Tetracycline Induces Stabilization of mRNA in
            <i>Bacillus subtilis</i>

Wei, Yi; Bechhofer, David H.

doi:10.1128/jb.184.4.889-894.2002

Cited by 31 publications

(27 citation statements)

References 32 publications

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“…Also, expression of the transcriptional fusion was slightly higher in TET-treated cells. Some antibiotics, including TET, are known to increase the stability of mRNA (2,7,27,36). Thus, it is possible that the apparent increased mRNA level in cells growing in the presence of TET could be due to this type of stabilization rather than to a second stage of regulation at the transcriptional level.…”

Section: Discussionmentioning

confidence: 97%

Regulation of a Bacteroides Operon That Controls Excision and Transfer of the Conjugative Transposon CTnDOT

2004

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CTnDOT is a conjugative transposon (CTn) that is found in many Bacteroides strains. Transfer of CTnDOT is stimulated 100-to 1,000-fold if the cells are first exposed to tetracycline (TET). Both excision and transfer of CTnDOT are stimulated by TET. An operon that contains a TET resistance gene, tetQ, and two regulatory genes, rteA and rteB, is essential for control of excision and transfer functions. At first, it appeared that RteA and RteB, which are members of a two-component regulatory system, might be directly responsible for the TET effect. We show here, however, that neither RteA nor RteB affected expression of the operon. TetQ, a ribosome protection type of TET resistance protein, actually reduced operon expression, possibly by interacting with ribosomes that are translating the tetQ message. Fusions of tetQ with a reporter gene, uidA, were only expressed at a high level when the fusion was cloned in frame with the first six codons of tetQ. However, out of frame fusions or fusions ending at the other five codons of tetQ showed much lower expression of the uidA gene. Moreover, reverse transcription-PCR amplification of tetQ mRNA revealed that despite the fact that the uidA gene product, ␤-glucuronidase (GUS), was produced only when the cells were exposed to TET, tetQ mRNA was produced in both the presence and absence of TET. Computer analysis of the region upstream of the tetQ start codon predicted that the mRNA in this region could form a complex RNA hairpin structure that would prevent access of ribosomes to the ribosome binding site. Mutations that abolished base pairing in the stem that formed the base of this putative hairpin structure made GUS production as high in the absence of TET as in TET-stimulated cells. Compensatory mutations that restored the hairpin structure led to a return of regulated production of GUS. Thus, the tetQ-rteA-rteB operon appears to be regulated by a translational attenuation mechanism.Many Bacteroides strains carry conjugative transposons (CTns) that are closely related to a CTn called CTnDOT (25,29,39). An interesting and unusual feature of many members of the CTnDOT family is that the antibiotic tetracycline (TET) stimulates both excision of the CTn from the chromosome and conjugal transfer of the excised element (24, 31, 34). In fact, without TET stimulation of donor cells, virtually no transfer occurs (33,38). Previous studies identified a central regulatory region on CTnDOT that is required for TET induction of transfer functions. This region contains a three-gene operon that consists of the TET resistance gene tetQ and two regulatory genes, rteA and rteB (Fig. 1) (29, 34). RteA and RteB are most closely related to the sensor and response regulator components, respectively, of two-component regulatory systems. RteA presumably activates RteB, and activated RteB stimulates the expression of a nearby gene, rteC, which is also required for expression of excision and transfer genes (32-34, 38) (Fig. 1).The regulatory cascade mediated by RteA, RteB, and RteC is not, however,...

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Section: Discussionmentioning

confidence: 97%

Regulation of a Bacteroides Operon That Controls Excision and Transfer of the Conjugative Transposon CTnDOT

2004

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“…We wished to determine the decay pattern for mRNAs encoded by endogenous genes in strains that were deficient in PNPase and other exoribonucleases. The first gene chosen for analysis was the veg gene, which encodes a small mRNA whose decay we have studied previously (34). Total RNA was isolated from a set of strains that were deficient either in PNPase alone or in combinations of three of the four B. subtilis 3Ј-to-5Ј exoribonucleases (triple mutants) ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

