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S-Adenosylmethionine Directly Inhibits Binding of 30S Ribosomal Subunits to the SMK Box Translational Riboswitch RNA

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S-Adenosylmethionine Directly Inhibits Binding of 30S Ribosomal Subunits to the SMK Box Translational Riboswitch RNA S-adenosylmethionine directly inhibits binding of 30S ribosomal subunits to the SMK box translational riboswitch RNA Ryan T. Fuchs*, Frank J. Grundy*, and Tina M. Henkin*†‡ *Department of Microbiology and †RNA Group, Ohio State University, 484 West 12th Avenue, Columbus, OH 43210 Edited by Jeffrey W. Roberts, Cornell University, Ithaca, NY, and approved January 19, 2007 (received for review November 10, 2006) The SMK box is a conserved riboswitch motif found in the 5؅ T.M.H., unpublished data). In contrast, the S box riboswitch is untranslated region of metK genes [encoding S-adenosylmethi- rare in members of the Lactobacillales branch of the Gram- onine (SAM) synthetase] in lactic acid bacteria, including Entero- positive bacteria. We investigated the metK genes of Lactoba- coccus, Streptococcus, and Lactococcus sp. Previous studies cillales sp. and found a conserved putative riboswitch element, showed that this RNA element binds SAM in vitro, and SAM which we named the SMK box on the basis of its association with binding causes a structural rearrangement that sequesters the metK genes (19). Because metK expression in organisms that use Shine–Dalgarno (SD) sequence by pairing with an anti-SD (ASD) the S box is controlled by negative feedback from SAM, it was element. A model was proposed in which SAM binding inhibits likely that SAM was also the effector molecule for the SMK box. metK translation by preventing binding of the ribosome to the SD We demonstrated that the Enterococcus faecalis SMK box RNA region of the mRNA. In the current work, the addition of SAM was binds SAM but not S-adenosylhomocysteine (SAH), which shown to inhibit binding of 30S ribosomal subunits to SMK box differs from SAM by one methyl group (19). SAM binding RNA; in contrast, the addition of S-adenosylhomocysteine (SAH) causes a structural rearrangement in the RNA that includes had no effect. A mutant RNA, which has a disrupted SD-ASD pairing of a portion of the metK Shine–Dalgarno (SD) sequence pairing, was defective in SAM binding and showed no reduction of to an anti-SD sequence (ASD) (Fig. 1). Unlike other metabolite- ribosome binding in the presence of SAM, whereas a compensa- binding riboswitches, which usually contain an effector binding tory mutation that restored SD-ASD pairing restored the response domain that is separable from the regulatory domain of the to SAM. Primer extension inhibition assays provided further evi- RNA, the SMK box appears to require the SD-ASD pairing for dence for SD-ASD pairing in the presence of SAM. These results SAM binding, as mutations that disrupt the pairing result in loss strongly support the model that SMK box translational repression of SAM binding (19). The E. faecalis SMK box was shown to operates through occlusion of the ribosome binding site and that confer translational repression of a lacZ reporter in Bacillus SAM binding requires the SD-ASD pairing. subtilis under conditions where SAM pools are elevated, and mutations of the SMK box that result in loss of SAM binding in regulatory RNA ͉ RNA structure ͉ translational control ͉ SAM synthetase vitro result in loss of repression in vivo. These findings suggested that the SMK box RNA represents a new class of SAM-responsive any regulatory mechanisms have been discovered recently riboswitch that regulates metK expression at the level of trans- Min bacteria in which RNA transcripts sense a regulatory lation initiation by preventing binding of the ribosome to the signal (1–6). These regulatory RNAs, usually called RNA sen- RNA when SAM is present. However, there was no direct sors or riboswitches, act in cis to regulate expression of the evidence that the apparent sequestration of the SD sequence is downstream coding sequence(s) without a requirement for sufficient to inhibit ribosome binding. Binding of other regula- regulatory proteins. Typically, the regulatory signal is an effector tory molecules, including proteins and antisense RNAs, directly molecule (tRNA, noncoding RNA, or small molecule) that binds to or near the RBS can inhibit translation initiation by preventing the nascent RNA transcript, causing a change in the RNA ribosome binding (20, 21). Alternatively, regulatory molecules structure. In many systems of this type, the structural change can bind to an mRNA and ‘‘trap’’ the 30S subunit on the mRNA either causes or prevents formation of the helix of an intrinsic in an inactive initiation complex (22, 23). transcriptional terminator (reviewed in 1, 4). Similar structural In this study, we used nitrocellulose filter binding and ribo- rearrangements can sequester the ribosome binding site (RBS) somal toeprinting assays to test whether