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Riboswitches are regulatory systems in which changes in structural elements in the 5' region of the nascent RNA transcript (the 'leader region') control expression of the downstream coding sequence in response to a regulatory signal in the absence of a trans-acting protein factor. The S-box riboswitch, found primarily in low-G+C gram-positive bacteria, is the paradigm for riboswitches that sense S-adenosylmethionine (SAM). Genes in the S-box family are involved in methionine metabolism, and their expression is induced in response to starvation for methionine. S-box genes exhibit conserved primary sequence and secondary structural elements in their leader regions. We previously demonstrated that SAM binds directly to S-box leader RNA, causing a structural rearrangement that results in premature termination of transcription at S-box leader region terminators. S-box genes have a variety of physiological roles, and natural variability in S-box structure and regulatory response could provide additional insight into the role of conserved S-box leader elements in SAM.directed transcription termination. In the current study, in vivo and in vitro assays were employed to analyze the differential regulation of S-box genes in response to SAM. A wide range of responses to SAM were observed for the 11 S-box-regulated transcriptional units in Bacillus subtilis, demonstrating that S-box riboswitches can be calibrated to different physiological requirements.

The [S.sub.MK] box is a conserved riboswitch motif found in the 5' untranslated region of metK genes [encoding S-adenosylmethionine (SAM) synthetase] in lactic acid bacteria, including Enterococcus, Streptococcus, and Lactococcus sp. Previous studies showed that this RNA element binds SAM in vitro, and SAM binding causes a structural rearrangement that sequesters the Shine-Dalgarno (SD) sequence by pairing with an anti-SD (ASD) element. A model was proposed in which SAM binding inhibits metK translation by preventing binding of the ribosome to the SD region of the mRNA. In the current work, the addition of SAM was shown to inhibit binding of 30S ribosomal subunits to [S.sub.MK] box RNA; in contrast, the addition of S-adenosylhomocysteine (SAH) had no effect. A mutant RNA, which has a disrupted SD-ASD pairing, was defective in SAM binding and showed no reduction of ribosome binding in the presence of SAM, whereas a compensatory mutation that restored SD-ASD pairing restored the response to SAM. Primer extension inhibition assays provided further evidence for SD-ASD pairing in the presence of SAM. These results strongly support the model that [S.sub.MK] box translational repression operates through occlusion of the ribosome binding site and that SAM binding requires the SD-ASD pairing. regulatory RNA | RNA structure | translational control | SAM synthetase

Posttranslational modification is an efficient mechanism for controlling the activity of structural proteins, gene expression regulators, and enzymes in response to rapidly changing physiological conditions. Here we report in vitro and in vivo evidence that the acuABC operon of the gram-positive soil bacterium Bacillus subtilis encodes a protein acetyltransferase (AcuA) and a protein deacetylase (AcuC), which may control the activity of acetyl-coenzyme A (CoA) synthetase (AMP-forming, AcsA) in this bacterium. Results from in vitro experiments using purified proteins show that AcsA is a substrate for the acetyl-CoA-dependent AcuA acetyltransferase. Mass spectrometry analysis of a tryptic digest of acetylated AcsA (Acs[A.sup.Ac]) identified residue Lys549 as the sole modification site in the protein. Unlike sirtuins, the AcuC protein did not require NA[D.sup.+] as cosubstrate to deacetylate Acs[A.sup.Ac]. The function of the putative AcuB protein remains unknown.

Genes in the S-box family are regulated by binding of S-adenosylmethionine (SAM) to the 5' region of the mRNA of the regulated gene. SAM binding was previously shown to promote a rearrangement of the RNA structure that results in premature termination of transcription in vitro and repression of expression of the downstream coding sequence. The S-box RNA element therefore acts as a SAM-binding riboswitch in vitro. In an effort to identify factors other than SAM that could be involved in the S-box regulatory mechanism in vivo, we searched for trans-acting mutations in Bacillus subtilis that act to disrupt repression of S-box gene expression during growth under conditions where SAM pools are elevated. We identified a single mutant that proved to have one nucleotide substitution in the metK gene, encoding SAM synthetase. This mutation, designated metK10, resulted in a 15-fold decrease in SAM synthetase activity and a 4-fold decrease in SAM concentration in vivo. The metK10 mutation specifically affected S-box gene expression, and the increase in expression under repressing conditions was dependent on the presence of a functional transcriptional antiterminator element. The observation that the mutation identified in this search affects SAM production supports the model that the S-box RNAs directly monitor SAM in vivo, without a requirement for additional factors.

Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. [tRNA.sup.Gly]-dependent antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed. We found that addition of [tRNA.sup.Gly] can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.

Modulation of the structure of a leader RNA to control formation of an intrinsic termination signal is a common mechanism for regulation of gene expression in bacteria. Expression of the S box genes in Gram-positive organisms is induced in response to limitation for methionine. We previously postulated that methionine availability is monitored by binding of a regulatory factor to the leader RNA and suggested that methionine or S-adenosylmethionine (SAM) could serve as the metabolic signal. In this study, we show that efficient termination of the S box leader region by bacterial RNA polymerase depends on SAM but not on methionine or other related compounds. We also show that SAM directly binds to and induces a conformational change in the leader RNA. Both binding of SAM and SAM-directed transcription termination were blocked by leader mutations that cause constitutive expression in vivo. Overproduction of SAM synthetase in Bacillus subtilis resulted in delay in induction of S box gene expression in response to methionine starvation, consistent with the hypothesis that SAM is the molecular effector in vivo. These results indicate that SAM concentration is sensed directly by the nascent transcript in the absence of a trans-acting factor.

Research indicates that the regulation of gene expression, for methionine metabolism, is controlled by the S-box regulon comprised of transcriptional units in bacteria. Data show that in Bacillus subtilis the ykrTS and ykrWXYZ genes mediate the recycling of 5'-methyladenosine.

Research demonstrates that the catabolite responsive element cre2 of Bacillus subtilis promoter region binds to carbon catabolite protein A for transcription activation. Mutagenesis data reveal that the upstream promoter sequences, beyond the protein binding region, are involved in the transcriptional activation.

The Staphylococcus aureus ileS gene, which encodes isoleucyl-tRNA synthetase (IleRS), has a long mRNA leader region. This region has many of the characteristics of the gram-positive synthetase family, including the T box and leader region terminator and antiterminator. The terminator was observed to function in vivo and readthrough increased during growth in the presence of mupirocin, an inhibitor of IleRS activity.

Specifier sequence switches in the Bacillus subtilis tyrS gene encoding tyrosyl-transfer RNA (tRNA) synthetase were analyzed to characterize the the specificity of the tRNA-messenger RNA (mRNA) interaction. Specifier sequence mutations of the tryS leader indicated the ability of tRNAs specific for phenylalanine, cysteine, valine and isoleucine to interact with the tryS leader. Furthermore, nucleotide modifications affected the ability of the tRNA to interact with aminoacyl-tRNA synthetases.

Experimental analyses of the transcription patterns of acetoin utilization (acu ABC) and acetyl coenzyme A synthetase (acsA) genes of Bacillus subtilis and the function of ccpA gene in the transcription reveal that CcpA modulates the carbon catabolite in the genes and mediates glucose suppression of the two genes. The transcription of both genes are distinct and with a spacing of 20bp between the -35 promoter sequences. The promoter regions of the genes contain two cis-acting regulatory target sites for amyE, amyO-like sites. The gene expression is maximum in the stationary phase.

Experimental analyses of the tyrS gene of Bacillus subtilis reveal the presence of a second site which mediates the interactions of T box and the acceptor end of gram-positive aminoacyl tRNA synthetase and activates antitermination in the gene. TyrS gene mutations inhibit the mutations in the antiterminator in a process corresponding to base pairing, indicating that base pairing directly influences function and development of the antiterminator. The tRNA discriminator position functions as the second specificity determinant while uncharged tRNA functions as the effector in the bacteria.

Carbon secretion pathways require the presence of ccpa, as ccpa mutant inhibits Escherichia coli ackA gene's glucose-induced expression. Acetate kinase enzyme activity is entirely absent when the acetate kinase-encoding portion of Bacillus subtilis gene is subjected to insertional inactivation. The presence of acetate kinase facilitates excess carbohydrate release.

A transcription antitermination mechanism controls tRNA cells genes in gram positive Bacillus subtilis, with no alternation in the reaction to cognate amino acids. Secondary structure conservation in T box genes is unaffected by primary sequential alterations. The amino acid specific reaction is attributed to a particularly positioned triplet sequence. Reaction specificity is altered by changing the sequence specific codon. Antitermination specificity is significantly governed by tRNA in the B. subtilis.

