Transcription and decay of the lac messenger: role of an intergenic terminator.
Prior work has indicated that the polycistronic lacZYA mRNA of Escherichia coli is cleaved during decay at approximately intergenic sites (L. W. Lim and D. Kennell, J. Mol. Biol. 135: 369-390, 1979). In this work, we characterized the products by using probes specific for the different cistrons. This analysis indicated that six lac mRNA species are present in the following order of decreasing abundance: lacZ, -A, -ZYA, -ZY, -YA, and -Y. Very little lacYA and lacY mRNAs were present, whereas in cells induced to steady state, there was 10 times more lacZ than lacZYA mRNA. The lacZ mRNA appeared as a discrete species extending to a site in the lacZ-Y intergenic space (ca. residue 3150). This site is just distal to a potential rho-independent termination sequence. We examined the function of this sequence to determine whether it contributes to the distribution of the mRNAs. Although the termination sequence was shown to function in vitro, when it was recloned into an expression vector, no termination was seen in vivo. Moreover, direct examination of the kinetics of lac messenger synthesis revealed that after initiation, most transcription continued to the end of the operon. We conclude that during normal growth, the operon is transcribed in its entirety and that the individual lac mRNAs are formed by cleavage. These results confirm earlier work implying that the lac operon is transcribed in its entirety but are in conflict with several recent reports suggesting that internal termination occurs. Our findings indicate that the natural polarity of the operon (lacZ is expressed sixfold more strongly than lacA) is based on posttranslational effects and not on polarity of transcription.
Murakawa
G J
GJ
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024-1489.
Kwan
C
C
Yamashita
J
J
Nierlich
D P
DP
D001483:Base Sequence; D015152:Blotting, Northern; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D007763:Lac Operon; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D015347:RNA Probes; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D015347:RNA Probes; D012329:RNA, Bacterial; D012333:RNA, Messenger