Use of antibiotics to determine ribosome-messenger RNA interactions necessary for in vivo stability of specific messengers

Cohen, Paul S.; Lynch, Kevin R.; Walsh, Marcia L.; Hill, Janet M.; Ennis, Herbert L.

doi:10.1016/0022-2836(77)90179-6

Cited by 10 publications

(6 citation statements)

References 11 publications

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“…Tc, which stabilizes polysomes, was found to stabilize mRNAs (9,28). However, in these studies the concentrations of antibiotics used could rapidly inhibit all protein synthesis (e.g., Tc at 100 g/ml).…”

Section: Discussionmentioning

confidence: 92%

Tetracycline Induces Stabilization of mRNA in Bacillus subtilis

Wei

Bechhofer

2002

J Bacteriol

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The tet(L) gene of Bacillus subtilis confers low-level tetracycline (Tc) resistance. Previous work examining the >20-fold-inducible expression of tet(L) by Tc demonstrated a 12-fold translational induction. Here we show that the other component of tet(L) induction is at the level of mRNA stabilization. Addition of a subinhibitory concentration of Tc results in a two-to threefold increase in tet(L) mRNA stability. Using a plasmid-borne derivative of tet(L) with a large in-frame deletion of the coding sequence, the mechanism of Tc-induced stability was explored by measuring the decay of tet(L) mRNAs carrying specific mutations in the leader region. The results of these experiments, as well as experiments with a B. subtilis strain that is resistant to Tc due to a mutation in the ribosomal S10 protein, suggest different mechanisms for the effects of Tc on translation and on mRNA stability. The key role of the 5 end in determining mRNA stability was confirmed in these experiments. Surprisingly, the stability of several other B. subtilis mRNAs was also induced by Tc, which indicates that addition of Tc may result in a general stabilization of mRNA.The control of bacterial mRNA decay has been shown to be dependent, in part, on events at or near the 5Ј end. In Escherichia coli, the 5Ј-end dependence of mRNA decay is thought to be due to RNase E, the major decay-initiating endoribonuclease, whose cleavage activity is strongly influenced by the phosphorylation state and accessibility of the 5Ј end (8, 21).In Bacillus subtilis, an RNase E sequence homologue is not identifiable in the genome. Nevertheless, indirect evidence for B. subtilis RNase E activity has been obtained (10), and we have shown, using the erythromycin (Em) resistance gene ermC mRNA as a model, that access to the 5Ј end is critical for determining mRNA half life; Em-induced stalling of a ribosome near the 5Ј end of ermC mRNA results in stabilization of the message (3, 11). The specific set of codons in the ermC leader region coding sequence is required for Em-induced ribosome stalling and resultant ermC mRNA stabilization (17,22). Other B. subtilis mRNAs have not been found to be stabilized by addition of Em.The B. subtilis chromosome contains a tetracycline (Tc) resistance gene (32), designated tet(L), encoding a multifunctional membrane protein that is a physiologically important monovalent cation/proton antiporter that catalyzes Naprotein is also capable of Tc/H ϩ exchange, and the presence of chromosomal tet(L) confers low-level resistance to Tc. Expression of tet(L) is inducible by Tc (7, 26), and inducibility is a function of the 124-nucleotide (nt) tet(L) leader region, which encodes a 20-amino-acid open reading frame (Fig. 1).Using tet(L)-lacZ fusions containing various mutations in the tet(L) leader region, we developed a model for translational regulation that involves Tc-induced reinitiation of translation (27). Specifically, the translational signals for the Tet(L) coding sequence function poorly, so that basal-level translation is low. On the...

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Section: Discussionmentioning

confidence: 92%

Tetracycline Induces Stabilization of mRNA in Bacillus subtilis

Wei

Bechhofer

2002

J Bacteriol

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“…The control values were obtained in experiments similar to that described in Fig. 1. inhibit specific steps in the protein synthesis pathway (5). Cycloheximide inhibits the elongation step of protein synthesis (29).…”

Section: Resultsmentioning

confidence: 99%

“…We previously showed that it was possible to assess, for bacteria, the role protein synthesis plays in the degradation of particular mRNAs by using inhibitors specific for individual reactions in protein synthesis (5). This approach enabled us to determine the effects of ribosome-mRNA interactions on mRNA degradation and to localize sites on the mRNA which appear to be particularly important for decay.…”

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confidence: 99%

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Kelly¹,

Shaw²,

Ennis³

1987

Mol. Cell. Biol.

