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The SMK box is a conserved riboswitch motif found in the 5؅ T.M.H., unpublished data).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 of metK genes [encoding S-adenosylmethi- rare in members of the Lactobacillales branch of the Gram- onine (SAM) synthetase] in lactic acid , 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 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 , 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 . Typically, the regulatory signal is an effector tory molecules, including proteins and antisense RNAs, directly molecule (tRNA, noncoding RNA, or ) 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 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. faecalis metK-lacZ translational fusion exhibited a pattern of effect of SAM is not a result of SAM preferentially binding to regulation in vivo in Escherichia coli, similar to that previously tRNAfmet over the metK RNA because tRNAfmet did not retain reported (ref. 19; data not shown), binding of highly purified E. a measurable amount of SAM in a binding assay (data not coli 30S subunits to SMK RNA was tested. The heterologous shown). These results suggest that SAM can compete with 30S ribosomal subunits were selected because the SMK system is subunit binding to the RNA, but the addition of the initiator absent in E. coli, reducing the probability that any additional tRNA to the complex results in a stable complex that is resistant cellular factors potentially required for regulation would co- to challenge by SAM and is therefore committed to translation. purify with the ribosomal subunits. End-labeled in vitro- The effect of mutations in the SD-ASD pairing on ribosome generated SMK box RNA was heated, slow-cooled, and incubated binding was also tested. The U22G substitution in the ASD, in the presence or absence of SAM. E. coli 30S ribosomal which was predicted to disrupt pairing with residue A93 imme- subunits were added, in the presence or absence of tRNAfmet, the diately downstream of the SD (positions 88–92), resulted in a samples were passed through a nitrocellulose filter, and the 40-fold reduction of SAM binding in vitro (Fig. 3a), as was amount of radiolabeled RNA retained by the filter was deter- previously observed for other mutations in the ASD element mined by scintillation counting. (ref. 19; R.T.F., unpublished data). The U22G mutation also Incubation of RNA corresponding to positions 15–118 relative resulted in complete loss of SAM-dependent inhibition of 30S to the E. faecalis metK transcription start-site with 30S ribosomal subunit binding (Fig. 3b). The A93C mutation, which is predicted subunits resulted in Ϸ30% retention of the RNA, whereas to disrupt SD-ASD pairing without affecting the SD sequence Ͻ0.03% retention was observed when ribosomal subunits were itself, similarly resulted in loss of SAM binding in vitro (Fig. 3a). not added (Fig. 2a). The addition of SAM resulted in a 4-fold Combination of the U22G and A93C substitutions, which is

Fuchs et al. PNAS ͉ March 20, 2007 ͉ vol. 104 ͉ no. 12 ͉ 4877 Downloaded by guest on October 1, 2021 Fig. 4. Primer extension inhibition analysis of SMK box RNA. E. faecalis metK RNAs containing positions 15–208 were generated by T7 RNAP transcription, gel-purified, and then annealed to a [␥-32P]ATP-labeled DNA primer comple- mentary to positions 180–203. Annealed RNAs were incubated in the presence of SAM or SAH followed by tRNA and 30S subunits. dNTPs and reverse Fig. 3. Effect of SMK box mutations on binding of SAM and 30S ribosomal subunits. (a) SAM binding. RNAs generated by T7 RNAP transcription were transcriptase were added, and reactions were quenched by the addition of gel-purified and then incubated in the presence of [methyl-14C]SAM. The gel-loading buffer. Radioactivity at the position of the U118 stop in lanes 7 and RNA-bound SAM was separated from unbound SAM by size-exclusion filtra- 8 was compared with the value for lane 6, which was normalized to 100. Lanes tion. Retention of SAM is expressed relative to that of the wild-type E. faecalis 10 and 11 were compared with lane 9, and lanes 13 and 14 were compared metK RNA extending from residues 15–118 (Fig. 1). (b) Nitrocellulose filter