycles the enzyme undergoes before leaving the promoter; specifically, with the G645A mutant, transcript release is favored at any ITS position, whereas with the lac ITS it is particularly frequent at positions five and six following the incorporation of uridine residues. Hence, the more abortive cycles per long transcript synthesis in vi...Continue Reading

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Citations

Jan 19, 2002·Journal of Virology·Rachel FearnsPeter L Collins
May 30, 1998·Proceedings of the National Academy of Sciences of the United States of America·P J LopezM Dreyfus
Apr 16, 2005·Proceedings of the National Academy of Sciences of the United States of America·Jean GuillerezMarc Dreyfus
May 5, 1998·Journal of Molecular Biology·P J LopezM Dreyfus
Sep 15, 1998·Journal of Molecular Biology·S A Joyce, M Dreyfus
Mar 30, 2017·Genomics·Jeremy I MarcusNikhil U Nair
May 10, 2020·Communications Biology·Patrick J ShillingDaniel O Daley
Oct 7, 2004·Analytical Biochemistry·Pamela R MollKlaus Richter

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