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Spore germination in Dictyostelium discoideum is a particularly suitable model for studying the regulation of gene expression, since developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. The previous isolation of three cDNA clones specific for mRNA developmentally regulated during spore germination allowed for the quantitation of the specific itlRNAs during this process. The three mRNAs specific to clones pLK109, pLK229, and pRK270 have half-lives much shorter (minutes) than those of constitutive mRNAs (hours). Using spore germination as a model, we studied the roles of ribosome-mRNA interactions and protein synthesis in mRNA degradation by using antibiotics that inhibit specific reactions in protein biosynthesis. Cycloheximide inhibits the elongation step of protein synthesis. Polysomes accumulate in inhibited cells because ribosomes do not terminate normally and new ribosomes enter the polysome, eventually saturating the mRNA. Pactamycin inhibits initiation, and consequently polysomes break down in the presence of this drug. Under this condition, the mRNA is essentially free of ribosomes. pLK109, pLK229, and pRK270 mRNAs were stabilized in the presence of cycloheximide, but pactamycin had no effect on their normal decay. Since it seems likely that stability of mRNA reflects the availability of sites for inactivation by nucleases, it follows that in the presence of cycloheximide, these sites are protected, presumably by occupancy by ribosomes. No ribosomes are bound to mRNA in the presence of pactamycin, and therefore mRNA degrades at about the normal rate. The data further indicate that a labile protein is probably not involved in mRNA decay or stabilization, since protein synthesis is inhibited equally by both antibiotics. We conclude that it may be important to use more than one type of protein synthesis inhibitor to evaluate whether protein synthesis is required for mRNA decay. The effect of protein synthesis ihhibition on mRNA synthesis and accumulation was also studied. mRNA synthesis continues in the presence of the inhibitors, albeit at a diminished rate relative to that of the uninhibited control.

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“…In K+-depleted cells, there appears to be a very close association between mRNA synthesis and its decay; i.e., the reduced rate of mRNA synthesis is mirrored by a reduced rate of mRNA decay ( Table 1). The same is true of specific messages in antibiotic-treated cells (3,19). This result indicates that a mechanism may be present in the cell that can regulate the concentration of mRNA much the same as the mechanism that exists for stable RNA in uninfected cells (9).…”

Section: Discussionmentioning

confidence: 85%

“…It may be possible to use K+ depletion as a simple method to prepare quantities of functional T4 mRNA's. It is known that inhibition of protein synthesis by itself is not sufficient to stabilize all T4 mRNA's, although treatment with individual inhibitors can result in stabilization of specific mRNA (3,19). K+ depletion may stabilize mRNA because (i) K+ may be required for an RNase that degrades mRNA because the chemical stability of phage T4 mRNA is regulated by a K+-dependent reaction (2) (several ion-dependent RNases have been described [10], but none as yet can be identified as the putative mRNase); (ii) the secondary structure of the mRNA in the absence of K+ may be such that it renders the molecule insensitive to nucleolytic attack in vivo; and (iii) K+ may be required for the proper in vivo organization of subcellular particles.…”

Section: Discussionmentioning

confidence: 99%

Role of potassium in the synthesis and decay of two specific bacteriophage T4 messages

Walsh¹,

Cohen²,

Ennis³

1977

J Virol

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The role of K+ in the in vivo metabolism of specific phage T4 messengers was studied. By using a mutant of Escherichia coli defective in its ability to accumulate K+ from the growth medium, it was possible to rapidly deplete cells of their intracellular K+ and in this way determine K+-dependent reactions in vivo. The rate constants for accumulation, synthesis, and decay of the early enzymes deoxynucleotide kinase and a-glucosyl transferase were determined. It was shown that there is a very close association between mRNA synthesis and its decay, indicating that a mechanism may be present in the cell that can regulate the concentration of these RNAs. Since the mRNA's for these enzymes are very stable in cells depleted of K+, K+ depletion may be a useful method for the isolation of functional T4 mRNA.

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Use of antibiotics to determine ribosome-messenger RNA interactions necessary for in vivo stability of specific messengers

Cited by 10 publications

References 11 publications

Tetracycline Induces Stabilization of mRNA in Bacillus subtilis

Tetracycline Induces Stabilization of mRNA in Bacillus subtilis

Role of protein synthesis in decay and accumulation of mRNA during spore germination in the cellular slime mold Dictyostelium discoideum.

Role of potassium in the synthesis and decay of two specific bacteriophage T4 messages

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