The stability of Escherichia coli lacZ mRNA depends upon the simultaneity of its synthesis and translation.
We have used either Escherichia coli or T7 RNA polymerase to transcribe in E. coli a series of lacZ genes that differ in the nature of their ribosome binding sites (RBS). Each T7 RNA polymerase transcript yields from 15- to 450-fold less beta-galactosidase than its E. coli polymerase counterpart, the ratio being larger when weaker RBS are used. The low beta-galactosidase yield from T7 transcripts reflects their low stability: the ams-1/rne-50 mutation, which inactivates RNase E, nearly equalizes the beta-galactosidase yields from T7 and E. coli RNA polymerase transcripts. T7 RNA polymerase transcribes the lacZ gene approximately 8-fold faster than the E. coli enzyme. We propose that this higher speed unmasks an RNase E cleavage site which is normally shielded by ribosomes soon after its synthesis when the slower E. coli enzyme is used. This leads to degradation of the T7 transcript, unless the leading ribosome comes in time to shield the cleavage site: the weaker the RBS, the lower this probability and the more severe the inability of T7 RNA polymerase transcripts for beta-galactosidase synthesis.
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Laboratoire de Génétique Moléculaire (CNRS D1302), Ecole Normale Supérieure, Paris, France.
Dreyfus
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D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002874:Chromosome Mapping; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D012329:RNA, Bacterial; D012333:RNA, Messenger; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase