The relation between translation and mRNA degradation in the lacZ gene.
The technique of gene fusion, in which the gene of interest, severed from its 3' end, is in-phase fused to a reporter gene--usually lacZ--is widely used to study translational regulation in Escherichia coli. Implicit in these approaches is the assumption that the activity of the ribosome binding site (RBS) fused in-phase with lacZ, does not per se modify the steady-state level of the lacZ mRNA. Herein, we have tested this hypothesis, using a model system in which the RBS of the lamB gene is fused to lacZ. Several point mutations affecting translation initiation have been formerly characterized in this RBS, and we used Northern blots to study their effect upon the lacZ mRNA pattern. Two series of constructs were assayed: in the first one, a 51-bp fragment centered around the lamB initiator codon, was inserted in front of lacZ within the natural lactose operon, whereas in the second the lacZ gene was fused to the genuine malK-lamB operon just downstream from the lamB RBS. We observed that in the first series, the concentration and average molecular weight of the lacZ mRNA dropped sharply as the efficiency of the RBS decreased. This apparently arose from a decreased stability of the message, since the mRNA patterns are equalized when the endonuclease RNase E is inactivated. We suggest that in this case the rate limiting step in the decay process is an RNase E cleavage that is outcompeted by translation.(ABSTRACT TRUNCATED AT 250 WORDS)
Yarchuk
O
O
Laboratoire de Génétique moléculaire de l'Ecole Normale Supérieure (CNRS D1302), Paris, France.
Iost
I
I
Dreyfus
M
M
D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007763:Lac Operon; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D012329:RNA, Bacterial; D012333:RNA, Messenger; D004722:Endoribonucleases; C022537:ribonuclease E