SAM inhibits the to regulate at the level of translation initiation (7–9). Binding of binding of 30S ribosomal subunits to SMK box RNA in vitro. The the effector to the RNA can also catalyze self-cleavage of the results show that the metK RNA start codon is properly posi- RNA transcript, as in the glmS system (10). Thermosensor tioned in the P site of the 30S subunit in the absence of SAM, riboswitches, in contrast, have no effector binding domain but and that preincubation of the RNA with SAM reduced 30S simply respond by temperature-dependent modulation of the subunit binding to the RNA and resulted in inhibition of RNA structure (11–13). Riboswitches generally exhibit con- processivity of reverse transcriptase near the SD region, which served sequence and structural elements that are responsible for supports our previous observation that this area becomes more high specificity for their cognate molecular signal. structured in the presence of SAM (19). These findings confirm Many members of the Bacillus/Clostridium group of bacteria our model for SMK box RNA regulation, where SAM-dependent use the S box riboswitch system to control expression of genes involved in methionine metabolism (14). Included in this regulon is the metK gene, which encodes S-adenosylmethionine (SAM) Author contributions: F.J.G. and T.M.H. designed research; R.T.F. performed research; synthetase, the enzyme responsible for the synthesis of SAM R.T.F., F.J.G., and T.M.H. analyzed data; and R.T.F. and T.M.H. wrote the paper. from methionine and ATP. The S box system uses SAM as the The authors declare no conflict of interest. effector molecule both in vivo (15, 16) and in vitro (15, 17, 18), This article is a PNAS direct submission. and regulation occurs primarily at the level of premature ter- Abbreviations: SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; RNAP, RNA mination of transcription (14, 15). Translational regulation has polymerase; SD, Shine–Dalgarno; ASD, anti-SD; RBS, ribosome binding site. also been predicted in certain S box genes, predominantly those ‡To whom correspondence should be addressed. E-mail: [email protected]. found in Gram-negative organisms (refs. 2 and 6; F.J.G. and © 2007 by The National Academy of Sciences of the USA 4876–4880 ͉ PNAS ͉ March 20, 2007 ͉ vol. 104 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609956104 Downloaded by guest on October 1, 2021 Fig. 1. Structural model of the SMK box. Putative forms of E. faecalis metK RNA BIOCHEMISTRY in the absence (a) or presence (b)ofSAM(*). Red, residues of the ASD sequence; blue, residues of the SD sequence; purple, AUG start codon; green arrows, Fig. 2. Nitrocellulose filter binding assays. RNAs generated by T7 RNAP location of stops observed in primer extension assays; black arrows, mutations in transcription were gel purified and end-labeled with [␥-32P]ATP. Labeled RNAs the E. faecalis SMK box. The U14G substitution was introduced by including were incubated with SAM or SAH followed by tRNA and 30S subunits (a)or tandem G residues at the start-site for T7 RNAP transcription. Numbering of the tRNA and 30S subunits followed by SAM or SAH (b). Samples were passed sequence is relative to the predicted E. faecalis metK transcription start-site. The through a nitrocellulose filter, and material retained by the filter was quan- U22G and A93C substitutions were predicted to disrupt the SD-ASD pairing. tified by scintillation counting. Less than 0.03% of input RNA was retained by Dotted lines represent potential pairings supported by phylogenetic and muta- the filters in the absence of 30S subunits. tional analyses (ref. 19; A. Smith, R.T.F., F.J.G., and T.M.H., unpublished results). The position of SAM within the RNA is not known. reduction in retention of the RNA by the 30S subunits, whereas the addition of SAH had no effect. Similar results were obtained SD-ASD pairing directly inhibits translation initiation by block- when the RNA was preincubated with 30S subunits before the ing access of the ribosome to the RBS. addition of SAM (Fig. 2b), suggesting that the RNA-ribosome complex can be disrupted by the addition of SAM. Retention of Results the RNA increased 2-fold when tRNAfmet was included presum- SAM Inhibits 30S Subunit Binding to SMK Box RNA. Nitrocellulose ably because the tRNA stabilizes the interaction of the 30S filter binding assays were performed to determine whether the subunit with the metK RNA. Incubation of the RNA with SAM proposed pairing of the SD to the ASD in E. faecalis metK RNA before the addition of 30S subunits and tRNAfmet reduced is sufficient to inhibit binding of 30S ribosomal subunits to the retention of RNA Ϸ2-fold, but SAM did not significantly reduce mRNA. This assay took advantage of the ability of nitrocellulose retention of the RNA when both 30S subunits and tRNAfmet filters to bind 30S subunits but not free nucleic acids. Because an were added before the addition of SAM (Fig. 2b). The loss of the E.
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