Transcription termination control of the S box system: Direct measurement of S -adenosylmethionine by the leader RNA

Abstract

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...

In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli

“…Increased levels both of MetR and homocysteine would presumably also occur in both MetJand MetK-deficient cells, both of which are induced for metA, B and C with or without in vivo SAMase activity. These and other conflicting and unresolved possibilities surrounding SAMase-mediated induced met gene induction can only be resolved by further investigation of the role of MetR activity and/or homocysteine levels, or by searching for other unidentified regulatory elements that influence met gene activity such as revealed by the role of SAM in the S-box system of Bacillus subtilis (Murphy McDaniel et al, 2003).…”

Confirmation of a second natural preQ1 aptamer class in Streptococcaceae bacteria

“…Four different aptamer classes recognizing Sadenosylmethionine (AdoMet or SAM) have been identified to date. SAM-I or S-box (Epshtein et al 2003;McDaniel et al 2003;Winkler et al 2003), SAM-II (Corbino et al 2005), SAM-III or SMKbox (Fuchs et al 2006), and SAM-IV ) often regulate the same genes in different organisms (e.g., SAM synthase, homoserine O-succinyltransferase). The discovery of a second natural preQ 1 aptamer class shows that SAM is not a special case and that additional metabolites may be bound by multiple distinct RNA architectures to control similar sets of genes in different organisms.…”

Folding of the SAM-I riboswitch

“…3,11,24,36 The large diversity of SAM riboswitches likely reflects the importance of regulating SAM-related metabolic pathways. 3 SAM riboswitches exhibit very high affinity for SAM and strongly discriminate against SAM-related analogs such as S-adenosylhomocysteine (SAH), 33,34 which differs from SAM by the absence of a single methyl group and the associated positive charge on the sulfur atom. A study has reported the existence of SAH-sensing riboswitches, 37 indicating that SAM/SAH riboswitches exploit chemical differences between closelyrelated metabolites to prevent cellular misregulation, which is consistent with recently obtained crystal structures.…”

“…11,[30][31][32][33][34] These riboswitches are located upstream of genes involved in the metabolism, biosynthesis and transport of methionine or SAM. 11,35 The SAM riboswitch class is one of the most abundant, 36 especially in Gram-positive organisms, and is unique in that six distinct SAM riboswitch families have been reported to date that bind SAM in diverse ways.…”