Multiple determinants of functional mRNA stability: sequence alterations at either end of the lacZ gene affect the rate of mRNA inactivation

Abstract

The Escherichia coli lacZ gene was used as a model system to identify specific sequence elements affecting mRNA stability. Various insertions and substitutions at the ribosome-binding site increased or decreased the rate of mRNA inactivation by up to fourfold. Deletion of a dyad symmetry, which may give rise to a very stable secondary structure in the mRNA immediately downstream of the gene, decreased the functional stability of the lacZ message. The magnitude of the latter effect was strongly dependent on the sequences at the ribosomebinding site, ranging from practically no effect for the most labile transcripts to a threefold decrease in stability for the most stable one. The results suggest that the wild-type lacZ message is inactivated predominantly by attacks near the ribosome-binding site, presumably in part because the putative secondary structure downstream of the gene protects against 3'-exonucleolytic attack. Taken together, the data for all of the modified variants of lacZ were shown to be quantitatively compatible with a general model of mRNA inactivation involving multiple independent target sites.In Escherichia coli, individual mRNAs are inactivated with half-lives ranging from 40 s to 20 min (6, 34), and much work has been done to identify and characterize the factors that are responsible for this great variation of mRNA stability (for reviews, see references 4, 7, and 22). Decay of some mRNAs like those of the ompA, pnp, and nusA genes appears to be initiated by endonucleolytic cleavages near the 5' end (30, 39, 40), whereas the mRNA of the X int gene is inactivated by endonucleolytic processing downstream of the gene, followed by exonucleolytic attack from the generated 3' end (38, 41). RNase III is responsible for endonucleolytic processing of the pnp, nusA, and int transcripts, but it is probably only a small class of mRNAs that are inactivated by this enzyme (3). The 3'-exonucleases RNase II and polynucleotide phosphorylase have been implicated in mRNA decay since the early 1960s (reviewed in references 2 and 11) and appear to be the major enzymes involved in chemical degradation of mRNA (13). However, for most mRNAs the target site of the initial inactivating event and the enzymes involved are unknown.Processive degradation by RNase II and polynucleotide phosphorylase is impaired by secondary structures in the substrate (18,31,42), and several mRNAs have been shown to be stabilized by stable secondary structures at the 3' end (12,19,32,44). Similarly, specific sequence elements which act as mRNA stabilizers when introduced near the 5' end of heterologous transcripts have been identified (5, 14, 16), but in these cases the mechanism responsible for the stabilization is not clear. I used a hybrid lacZ-galK operon as a model system to investigate how the functional stability of the lacZ message may be determined by a combination of specific sequence elements. In agreement with previous studies (9, 21), the results suggest that the lacZ message is inactivated predominantly by attacks near the r...

An efficient Shine-Dalgarno sequence but not translation is necessary for lacZ mRNA stability in Escherichia coli

“…The rates of decay of several bacterial transcripts, including ompA, lacZ, and ermC, are controlled by determinants located at the 5' ends of the mRNAs (2,3,5,12,29,30). Decay is initiated at the 5' ends of some transcripts, for example, lacZ and ompA (6,23,24).…”

Roles of a ribosome-binding site and mRNA secondary structure in differential expression of Shiga toxin genes

“…Previous studies have reported that various insertions and substitutions at the RBS affect the rate of mRNA degradation (30) and that the hairpin structures impart stability to selected regions of polycistronic mRNAs (12). However, Northern blot analysis of total cellular RNA isolated from strains containing the six operon with the RBS and STL mutations showed that there was no effect on the steadystate level of six mRNA.…”

Control of mRNA processing and decay in prokaryotes

“…Several studies demonstrated that mRNA leader segments confer stability to otherwise labile transcripts (Yamamoto & Imamoto, 1975;Gorski et al, 1985;Belasco et al, 1986;Gerdes et al, 1988;Rdgnier & Grunberg-Manago, 1990;Petersen, 1991). The ompA leader has been shown to stabilize efficiently a downstream bla message fused to it; interestingly, this stabilization required both the presence of an upstream segment of the ompA leader, which contains secondary structures, and tr'anslation of the bla message from the ompA ribosome-binding site (Belasco etal., 1986;Emory & Belasco, 1990).…”

“…For such a reason, the segment corresponding to the lacZ message was more stable than the downstream lacY message, and it was inactivated predominantly by endonucleolytic attacks occurring near the ribosome-binding site. The general model of mRNA inactivation derived by Petersen (1991) also implies that chemical mRNA decay, endoribonucleolytic or exoribonucleolytic, depends on the presence and distribution of endonucleolytic target sites and protective RNA secondary structures at the 3 ~ end. In 1982 Higgins et al identified a novel intercistronic regulatory element of prokaryotic operons: the repetitive extragenic palindromic (REP) sequence.…”