Transcribing of Escherichia coli genes with mutant T7 RNA polymerases: Stability of lacZ mRNA inversely correlates with polymerase speed

Olga V. Makarova, Evgeny M. Makarov, Rui Sousa, Marc Dreyfus

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Abstract

When in Escherichia coli the host RNA polymerase is replaced by the 8- fold faster bacteriophage T7 enzyme for transcription of the lacz gent, the β-galactosidase yield per transcript drops as a result of transcript destabilization. We have measured the β-galactosidase yield per transcript from T7 RNA polymerase mutants that exhibit a reduced elongation speed in vitro. Aside from very slow mutants that were not sufficiently processive to transcribe the lacz gent, the lower the polymerase speed, the higher the β- galactosidase yield per transcript. In particular, a mutant which was 2.7- fold slower than the wild-type enzyme yielded 3.4- to 4.6-fold more β- galactosidase per transcript. These differences in yield vanished in the presence of the rne-50 mutation and therefore reflect the unequal sensitivity of the transcripts to RNase E. We propose that the instability of the T7 RNA polymerase transcripts stems from the unmasking of an RNase E-sensitive site(s) between the polymerase and the leading ribosome: the faster the polymerase, the longer the lag between the synthesis of this site(s) and its shielding by ribosomes, and the lower the transcript stability.

Original languageEnglish (US)
Pages (from-to)12250-12254
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume92
Issue number26
DOIs
StatePublished - Dec 19 1995

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