Transcription and decay of the lac messenger: role of an intergenic terminator

Abstract

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.There have been extensive studies of the transcription and decay of the lac operon mRNA (reviewed in reference 18). According to current understanding, the lacZYA mRNA is transcribed polycistronically and then is cleaved at approximately intergenic sites. These cleavages, near the 5' ends of the lacY and lacA transcripts and at an additional site near the 5' end of the lacZ transcript, inactivate the distal mRNAs. The further chemical decay of these mRNAs then occurs as a net 5'-to-3' process. In this process, endonucleolytic cleavages occur on the mRNA that is exposed as ribosomes run off inactivated messages. The resulting fragments are then removed by the 3'-to-5' exonucleases, RNase II, polynucleotide phosphorylase and possibly other enzymes (1,14,18). The endonucleolytic cleavage sites on the lacZ and lacY species have been characterized but the enzyme(s) responsible for the endonucleolytic cleavages has not been identified (7,34). It appears, however, that RNase III is not involved in lac mRNA decay, although it does inactivate a few other mRNA species (2, 18, 27a). A newly described endonuclease has some properties inferred for the enzyme that cleaves the lac transcripts (6).The cleavage of the lacZYA mRNA was first deduced from the observation that the lacA mRNA decays about twice as fast as lacZ mRNA (19). Subsequently, the cleavag...

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