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. A variety of mechanisms for control of gene expression by premature termination of transcription have been uncovered in bacteria (1, 2). Genes regulated in this way contain a transcription termination signal in the mRNA region upstream of the coding sequence of the regulated gene. The activity of this terminator can be controlled by modification of the activity of RNA polymerase (RNAP), blocking access of transcriptiontermination factor Rho, or by modulation of the leader RNA structure, commonly through alternate folding patterns. RNA folding can be controlled in turn through interaction with some regulatory factor, such as a translating ribosome, as in the Escherichia coli trp operon, or an RNA binding protein, as in the Bacillus subtilis trp operon or the E. coli bgl system. Modulation of RNA structure by an effector, in the absence of accessory proteins, has been demonstrated for the T box system, in which uncharged tRNA interacts directly with the leader RNA to promote antitermination (3, 4). Similar regulation by small molecules has recently been demonstrated for riboflavin and thiamin biosynthesis genes, by using flavin mononucleotide and thiamin pyrophosphate, respectively (5-7).The S box regulatory mechanism represents a system in which synthesis of the full-length transcript is determined by controlling whether the leader folds into the stem loop of an intrinsic terminator or a competing antiterminator structure (ref. 8 and Fig. 1). Formation of the antiterminator is inhibited by an alternative helical structure, which functions as an antiantiterminator. An anti-antiterminator element is also found in the B. subtilis pyr operon, in which leader RNA structure is controlled by binding of a regulatory protein (9). We initially identified the S box family by recognizing a high...

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.

SummaryThe S box transcription termination control system regulates expression of genes involved in methionine metabolism. Expression of the S box regulon, comprised of 11 transcriptional units in Bacillus subtilis , is induced in response to starvation for methionine. We previously demonstrated that S -adenosylmethionine (SAM) is the molecular effector sensed by the S box leader RNAs during transcription. A secondary structure model for S box leader RNAs was developed based on conservation of primary sequence elements and sequence covariation in helical domains. Covariation of nucleotides in two distantly spaced unpaired regions in the S box leader RNAs suggested that these two domains might interact in the RNA tertiary structure. In this study, site-directed mutagenesis of the covarying residues in two B. subtilis S box leader sequences was employed to test the hypothesis that base-pairing between these regions may be important. The effect of these mutations on in vivo expression, transcription termination in vitro , SAM binding, and leader RNA structure strongly supported the model that interaction between these two regions plays a key role in S box leader function. This provides the first insight into the three-dimensional arrangement of structural elements within the S box RNAs.

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.The S-box regulatory system is used in low-GϩC grampositive organisms, including members of the Bacillus/Clostridium/Staphylococcus group, to regulate expression of genes involved in biosynthesis and transport of methionine and S-adenosylmethionine (SAM) (9)(10)(11)29). Genes in the S-box family exhibit a pattern of conserved sequence and structural elements in the 5Ј region of the mRNA, upstream of the start of the regulated coding sequence(s). These conserved elements include an intrinsic terminator and a competing antiterminator that can sequester sequences that otherwise form the 5Ј portion of the terminator helix; residues in the 5Ј region of the antiterminator can also pair with sequences located further upstream, and this pairing results in formation of a structure (the anti-antiterminator) that sequesters sequences necessary for formation of the antiterminator. The anti-antiterminator helix (helix 1) (Fig. 1) is located at the base of a complex structure comprised of helices 1 to 4. Genetic analyses of S-box leader RNAs supported the model that formation of the antiantiterminator and transcription termination occur during growth under conditions where methionine is abundant, while starvation for methionine results in destabilization of the anti-antiterminator, allowing antiterminator formation and readthrough of the transcription termination site (9). Mutational analysis also suggested that the helix 1 to 4 region is likely to be the target for binding of a negative regulatory factor or factors (9, 38) and that pairing between residues in the terminal loop of helix 2 and the region between helices 3 and 4 is important for termination during growth in h...