Transcribing of Escherichia coli Genes with Mutant T7 RNA Polymerases: Stability of lacZ mRNA Inversely Correlates with Polymerase Speed

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 92; no. 26; pp. 12250 - 12254
• Main Authors: Makarova, O V, Makarov, E M, Sousa, R, Dreyfus, M
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 19-12-1995; National Acad Sciences; National Academy of Sciences
• Subjects: Bacteriology; beta-Galactosidase - biosynthesis; beta-Galactosidase - genetics; Blotting, Northern; DNA-Directed RNA Polymerases - metabolism; Enzyme stability; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Genes; Genes, Bacterial; Genetic mutation; Kinetics; Lac operon; Messenger RNA; Mutagenesis, Site-Directed; Operator regions; phage T7; Point Mutation; Recombinant Proteins - metabolism; Ribonucleic acid; Ribosomes; RNA; RNA, Messenger - biosynthesis; RNA, Transfer, Arg - biosynthesis; Transcription, Genetic; Transfer RNA; Viral Proteins
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.92.26.12250

When in Escherichia coli the host RNA polymerase is replaced by the 8-fold faster bacteriophage T7 enzyme for transcription of the lacZ gene, 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 gene, 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.