Initiation, elongation and inactivation of lac messenger RNA in Escherichia coli studied by measurement of its β-galactosidase synthesizing capacity in vivo

Jacquet, Michel; Képès, Adam

doi:10.1016/0022-2836(71)90181-1

Cited by 67 publications

(37 citation statements)

References 23 publications

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“…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 ribosome-binding site, whereas a putative secondary mRNA structure immediately downstream of the coding sequence is proposed to play a more passive role, providing protection against 3'-exonucleolytic attack. Based on the experimental data, a general model of mRNA inactivation which involves multiple independent target sites and allows for quantitative assessment of their relative importance is proposed.…”

supporting

confidence: 92%

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

Petersen

1991

J Bacteriol

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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...

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supporting

confidence: 92%

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

Petersen

1991

J Bacteriol

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“…Uninfected cells had to be used because synthesis of j3-galactosidase is shut off between 10 and 20 min after S13 infection (BorrAs and E. S. Tessman, unpublished data). After an initial 3-min lag, the ability to synthesize f3-galactosidase decays with a half-life of 1.6 0.3 min in good agreement with other determinations (10)(11)(12). The functional decay of ,B-galactosidase mRNA occurs at nearly the same rate as the structural decay of bulk cell mRNA (1.9 0.1 min).…”

supporting

confidence: 84%

“…It is conceivable that the more rapid functional decay arises from some unknown effect of rifampicin on the rate of mRNA translation. However, experiments (11) in which the synthesis of ,3-galactosidase after dilution of the inducer was compared with synthesis after addition of rifampicin have shown that the amount of protein translated from previously initiated mRNA chains is not modified by the drug. This result suggests that translation itself is not impaired by the presence of rifampicin.…”

mentioning

confidence: 99%

Difference Between Functional and Structural Integrity of Messenger RNA

Puga

Borrás

Tessman

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

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Messenger RNA molecules that are structurally stable, as measured by their ability to hybridize to DNA, may nevertheless be considerably less stable in retaining their ability to function in protein synthesis. The structure of the majority of the mRNA of phage S13 decays with a half-life of 10.6 4-0.5 min. In contrast, much of the function of the mRNA that is involved in synthesis of a capsid protein (product of the F gene) decays rapidly with a half-life of 1.4 4 0.8 min; a residual amount of function decays with a half-life of 14.0 4 4.0 min. The measurements were made in the presence of rifampicin, which was used to prevent the formation of new mRNA. A proposed model for the functional decay is based on the polycistronic nature of the mRNA. Degradation of the mRNA would proceed in two steps: the first step would be a fast attack at a region near the 5'-terminus of each molecule that would eliminate the function of the proximal message; the second step would be a slow attack on the remaining messenger molecule precipitating a subsequent rapid degradation of the physical structure.Studies on degradation of messenger RNA of bacteriophages OX174 (1, 2), M13 (3), and T7 (4, 5) have shown that their mRNA is relatively resistant to decay. At least in T7 infection only the viral mRNA is stabilized, not the mRNA of the infected host, thus ruling out a viral-directed general interference with mRNA degradation.For the above three viruses, only decay of the ability of the mRNA to hybridize to DNA was measured, and no information was obtained on decay of function of the mRNA. But the functional half-life is critical in protein synthesis. Alteration or removal of perhaps just one nucleotide could rapidly eliminate the function of mRNA without significantly reducing the amount of mRNA detected by hybridization. As a result, a structurally long-lived messenger could have a quite short functional life.Since it is likely that degradation of mRNA plays an important role in regulation of protein synthesis, we have studied the decay of both biological function and structural integrity in the S13-OX174 phage system. Functional decay was studied by examination of the rate of protein synthesis during inhibition of new RNA synthesis by rifampicin. The rate of protein synthesis was measured by the uptake of a labeled amino acid during short pulses into the major phage (1, 2) and with the small filamentous phage M13 (3). We examined early and late S13 mRNA to see if both have the same decay rate, particularly to examine the possibility that after infection some phage-specified protein may be synthesized that selectively protects phage mRNA against degradation; if this were true, the early messenger that would have to code for such a protein could not be protected, and would be degraded as fast as the host mRNA. We also examined the effect of messenger concentration on its degradation by using a mutant in gene A, blocked in DNA replication, that produces four-times less mRNA than the wild type (6). Thus, we could test the possi...

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“…The enzyme has a very high turnover for the polymerisation of ribonucleotides, both in vitro (6) and in vivo (7). As a result, in E. coli it runs far ahead of the ribosomes which translate its nascent transcript, a situation which is quite unusual in this organism (8). It is not known whether this desynchronisation affects gene expression, although we recently hypothesized that it might decrease the number of polypeptides synthesized per transcript by depressing mRNA stability and/or translatability (7).…”

Section: Introductionmentioning

confidence: 99%

The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient inEscherichia colibut its transcripts are poorly expressed

1994

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In gene expression studies, promoters are often fused to a protein-encoding reporter gene, the expression of which is then taken as an indirect measure of their strength. Here, we advocate the use of a tRNA reporter for the direct quantification of promoter strength. Using this method, we have studied the bacteriophage T7 gene 10 promoter in an E.coli strain that produces saturating amounts of T7 RNA polymerase. At 370C in aminoacid-glycerol medium, we show that this promoter ranks amongst the strongest known, directing ca 1.1 transcription events per second, 2.2-fold more than the promoters for rRNA operons, or 15-fold more than the induced lac promoter. Surprisingly, compared to the lac promoter, the T7 promoter is far less efficient in driving the expression of protein-encoding genes such as cat, neo or lacZ. Therefore, the polypeptide yield per transcript is lower when the T7 RNA polymerase is used instead of the E.coli RNA polymerase. The former enzyme travels faster than the translating ribosomes, and we suggest that this desynchronization lowers the polypeptide yield per transcript. INTRODUCTIONBacteriophages T3, T7 and SP6 encode closely related RNA polymerases which transcribe the viral genome from highly specific promoters, during the late phase of infection (1). The prototype of the group, the T7 RNA polymerase, can be stably expressed in E. coli, and genes that are fused to a T7 late promoter can be expressed to high levels when introduced in such hosts (2, 3). In spite of this practical bearing, relatively little is known about the in vivo behaviour of these RNA polymerases. Indirect evidence suggests that the T7 RNA polymerase interacts very efficiently with its promoter in E. coli (4), whereas in vitro the interaction appears relatively weak (1,5). The enzyme has a very high turnover for the polymerisation of ribonucleotides, both in vitro (6) and in vivo (7). As a result, in E. coli it runs far ahead of the ribosomes which translate its nascent transcript, a situation

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Initiation, elongation and inactivation of lac messenger RNA in Escherichia coli studied by measurement of its β-galactosidase synthesizing capacity in vivo

Cited by 67 publications

References 23 publications

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

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

Difference Between Functional and Structural Integrity of Messenger RNA

The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient inEscherichia colibut its transcripts are poorly expressed

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