with lane 12 in a similar manner. RT, readthrough to 5’ end. binding assays. RNAs end-labeled with [␥-32P]ATP were incubated with SAM or SAH followed by tRNA and 30S subunits. Samples were passed through a nitrocellulose filter, and material retained by the filter was quantified by extended by reverse transcriptase. Extension by reverse tran- scintillation counting. Less than 0.03% of input RNA was retained by the filters scriptase is inhibited when the enzyme encounters a bound in the absence of 30S subunits. obstacle (e.g., the 30S subunit) or significant secondary struc- ture. The extension products were resolved by PAGE and identified by comparison to a DNA sequencing ladder. predicted to restore pairing, restored both SAM binding and Reactions containing the E. faecalis wild-type SMK RNA sensitivity to SAM in the filter binding assays, indicating that the (containing residues 15–208 relative to the transcription start- A93C mutation could suppress the defect in response to SAM site) resulted in two extension products, one corresponding to a conferred by the U22G substitution. These results show that the halt at position G77 and the other resulting from full-length reduction of 30S subunit binding in the presence of SAM occurs extension to the 5Ј end of the RNA (Fig. 4, lane 1). The G77 only when the RNA can bind SAM. The increase in 30S subunit product was observed in all reactions and is probably due to binding exhibited by the U22G mutant RNA in the absence of secondary structure in the core region of the RNA (Fig. 1). A 5Ј SAM compared with either the wild-type or U22G/A93C double truncated RNA that began with residue 20, which retained SAM mutant RNA (Fig. 3b) suggests that SD-ASD pairing occurs to binding (Fig. 3a), also exhibited the G77 product (data not some extent even in the absence of SAM and that SAM binding shifts the equilibrium to the paired form. These experiments shown); this indicates that the conserved predicted pairing support the model that the SAM-dependent SD-ASD pairing between positions 15–20 and 68–75 (ref. 19; Fig. 1a), which is absent in the 20–208 RNA, is dispensable for SAM binding and can inhibit 30S ribosomal subunit binding to SMK box RNA. Lactobacillus plantarum metK, which also contains a SAM is not responsible for the G77 extension product. Incubation of the 15–208 RNA in the presence of SAM binding SMK box element (19), showed the same pattern of 30S subunit binding as the E. faecalis S box RNA, although the resulted in a new extension product corresponding to position MK Ј binding efficiency was somewhat lower (Fig. 3b). This result A94 (Fig. 4, lane 2), which is two nt 3 of the end of the SD (residues 88–92) and comprises the first position of the predicted suggests that other SMK box RNAs exhibit a similar effect of SAM on ribosome binding. SD-ASD pairing (19; Fig. 1b). Studies with avian myeloblastosis virus reverse transcriptase have shown that termination can

SMK Box RNA Is Positioned Properly Within the 30S Subunit. Although occur when the enzyme encounters secondary structure, and the the filter binding studies indicated that 30S subunits bound the majority of these truncated products correspond to the first E. faecalis metK RNA, there was still a question as to whether paired base in the structure or the last position before a binding was localized to the RBS. Primer extension inhibition cross-link (25, 26). Incubation of the RNA in the presence of (‘‘toeprint’’) assays (24) were used to localize the position of the SAH instead of SAM did not result in formation of the A94 30S subunit on the RNA and assess the effect of SAM. A DNA product (Fig. 4, lane 3). The A94 extension product was also primer complementary to a region of the E. faecalis metK RNA absent in reactions using the U22G mutant RNA (which did not downstream of the SD was annealed to the RNA and then bind SAM) and was present in the U22G/A93C double mutant

4878 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0609956104 Fuchs et al. Downloaded by guest on October 1, 2021 in which both the SD-ASD pairing and SAM binding were with a high-salt wash from a heterologous host in which the SMK restored (Fig. 4, lanes 10, 13). These observations support the box mechanism is not found, demonstrating that the observed conclusion that the A94 product depends on the RNA-SAM effect of SAM on ribosome binding does not require additional binding interaction and is likely to correspond to the SD-ASD cellular factors. The SMK box is therefore the first translational pairing. control riboswitch for which the translational mechanism is The addition of 30S subunits to a reaction containing the clearly established by biochemical analyses. 15–208 RNA resulted in a set of low-abundance products Inhibition of 30S subunit binding to SMK box RNA was clustered around U116–U122 (Fig. 4, lane 4). If both 30S observed regardless of whether SAM was added before or after subunits and tRNAfmet were included, Ϸ90% of the primer the ribosomal subunits. However, the addition of the initiator extension products corresponded to position U118, which is 16 tRNA to the complex resulted in a complex that was resistant to nt downstream of the A in the AUG start codon (Fig. 1). This SAM, indicating that the complex was now committed to trans- distance is representative of the position at which reverse lation. This suggests that competition between the ribosome and transcriptase stops when the start codon of an mRNA is posi- SAM for binding to the mRNA occurs at the initial stages of tioned in the P site of the 30S subunit (27). The abundance of formation of the initiation complex. One possible benefit of extension products corresponding to U118 was reduced Ϸ2-fold regulating in this manner is that the cell can quickly respond to in the presence of SAM, whereas SAH had no effect. These a sudden drop in SAM levels by release of SAM from SMK box results indicate that E. coli 30S subunits bind to the E. faecalis mRNAs, allowing access to the RBS and expression of the gene. metK RBS and that ribosome binding is inhibited in the presence The importance of a rapid response may reflect the fact that of SAM. metK is an essential gene for which expression is required at some In contrast to what was observed for the wild-type RNA, the level during all growth conditions. The possibility that the SMK addition of SAM had no effect on the abundance of the U118 box represents a true reversible switch will require further product for the U22G mutant RNA (Fig. 4, lanes 9–11), which analysis of regulation in vivo because reversibility depends on is defective in SAM binding. Introduction of the compensating maintenance of a stable pool of the metK transcript. A93C mutation, which suppressed the SAM binding defect of the Multiple mechanisms of translational regulation have been iden- U22G mutation (Fig. 3a), resulted in restoration of sensitivity to tified in bacteria. For example, the E. coli CsrA regulates the SAM (Fig. 4, lanes 12–14), providing further support for the expression of cstA by directly binding the cstA RBS and preventing model that the SD-ASD pairing is required for both SAM ribosome binding (20). A similar mechanism is used by the trans-

binding and SAM-dependent inhibition of ribosome binding. acting OxyS RNA, which pairs with the RBS of the fhlA mRNA BIOCHEMISTRY This confirms the results from the filter binding assays where (21). Thermosensor RNAs, like E. coli rpoH and Listeria monocy- SAM inhibited the binding of 30S subunits to SMK box RNA only togenes prfA, use intramolecular interactions that sequester the RBS when the RNA can bind SAM. at low temperatures and expose the RBS at higher temperatures (11–13). In contrast, regulation of the ribosomal proteins encoded Discussion by the ␣ operon in E. coli results from ‘‘entrapment’’ of the 30S In this study, we report that E. coli 30S subunits bind to SMK box subunit in an unproductive complex by binding of the S4 repressor RNA in the absence of other factors and that SAM specifically protein to the mRNA (23). Two examples of a ‘‘typical’’ metabolite- inhibits ribosome binding to the RNA. Primer extension analysis binding riboswitch translational mechanism are found in the ypaA demonstrated that in the absence of SAM, the 30S subunits are gene in B. subtilis and the thiM gene in E. coli (9, 28, 29). In each positioned with the AUG start codon of the RNA within the case, the effector molecule, flavin mononucleotide for ypaA and ribosomal P site, whereas the addition of SAM resulted in a pyrophosphate for thiM, binds to an domain in significant reduction in the ribosomal toeprint. Under these the 5Ј untranslated region of the mRNA. This binding causes a conditions, the extension proceeded through the ribosomal downstream structural shift that sequesters the RBS, the regulatory toeprint site and yielded a new product corresponding to the 3Ј target, and sequestration of the RBS was proposed to block end of the SD-ASD pairing. These findings further support the ribosomal access, although no biochemical analyses were per- model that SAM binding promotes pairing of the SD to the ASD, formed to test the proposed mechanism. The SMK box also uses which prevents translation initiation by blocking access of the effector-dependent occlusion of the RBS to inhibit translation ribosome to the RBS. initiation but differs from these other systems in that the regulatory Most studies on the mechanism of action of riboswitches have target is an intrinsic part of the effector binding domain, so that focused on transcription termination systems. In systems of this disruption of the SD-ASD pairing results in loss of SAM binding type, binding of the effector to the riboswitch RNA causes the (Fig. 3a; ref. 19). The SMK riboswitch therefore represents a special RNA to form an intrinsic transcriptional terminator or a com- class of metabolite-binding regulatory RNAs. peting antiterminator element. Structural probing, in vitro tran- The SMK box represents one of the simplest known metabolite- scription, and mutational analyses have provided support for this binding riboswitch elements. The crystal structure of the much mode of action for several of these riboswitch systems (reviewed more complex S box RNA from Thermoanaerobacter tengcon- in 1, 4). By contrast, most translational riboswitches have been gensis has recently been reported (30), and the RNA was shown analyzed in less detail, with most of the experimental work to envelop and interact with almost every functional group in focused on regulation in vivo and effector-dependent modula- SAM. The location and structure of the SAM binding pocket in tion of RNA structure in vitro. Primer extension assays of the E. the SMK box RNA is not yet known, and the structural dissim- coli btuB riboswitch showed that the ability of 30S subunits to ilarity of SMK and S box RNAs suggests that the molecular basis bind btuB RNA is reduced when is present, for SAM recognition may be very different. providing a clear indication that regulation occurs at the level of The majority of riboswitches identified in low GϩC Gram- translation initiation (7). One caveat is that the ribosomes used positive organisms appear to regulate gene expression by pre- in the btuB experiments were prepared with a low-salt wash, mature transcription termination, whereas riboswitches from which does not remove all ribosome-associated factors; binding high GϩC Gram-positive and Gram-negative organisms tend to was not observed with ribosomal subunits prepared with a regulate at the level of translation (1, 2, 5, 6). This divergence is high-salt wash, which was interpreted by the authors to indicate readily observed with riboswitch elements that are found in both that other factors could be involved in translational control of groups of organisms but regulate by different mechanisms btuB mRNA (7). The current study demonstrated a SAM- depending on the host (e.g., the -binding L box riboswitch; dependent effect on binding of 30S ribosomal subunits prepared ref. 31). The SMK box, which is found only in the Lactobacillales

Fuchs et al. PNAS ͉ March 20, 2007 ͉ vol. 104 ͉ no. 12 ͉ 4879 Downloaded by guest on October 1, 2021 group of low GϩC Gram-positive bacteria, is unusual in that it (10 mM Tris⅐HCl, pH 7.5, 10 mM MgCl2,60mMNH4Cl, and 6 appears to function only at the level of translation. A third class mM 2-mercaptoethanol) were heated to 65°C and slow-cooled to of SAM-responsive riboswitch was recently identified in ␣-pro- 40°C. SAM (160 ␮M), SAH (160 ␮M), or water was added, and teobacteria, but the mechanism of action has not yet been the samples were incubated for 10 min at 37°C. 30S ribosomal reported (32). The experiments in the current study were subunits (60 nM) isolated from E. coli MRE600 by using a performed with E. coli components, whereas in vivo experiments high-salt wash (35) and provided by K. Fredrick (Ohio State in previous work were performed in B. subtilis (19). These results University) were added, followed by a 30-min incubation at 37°C. fmet show that SMK box riboswitches have the potential to function as tRNA (120 nM; Sigma–Aldrich, St. Louis, MO) was included gene regulators in a variety of nonnative organisms, including as indicated. Samples were then loaded onto a nitrocellulose both Gram-positive and Gram-negative species. It will be im- filter (0.45-␮M pore size; Whatman, Clifton, NJ) that had been portant to demonstrate that the mechanism operates as pre- soaked in 1ϫ binding buffer. A vacuum was applied, and filters dicted in the native host. were washed five times with 300 ␮lof1ϫ binding buffer. Radioactivity retained by the filters was measured in a Packard Materials and Methods Tri-Carb 2100TR liquid scintillation counter. DNA Constructs. DNA templates for transcription by T7 RNA polymerase (RNAP) were generated by combining pairs of Primer Extension Inhibition Assays. A DNA primer complementary complementary oligonucleotides as previously described (33). to positions 180–203 of the E. faecalis metK transcript was 5Ј Sequence changes were introduced by replacement of a pair of end-labeled as described above, except that a G-25 column was oligonucleotides with a pair containing the changes. The metK used to remove excess [␥-32P]ATP. The labeled oligonucleotide constructs were derived from the sequences for E. faecalis strain (10 nM) was annealed to T7 RNAP-transcribed RNA (10 nM) V583 (NCBI AE016830) and L. plantarum strain WCFS1 (NCBI corresponding to positions 15–208 of the metK transcript in 1ϫ AL935263). All constructs were confirmed by DNA sequencing. binding buffer by heating to 65°C and slow cooling to 40°C. SAM (160 ␮M), SAH (160 ␮M), or water was added, and the samples T7 RNAP Transcription Reactions. RNA was generated by using were incubated for 10 min at 37°C. 30S ribosomal subunits [60 a MEGA-shortscript T7 RNAP high-yield transcription kit nM; isolated from E. coli MRE600 by using a high-salt wash (35) (Ambion, Austin, TX). The RNA products were gel-purified as and provided by K. Fredrick (Ohio State University)] were described previously (33). added, with or without tRNAfmet (120 nM; Sigma–Aldrich), followed by a 30-min incubation at 37°C. Avian myeloblastosis ␮ ϫ SAM Binding Assays. T7 RNAP-transcribed RNA (3 M) in 1 virus reverse transcriptase (1 unit per rxn, Thermoscript RT- transcription buffer (34) was heated to 65°C and slow-cooled to PCR; Invitrogen, Carlsbad, CA) and dNTPs (375 ␮M) were 14 Ϫ1 40°C. [methyl- C]SAM [ICN; 52 mCi mmol (1.92 GBq added followed by 10-min incubation at 37°C. Reactions were Ϫ1 ␮ mmol )] was added to a final concentration of 8 M. After quenched by the addition of gel-loading buffer (Ambion) and incubation for 10 min at room temperature, samples were passed resolved by using 10% denaturing PAGE. Products were visu- through a Nanosep 10K Omega filter and washed four times with alized by using PhosphorImager analysis (Molecular Dynamics, ␮ ϫ 75 lof1 transcription buffer. Radioactivity retained by the filters Sunnyvale, CA) and quantified by using ImageQuant 5.2 soft- was measured in a Packard Tri-Carb 2100TR liquid scintillation ware. A DNA sequencing ladder was generated by using a DNA counter. Sequenase 2.0 Kit (USB), a DNA template containing positions 15–220 of the E. faecalis metK gene, and the same downstream Filter Binding Assays. T7 RNAP-transcribed RNAs corresponding primer as was used in the primer extension assays. to positions 15–118 of the E. faecalis metK transcript were 5Ј ␥ 32 Ϫ1 end-labeled with [ - P]ATP (7,000 Ci mmol ) by using a We thank K. Fredrick and S. Walker (Ohio State University) for KinaseMax kit (Ambion) and passed through a MicroSpin G-50 assistance with toeprint experiment conditions and for providing 30S column (Amersham Biosciences, Piscataway, NJ) to remove subunits and tRNAfmet. This work was supported by the National excess [␥-32P]ATP. Labeled RNAs (10 nM) in 1ϫ binding buffer Institutes of Health Grant GM63615.

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