The SMK box is a new SAM-binding RNA for translationalregulation of SAM synthetaseRyan T Fuchs, Frank J Grundy & Tina M Henkin

We have identified the SMK box as a conserved RNA motif in the 5¢ untranslated leader region of metK (SAM synthetase) genesin lactic acid bacteria, including Enterococcus, Streptococcus and Lactococcus species. This RNA element bound SAM in vitro,and binding of SAM caused an RNA structural rearrangement that resulted in sequestration of the Shine-Dalgarno (SD) sequence.Mutations that disrupted pairing between the SD region and a sequence complementary to the SD blocked SAM binding, whereascompensatory mutations that restored pairing restored SAM binding. The Enterococcus faecalis SMK box conferred translationalrepression of a lacZ reporter when cells were grown under conditions where SAM pools are elevated, and mutations that blockedSAM binding resulted in loss of repression, demonstrating that the SMK box is functional in vivo. The SMK box therefore representsa new SAM-binding riboswitch distinct from the previously identified S box RNAs.

Direct sensing of regulatory signals by nascent RNA transcripts hasrecently emerged as a common mechanism for regulation of geneexpression in bacteria1–3. RNAs of this type, termed riboswitches orRNA sensors, sense their signals without a regulatory protein or trans-lating ribosome. Binding of the effector molecule (such as tRNA, smallnoncoding RNAs or metabolites) modulates the structure of the nas-cent transcript, which affects expression of the downstream codingsequence(s). In many systems, binding of the effector determineswhether the RNA folds into the helix of an intrinsic terminator,resulting in premature termination of transcription. Similar RNA re-arrangements can mediate translational regulation by sequestration ofthe ribosome binding site. Each class of riboswitch RNA has a con-served pattern of primary sequence and structural elements necessaryfor highly specific recognition of the cognate regulatory signal and abinding affinity appropriate to the physiological effector concentration.

The S box RNAs are a well-characterized class of riboswitch RNAs.The S box motif was originally recognized upstream of genes involvedin methionine metabolism in many Gram-positive bacteria with lowG + C content4,5. Genetic analysis of S box genes in Bacillus subtilis hasshown that repression of downstream gene expression occurs at thelevel of premature transcription termination. The conserved elementsinclude a transcriptional terminator and a competing antiterminator,located upstream of the regulated coding sequences, and mutationalanalyses have supported a model in which growth in the presence ofmethionine stabilizes an anti-antiterminator element that preventsformation of the antiterminator structure, resulting in prematuretermination of transcription. Biochemical studies have demonstratedthat the molecular effector recognized by S box RNAs is S-adenosyl-methionine (SAM) and that SAM binding stabilizes the anti-anti-terminator element and promotes transcription termination in a

purified in vitro transcription system6–8. SAM is likely to be theeffector in vivo, as growth under conditions that result in increasedSAM pools leads to repression of S box gene expression6. A tertiaryinteraction within the S box element is crucial for SAM binding andSAM-dependent transcription termination9, which suggests that theSAM-binding pocket requires a complex three-dimensional arrange-ment. SAM recognition by S box RNAs is highly specific, but themolecular basis for binding specificity is not yet understood. S boxmotifs in a few Gram-negative and high G + C Gram-positiveorganisms appear to function at the level of translation initiation bysequestration of the translation initiation region1,10.

Although the S box mechanism commonly regulates methionine-related genes in organisms in the Bacillus/Clostridium group, it has notbeen found in most members of the Lactobacillales order10,11, with theexception of the genera Leuconostoc and Oenococcus (F.J.G. andT.M.H., unpublished data). The metK gene (which encodes SAMsynthetase, the enzyme responsible for synthesis of SAM frommethionine and ATP) is usually an S box gene in organisms inwhich this mechanism is used. As SAM is the molecular effectorcontrolling S box gene expression, regulation of metK by this mechan-ism provides a sensitive feedback response to the intracellular con-centration of SAM. We therefore were interested in determiningwhether SAM is the molecular effector for metK regulation in bacteriathat lack the S box mechanism. We find that metK genes fromLactobacillales species contain a conserved 5¢ element with no simi-larity to S box RNAs and that RNAs containing this element bindSAM in vitro and show SAM-dependent structural changes. We alsodemonstrate that this element confers translational repression in vivo.These results identify a new class of SAM-responsive riboswitch RNAs,which we designate the SMK box.

Received 11 November 2005; accepted 6 January 2006; published online 19 February 2006; doi:10.1038/nsmb1059

Department of Microbiology, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA. Correspondence should be addressed to T.M.H.(henkin.3@osu.edu).

