Transcribing of Escherichia coli genes with mutant T7 RNA polymerases: stability of lacZ mRNA inversely correlates with polymerase speed.
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 beta-galactosidase yield per transcript drops as a result of transcript destabilization. We have measured the beta-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 beta-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 beta-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.
Makarova
O V
OV
Laboratoire de Génétique Moléculaire, Centre National de la Recherche Scientifique, URA 1302, Ecole Normale Supérieure, Paris, France.
Makarov
E M
EM
Sousa
R
R
Dreyfus
M
M
D015152:Blotting, Northern; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D012333:RNA, Messenger / Q000096:biosynthesis*; D012347:RNA, Transfer, Arg / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins; D001616:beta-Galactosidase / Q000096:biosynthesis* / Q000235:genetics
D012333:RNA, Messenger; D012347:RNA, Transfer, Arg; D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase