Analysis of <i>cis</i>‐acting sequence and structural elements required for antitermination of the <i>Bacillus subtilis tyrS</i> gene

Rollins, Sean M.; Grundy, Frank J.; Henkin, Tina M.

doi:10.1046/j.1365-2958.1997.4851839.x

Cited by 66 publications

(111 citation statements)

References 27 publications

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“…For example, a single species of tRNA Tyr , with an anticodon to match the UAC tyrS specifier, has been identified; this tRNA must translate UAU tyrosine codons, by wobble pairing, yet a UAU specifier sequence mutation results in a severe decrease in tyrS readthrough (30). In contrast, no variant of tRNA Val with the appropriate anticodon has been reported, yet the switch to a valine specifier was successful.…”

Section: Discussionmentioning

confidence: 99%

Specificity of tRNA-mRNA interactions in Bacillus subtilis tyrS antitermination

et al. 1997

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The Bacillus subtilis tyrS gene, encoding tyrosyl-tRNA synthetase, is a member of the T-box family of genes, which are regulated by control of readthrough of a leader region transcriptional terminator. Readthrough is induced by interaction of the cognate uncharged tRNA with the leader; the system responds to decreased tRNA charging, caused by amino acid limitation or insufficient levels of the aminoacyl-tRNA synthetase. Recognition of the cognate tRNA is mediated by pairing of the anticodon of the tRNA with the specifier sequence of the leader, a codon specifying the appropriate amino acid; a second interaction between the acceptor end of the tRNA and an antiterminator structure is also important. Certain switches of the specifier sequence to a new codon result in a switch in the specificity of the amino acid response, while other switches do not. These effects may reflect additional sequence or structural requirements for the mRNA-tRNA interaction. This study includes investigation of the effects of a large number of specifier sequence switches in tyrS and analysis of structural differences between tRNA Tyr and tRNA species which interact inefficiently with the tyrS leader to promote antitermination.Many aminoacyl-tRNA synthetase and amino acid biosynthesis operons in gram-positive bacteria are regulated by a common transcription antitermination system (11,12,16,28). The genes in this group share a number of features in the 5Ј region in their transcripts, including a set of conserved primary sequence and structural elements located upstream of the start of the coding sequence. The mRNA leader region structural elements include three large stem-loops, designated stems I, II, and III, preceding a factor-independent transcriptional terminator. The most prominent of the sequence elements, the 14-base T box, forms a portion of an antiterminator structure by pairing with conserved sequences on the 5Ј side of the terminator. Expression of the genes in this group is therefore controlled at the level of readthrough of the leader region transcriptional terminator, by a switch between the antiterminator and terminator forms of the transcript.Expression of several genes in this family has been shown to be induced in response to limitation for the cognate amino acid and not by general amino acid starvation (5,6,18,27,29). Examination of a number of the leader regions revealed a precisely located single codon, specifying the appropriate amino acid (11); the effector signalling amino acid limitation was therefore proposed to be tRNA, with the specificity of the amino acid response dictated by codon-anticodon pairing. The induction specificity of the Bacillus subtilis tyrS gene was switched from a response to tyrosine limitation to a response to phenylalanine limitation by a single base substitution in the UAC tyrosine codon of the tyrS leader to a UUC phenylalanine codon (11). These studies indicated that the leader region codon, termed the "specifier sequence," acts as a critical determinant of the specificity of the amino a...

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Section: Discussionmentioning

confidence: 99%

Specificity of tRNA-mRNA interactions in Bacillus subtilis tyrS antitermination

et al. 1997

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“…These features include the capacity of the leader RNA to form alternative secondary structures, one of which can serve as an intrinsic transcription terminator or as an anti-SD (ASD) helix that pairs with an SD sequence, blocking translation initiation. The major structures formed within the T-box RNA, in addition to the segments that can form the terminator/antiterminator (or ASD/anti-ASD) elements, are designated stem I, stem II, the stem IIA/stem IIB pseudoknot, and stem III (47,88) (Fig. 1A).…”

Section: General Features Of T-box Riboswitch Rnamentioning

confidence: 99%

“…T-box leader RNAs are further characterized by the existence of a set of conserved primary sequence elements at specific locations relative to the structural features (43). Mutational studies have demonstrated that many of the sequence and structural features conserved in T-box leader RNAs are important for function (49,88,112), but overall primary sequence conservation is low. Interestingly, the two main features of tRNAs (the anticodon and the base immediately preceding the CCA at its 3Ј end) that are recognized by the T-box RNAs are also usually central elements for recognition by the cognate aaRS (14).…”

Section: General Features Of T-box Riboswitch Rnamentioning

confidence: 99%

Biochemical Features and Functional Implications of the RNA-Based T-Box Regulatory Mechanism

Gutiérrez-Preciado

Henkin

Grundy

et al. 2009

Microbiol Mol Biol Rev

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SUMMARY The T-box mechanism is a common regulatory strategy used for modulating the expression of genes of amino acid metabolism-related operons in gram-positive bacteria, especially members of the Firmicutes. T-box regulation is usually based on a transcription attenuation mechanism in which an interaction between a specific uncharged tRNA and the 5′ region of the transcript stabilizes an antiterminator structure in preference to a terminator structure, thereby preventing transcription termination. Although single T-box regulatory elements are common, double or triple T-box arrangements are also observed, expanding the regulatory range of these elements. In the present study, we predict the functional implications of T-box regulation in genes encoding aminoacyl-tRNA synthetases, proteins of amino acid biosynthetic pathways, transporters, and regulatory proteins. We also consider the global impact of the use of this regulatory mechanism on cell physiology. Novel biochemical relationships between regulated genes and their corresponding metabolic pathways were revealed. Some of the genes identified, such as the quorum-sensing gene luxS, in members of the Lactobacillaceae were not previously predicted to be regulated by the T-box mechanism. Our analyses also predict an imbalance in tRNA sensing during the regulation of operons containing multiple aminoacyl-tRNA synthetase genes or biosynthetic genes involved in pathways common to more than one amino acid. Based on the distribution of T-box regulatory elements, we propose that this regulatory mechanism originated in a common ancestor of members of the Firmicutes, Chloroflexi, Deinococcus-Thermus group, and Actinobacteria and was transferred into the Deltaproteobacteria by horizontal gene transfer.

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“…Both structures displayed the codon within the Specifier loop. However, the second most stable structure with a free energy (DG°at 298°K) of ÿ19.2 Kcal/mol most closely resembled the conformation supported by genetic analysis in vivo and chemical and enzymatic probing experiments in vitro (Rollins et al 1997;Winkler et al 2001;Putzer et al 2002;Yousef et al 2005). The other structure was only modestly more stable with a DG°of ÿ21.0 Kcal/mol.…”

Section: Monitoring Of Specifier/common Complex Formation By Spectrofmentioning

confidence: 80%

tRNA regulation of gene expression: Interactions of an mRNA 5′-UTR with a regulatory tRNA

Nelson¹,

Henkin²,

Agris³

2006

RNA

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Many genes encoding aminoacyl-tRNA synthetases and other amino acid–related products in Gram-positive bacteria, including important pathogens, are regulated through interaction of unacylated tRNA with the 5′-untranslated region (5′-UTR) of the mRNA. Each gene regulated by this mechanism responds specifically to the cognate tRNA, and specificity is determined by pairing of the anticodon of the tRNA with a codon sequence in the “Specifier Loop” of the 5′-UTR. For the 5′-UTR to function in gene regulation, the mRNA folding interactions must be sufficiently stable to present the codon sequence for productive binding to the anticodon of the matching tRNA. A model bimolecular system was developed in which the interaction between two half molecules (“Common” and “Specifier”) would reconstitute the Specifier Loop region of the 5′-UTR of the Bacillus subtilis glyQS gene, encoding GlyRS mRNA. Gel mobility shift analysis and fluorescence spectroscopy yielded experimental Kds of 27.6 ± 1.0 μM and 10.5 ± 0.7 μM, respectively, for complex formation between Common and Specifier half molecules. The reconstituted 5′-UTR of the glyQS mRNA bound the anticodon stem and loop of tRNAGly (ASLGlyGCC) specifically and with a significant affinity (Kd = 20.2 ± 1.4 μM). Thus, the bimolecular 5′-UTR and ASLGlyGCC models mimic the RNA–RNA interaction required for T box gene regulation in vivo.

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Analysis of cis‐acting sequence and structural elements required for antitermination of the Bacillus subtilis tyrS gene

Cited by 66 publications

References 27 publications

Specificity of tRNA-mRNA interactions in Bacillus subtilis tyrS antitermination

Specificity of tRNA-mRNA interactions in Bacillus subtilis tyrS antitermination

Biochemical Features and Functional Implications of the RNA-Based T-Box Regulatory Mechanism

tRNA regulation of gene expression: Interactions of an mRNA 5′-UTR with a regulatory tRNA

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