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RESULTSIdentification of the SMK boxWe have previously identified metK genes among members of theS box family in Gram-positive bacteria4 (F.J.G. and T.M.H., unpub-lished data). We therefore examined metK genes from other organismsfor features that might be involved in regulation. We found that allmetK genes from Enterococcus, Lactococcus, Lactobacillus and Strepto-coccus spp. (members of the order Lactobacillales) for which genomicsequences are available contain a conserved element in the regionbetween the promoter and the translation start site (Fig. 1). The only

other Lactobacillales for which metK sequence is available are Leuco-nostoc mesenteroides and Oenococcus oeni, in which the metK gene hasthe S box motif, suggesting that regulation is at the level of transcrip-tion termination in response to SAM.

The core conserved element of the non–S box metK genes has highprimary-sequence conservation at certain positions and conservedpositioning of these residues relative to predicted helical domainsthat covary to maintain base-pairing. Two possible alternativeconformations for the Enterococcus faecalis and Streptococcus gordoniimetK core regions are shown in Figure 2. Pairing between a portion of

Figure 1 Alignment of SMK box sequences from

metK genes in Lactobacillales. Sequences are

aligned to the E. faecalis sequence, which

extends from +9 relative to the predicted

transcription start site to the end of the AUG

start codon (+104). Abbreviations are as follows:

Efae, E. faecalis; Efcm, Enterococcus faecium;

Laci, Lactobacillus acidophilus; Lcas, Lacto-

bacillus casei; Ldel, Lactobacillus delbrueckii;

Ljoh, Lactobacillus johnsonii; Lpla, Lactobacillus

plantarum; Lsak, Lactococcus sakei; Llac,

Lactococcus lactis; Saga, Streptococcus

agalactiae; Sequ, Streptococcus equi; Sgor,

Streptococcus gordonii; Smit, Streptococcus

mitis; Spne, Streptococcus pneumoniae; Spyo,Streptococcus pyogenes; Ssui, Streptococcus suis;

Sthe, Streptococcus thermophilus; Sube,

Streptococcus uberus. Asterisks mark sequences tested for SAM binding. A consensus sequence is shown at bottom (magenta, 100% conserved residues;

black capitals, Z85% conserved; lower-case, Z45% conserved; R, G or A; Y, C or U; W, A or U). Numbering at top is for E. faecalis metK sequence. Numbers

within the alignment indicate the number of residues in the upper part of the core element (22 for E. faecalis) or in the region just upstream of the SD region

(0 for E. faecalis). Potential pairing as shown in Figure 2a,c (for E. faecalis and S. gordonii, respectively) is indicated with dashed arrows and italics; the

alternative pairing for the bottom part of the helix as shown in Figure 2b,d is indicated with underlining. Potential pairing between residues corresponding to

E. faecalis 90–95, overlapping the SD sequence (residues 88–92) and ASD sequence (21–25), is shown in blue, the AUG start codon (102–104) is shown

in red and the potential pairing of residues corresponding to E. faecalis metK A29, G30 and G31 with C67, C68 and U69 is shown in green.

Efae*EfcmLaciLcasLdelLjohLpla*LsakLlacSagaSequSgor*SmitSmutSpneSpyoSsuiStheSube

20 30 60 70 80 90SD

100

ASD

ASD

Consensus:SD

50

110

40

50

100

110

60

GG

100

30

40

60

30

C C

G

G

C

C

G

G

G

ASD

ASD

ASD

SD

ASD

SD

G

G

20

80

70

90

SD

GG

SD

90

C C

70

80

20

a b c d

Figure 2 Structural models of the SMK box. (a–d) Putative forms of SMK box from E. faecalis (a,b) or S. gordonii (c,d) in the absence (a,c) or presence

(b,d) of SAM. Green, covarying residues predicted to be unpaired in the –SAM form and paired in the +SAM form (thin dashed lines); red and blue, as in

Figure 1; arrows, mutations in the E. faecalis SMK box. The U14G substitution (and the corresponding change in the S. gordonii sequence) was introduced

by including tandem G residues at the start-site for T7 RNAP transcription. Numbering of the E. faecalis sequence is as in Figure 1.