Participation of 3′-to-5′ Exoribonucleases in the Turnover of Bacillus subtilis mRNA

et al. 2005

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Four 3-to-5 exoribonucleases have been identified in Bacillus subtilis: polynucleotide phosphorylase (PNPase), RNase R, RNase PH, and YhaM. Mutant strains were constructed that were lacking PNPase and one or more of the other three ribonucleases or that had PNPase alone. Analysis of the decay of mRNA encoded by seven small, monocistronic genes showed that PNPase was the major enzyme involved in mRNA turnover. Significant levels of decay intermediates, whose 5 ends were at the transcriptional start site and whose 3 ends were at various positions in the coding sequence, were detected only when PNPase was absent. A detailed analysis of rpsO mRNA decay showed that decay intermediates accumulated as the result of a block to 3-to-5 processivity at the base of stem-loop structures. When RNase R alone was present, it was also capable of degrading mRNA, showing the involvement of this exonuclease in mRNA turnover. The degradative activity of RNase R was impaired when RNase PH or YhaM was also present. Extrapolation from the seven genes examined suggested that a large number of mRNA fragments was present in the PNPase-deficient mutant. Maintenance of the free ribosome pool in this strain would require a high level of activity on the part of the tmRNA trans translation system. A threefold increase in the level of peptide tagging was observed in the PNPase-deficient strain, and selective pressure for increased tmRNA activity was indicated by the emergence of mutant strains with elevated tmRNA transcription.The steady-state amount of a particular mRNA in a cell is a function of its synthesis and degradation. Thus, it is understood that mRNA decay is an important factor in setting levels of gene expression. In a generally accepted model for Escherichia coli (4, 18, 21), mRNA decay initiates with endonuclease cleavage by RNase E, a 5Ј-end-dependent endoribonuclease (23). Such cleavage generates an upstream fragment with an unprotected 3Ј end, which is rapidly degraded by the 3Ј-to-5Ј exonucleases, RNase II or polynucleotide phosphorylase (PNPase), and a downstream fragment that begins with a monophosphate nucleoside, which is a much better substrate for subsequent binding and cleavage by RNase E than the 5Ј-terminal triphosphate nucleoside of the initial transcription product. Additional cycles of rapid endonucleolytic cleavage and 3Ј-to-5Ј degradation result in the observed all-or-nothing pattern of decay, i.e., Northern blot analysis of a specific mRNA generally shows the full-length mRNA but few or no mRNA decay fragments. Rapid decay of mRNA seems to be an essential function, as either RNase II or PNPase is required for viability; inactivation of both RNase II and PNPase is lethal (11). E. coli contains six other 3Ј-to-5Ј exoribonucleases (39). Very recently, one of these, RNase R, was reported by several groups to be involved in mRNA decay (1).We have been studying mRNA decay in Bacillus subtilis. It was shown years ago by Duffy and colleagues that mRNA decay in B. subtilis occurs primarily phosphorolytically, rather than...

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“…The effect of mutations that increase the free energy of interaction between the ribosome and the SD sequence can be mimicked by the addition of very low levels of tetracycline (0.25 g/ml) to B. subtilis cell cultures (230). This results in the stabilization of a number of cellular mRNAs including polC, sigA, veg, and tet(L) genes, the last of which encodes a membrane-bound ion transport protein that affords resistance to tetracycline itself.…”

Section: General Principlesmentioning

confidence: 99%

RNA Processing and Degradation inBacillus subtilis

Condon

2003

Microbiol Mol Biol Rev

141

134

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This review focuses on the enzymes and pathways of RNA processing and degradation in Bacillus subtilis, and compares them to those of its gram-negative counterpart, Escherichia coli. A comparison of the genomes from the two organisms reveals that B. subtilis has a very different selection of RNases available for RNA maturation. Of 17 characterized ribonuclease activities thus far identified in E. coli and B. subtilis, only 6 are shared, 3 exoribonucleases and 3 endoribonucleases. Some enzymes essential for cell viability in E. coli, such as RNase E and oligoribonuclease, do not have homologs in B. subtilis, and of those enzymes in common, some combinations are essential in one organism but not in the other. The degradation pathways and transcript half-lives have been examined to various degrees for a dozen or so B. subtilis mRNAs. The determinants of mRNA stability have been characterized for a number of these and point to a fundamentally different process in the initiation of mRNA decay. While RNase E binds to the 5′ end and catalyzes the rate-limiting cleavage of the majority of E. coli RNAs by looping to internal sites, the equivalent nuclease in B. subtilis, although not yet identified, is predicted to scan or track from the 5′ end. RNase E can also access cleavage sites directly, albeit less efficiently, while the enzyme responsible for initiating the decay of B. subtilis mRNAs appears incapable of direct entry. Thus, unlike E. coli, RNAs possessing stable secondary structures or sites for protein or ribosome binding near the 5′ end can have very long half-lives even if the RNA is not protected by translation

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Tetracycline Induces Stabilization of mRNA in Bacillus subtilis

Cited by 31 publications

References 32 publications

Regulation of a Bacteroides Operon That Controls Excision and Transfer of the Conjugative Transposon CTnDOT

Regulation of a Bacteroides Operon That Controls Excision and Transfer of the Conjugative Transposon CTnDOT

Participation of 3′-to-5′ Exoribonucleases in the Turnover of Bacillus subtilis mRNA

RNA Processing and Degradation inBacillus subtilis

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