The regulation of the Bacillus subtilis rpsD gene, which encodes ribosomal protein S4, was studied. The gene was monocistronic and subject to autogenous control. Repression by excess S4 protein required sequences in the leader region of the gene. Deletion analysis indicated that the rpsD regulatory target site was located within positions +30 to +190 of the mRNA. This region was unlikely to form a pseudoknot-like structure similar to that in the Escherichia coli alpha operon.

The tyrS gene, which codes for tyrosyl-tRNA synthetase in Bacillus subtilis, was isolated and characterized. Sequence analyses showed high similarities to tyrS genes in Escherichia coli and Bacillus stearothermophilus. The B. subtilis tyrS is negatively regulated by tyrosine. Primer extension analysis showed that tyrosine regulation is at post-transcription initiation. The response to tyrosine was found to be dependent on a terminator sequence in the mRNA, suggesting control via the antitermination mechanism. A conserved sequence was observed upstream of the terminator sequence and was designated as the T-box.

Isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible P(spac) promoter initiates amber suppressor variant of tyrosyl-tRNA in Bacillus subtilis. The rpsD-lacZ fusion expression is enhanced by IPTG. The rpsD-lacZ fusion contains an UAG amber codon. The inducible suppressor is used to characterize and isolate lethal mutants that require IPTG for growth.

Transcription of the Bacillus subtilis ackA gene encoding acetate kinase is induced during growth in glucose. This induction needs the catabolite control protein A (CcpA) and sequences upstream of the promoter, which include two sites which are similar to cre or catabolite response element. Investigation of the two sites indicated that cre2, the site closer to the promoter, and the region upstream of cre2, are required for CcpA-dependent transcriptional activation of ackA. However, the cre1 site is not needed.

The T box riboswitch regulates expression of amino acid-related genes in Gram-positive bacteria by monitoring the aminoacylation status of a specific tRNA, the binding of which affects the folding of the riboswitch into mutually exclusive terminator or antiterminator structures. Two main pairing interactions between the tRNA and the leader RNA have been demonstrated to be necessary, but not sufficient, for efficient antitermination. In this study, we used the Clostridium acetobutylicum alaS gene, which encodes alanyl-tRNA synthetase, to investigate the specificity of the tRNA response. We show that the homologous C. acetobutylicum tRNAAla directs antitermination of the C. acetobutylicum alaS gene in vitro, but the heterologous Bacillus subtilis tRNAAla (with the same anticodon and acceptor end) does not. Base substitutions at positions that vary between these two tRNAs revealed synergistic and antagonistic effects. Variation occurs primarily at positions that are not conserved in tRNAAla species, which indicates that these non-conserved residues contribute to optimal antitermination of the homologous alaS gene. This study suggests that elements in tRNAAla may have coevolved with the homologous alaS T box leader RNA for efficient antitermination. [ABSTRACT FROM AUTHOR]

Misincorporation of D-tyrosine (D-Tyr) into cellular proteins due to mischarging of tRNATyr with D-Tyr by tyrosyl-tRNA synthetase inhibits growth and biofilm formation of Bacillus subtilis. Furthermore, many B. subtilis strains lack a functional gene encoding D-aminoacyl-tRNA deacylase, which prevents misincorporation of D-Tyr in most organisms. B. subtilis has two genes that encode tyrosyl-tRNA synthetase: tyrS is expressed under normal growth conditions, and tyrZ is known to be expressed only when tyrS is inactivated by mutation. We hypothesized that tyrZ encodes an alternate tyrosyl-tRNA synthetase, expression of which allows the cell to grow when D-Tyr is present. We show that TyrZ is more selective for L-Tyr over D-Tyr than is TyrS; however, TyrZ is less efficient overall. We also show that expression of tyrZ is required for growth and biofilm formation in the presence of D-Tyr. Both tyrS and tyrZ are preceded by a T box riboswitch, but tyrZ is found in an operon with ywaE, which is predicted to encode a MarR family transcriptional regulator. Expression of tyrZ is repressed by YwaE and also is regulated at the level of transcription attenuation by the T box riboswitch. We conclude that expression of tyrZ may allow growth when excess D-Tyr is present. [ABSTRACT FROM AUTHOR]