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the SD sequence and a complementary sequence domain that couldpotentially block the SD (termed the anti-SD or ASD) was identifiedin the same contextual arrangement in each sequence, which suggeststhat regulation could occur at the level of translation initiation. Noneof the sequences contained elements resembling a transcriptionalterminator. The minimal upper portion of the core element wasdefined as a 4-base-pair helix (corresponding to the pairing of residues33–36 to residues 59–62 in E. faecalis metK). The size of the helicaldomain above the core element ranged from 5 to 211 nucleotides (nt),and the distance between the core upstream element and theSD sequence ranged from 4 to 200 nt. The larger sequences showedextensive potential base-pairing within the inserted regions. Therewas no obvious similarity between this RNA element and thepreviously described S box RNA pattern4. The conservation of thecore element, including the possible pairing of the ASD sequencewith the SD sequence, suggested that this RNA elementcould participate in metK regulation. Searches for similar elementsupstream of other genes involved in methionine biosynthesis inthese organisms were not successful. We chose the E. faecalis metKgene for further analysis.

E. faecalis metK-lacZ expression in vivo in B. subtilisThe putative metK regulatory element was predicted to have thepotential to sequester the SD sequence (Fig. 2). To investigate effectson translation independent of possible transcriptional regulation, wepositioned the E. faecalis metK leader sequence downstream of theB. subtilis glyQS promoter (Pgly) and generated a metK-lacZ trans-lational fusion in which lacZ expression was dependent on the metKribosome binding site. The fusion was integrated as a single copy intothe B. subtilis chromosome, and b-galactosidase levels were monitoredduring growth in the presence or absence of methionine. TheB. subtilis host was chosen for direct comparison of the metKelement to previously characterized S box elements under identicalgrowth conditions. Expression of the Pgly-metK-lacZ translationalfusion was low during growth in the presence of methionine andwas induced by starvation for methionine (Table 1), with a pattern ofinduction similar to that observed for a B. subtilis yitJ-lacZ S boxtranscriptional fusion4. The lower stringency of regulation of thePgly-metK-lacZ construct may be due to higher activity from theglyQS promoter than the native promoter and may also reflect the

requirement for basal SAM synthetase activity even when methionineis abundant.

In contrast to the Pgly-metK-lacZ translational fusion, ananalogous Pgly-metK-lacZ transcriptional fusion, in which translationof the lacZ reporter was independent of the metK ribosomebinding site, showed high constitutive expression (Table 1). The lossof repression of the transcriptional fusion during growth inmethionine suggests that regulation of the E. faecalis metK gene islikely to occur at the level of translation, as repression was observedonly when expression was dependent on the metK translationinitiation region.

Binding of SAM by the E. faecalis metK leader RNAmetK genes in many Gram-positive organisms are regulated by directbinding of SAM to the leader RNA. We therefore considered SAM alikely effector for metK regulation in the Lactobacillales, and we testedthe E. faecalis metK leader RNA for SAM binding under conditionssimilar to those used for S box RNAs6. RNA containing sequencesfrom position 15 to 118 relative to the predicted transcription start sitewas generated by in vitro transcription with T7 RNA polymerase(RNAP) and incubated with radiolabeled SAM. Unbound SAM wasremoved by filtration, and radiolabeled SAM retained by the filter wasquantitated. SAM bound the 15–118 RNA, and binding was blocked by

addition of excess unlabeled SAM (Fig. 3a);in contrast, unlabeled S-adenosylhomo-cysteine (SAH) did not compete, indicatingthat SAM binding is specific. The efficiencyand specificity of SAM binding was similarto that of S box RNAs6. The metK RNAtherefore binds SAM in the absence of anyother cellular factor.

Conservation of the core RNA elementupstream of metK genes in other membersof the Lactobacillales suggested that theseRNAs might have a similar function. RNAscorresponding to the 5¢ regions of the Lacto-bacillus plantarum and S. gordonii metK genesbound SAM (Fig. 3a), demonstrating thatSAM binding is a general property of thisclass of RNA elements. As this SAM-bindingelement is unlike the S box SAM-bindingmotif in sequence or structure and appearsto be specific to metK genes, we designate itthe SMK box.

RNA0

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50

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%)

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18

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5

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G91

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68G

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CompetitorCompound

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SAMSAH

a b c

Figure 3 Binding of SAM to SMK box RNAs. RNAs generated by T7 RNAP transcription were gel-purified, denatured and refolded in the presence of [14C]SAM, and the RNA-bound SAM was separated

from unbound SAM by size-exclusion filtration. Retention of SAM is expressed relative to that of the

wild-type E. faecalis metK RNA extending from residue 15 to 118 (Fig. 2). No SAM was retained by

the filter in the absence of RNA. Black or gray bar denotes a sample incubated with a 50-fold molar

excess of unlabeled SAM or SAH, respectively. (a) SAM binding by E. faecalis, L. plantarum and

S. gordonii RNAs. (b) Deletion analysis of the E. faecalis SMK box. (c) Effect of point mutations

on SAM binding by the E. faecalis SMK box RNA. All constructs contained residues 15–118.

Table 1 Expression of Pgly-metK-lacZ fusions in vivo

b-galactosidase

activitya (Miller units25)

Fusion Fusion type –Met +Met

Induction

ratiob

Pgly-metK-lacZ Translational 120 24 5.0

Pgly-metK-lacZ Transcriptional 380 340 1.1

Pgly-metK-lacZ C24G Translational 110 95 1.2

Pgly-metK-lacZ G30C G31C Translational 220 160 1.4

yitJ-lacZ Transcriptional 270 0.40 680

aCells were grown in minimal medium24 in the presence of methionine, then were split andgrown in the presence (+Met) or absence (–Met) of methionine. b-galactosidase activity isshown for samples taken 4 h after the cultures were split. bInduction ratio is the b-galacto-sidase activity in cells grown in the absence of methionine divided by the activity in cellsgrown in the presence of methionine.

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Mutational analysis of the E. faecalis SMK boxDeletion analysis was used to determine the limits of the elementrequired for SAM binding (Fig. 3b). Deletion of residues at the 5¢ endof the element, to generate an RNA extending from position 32 to 118,resulted in a 50-fold reduction in SAM binding. RNAs extending fromposition 15 to position 95 or 105 retained 60%–85% of the bindingactivity of the 15–118 RNA, whereas an RNA extending to position 90,in which the 3¢ portion of the SD sequence was removed, showed nodetectable SAM binding. These results indicate that the entire coreelement and the SD region of the RNA are necessary for stableinteraction with SAM.

The requirement for sequences in the SD region for SAM bindingsuggested that interaction between this element and upstreamsequences may be important. Potential pairing of residues correspond-ing to U21–C25 with residues G90–A94 was found in each sequence.The downstream sequence overlaps the SD sequence (positions88–92), and the residues corresponding to the predicted U21-A94pairing covaried to G-C in 14 of 19 sequences. Introduction of theC24G and G91C substitutions to disrupt the pairing resulted in loss ofSAM binding, and combining the C24G and G91C substitutions torestore pairing also restored SAM binding, despite 100% conservationof the residues corresponding to C24 and G91 (Fig. 3c). Therestoration of binding activity in the C24G G91C double mutantsuggests that conservation of G91 is dictated by its participation in theSD sequence and not by any requirement for interaction with SAM;conservation of C24 is presumably dictated in turn by the requirementto retain pairing with G91.

Residues corresponding to positions G30 and C68 of the E. faecalismetK sequence covary to A-U in Streptococcus metK genes (Fig. 1).The flanking residues are conserved and can also pair (Fig. 2b,d).Residues 68 and 69 are predicted to pair with residues 20 and 19,respectively, in the alternative structure (Fig. 2a,c). The G30C G31Cand C67G C68G constructs, which are predicted to result in disrup-tion of the pairing shown in Figure 2b, showed a major reduction inSAM binding (Fig. 3c). Combination of both sets of mutations to

restore pairing resulted in partial restoration of SAM binding, whichsupports the model that pairing of these residues is important forSAM binding. The lack of complete restoration of normal bindingactivity may be due to an additional requirement for pairing of C68with G20 in the alternative form (Fig. 2a) or to other requirements formaintenance of primary sequence in this region.

The importance of residues C24, G30 and G31 for binding of SAMto the SMK box RNA led us to test the effects of the C24G and G30CG31C mutations on expression of a Pgly-metK-lacZ translationalfusion. These mutations resulted in loss of repression during growthin methionine (Table 1), which demonstrates that these residues areimportant both for SAM binding in vitro and for regulation of metKexpression in vivo.

RNase H mapping of the SMK boxAccessibility of specific regions of the E. faecalis SMK region toantisense DNA oligonucleotides (Fig. 4a) was monitored by cleavagewith RNase H, which targets RNA-DNA hybrids. Binding of oligo-nucleotides complementary to residues 25–32 (5¢ side of the coreelement), residues 63–72 (3¢ side of the core element), and residues85–94, 86–93 or 90–97 (the SD region) to transcripts generated byT7 RNAP transcription in the absence of SAM resulted in efficientcleavage by RNase H (Fig. 4b, lanes 3, 5, 7, 9 and 11). In contrast,transcripts generated in the presence of SAM were resistant to cleavagedirected by these oligonucleotides (Fig. 4b, lanes 4, 6, 8, 10 and 12),which suggests that binding of SAM sequesters the targeted regionsinto a structure that inhibits binding of the oligonucleotides.Transcripts generated in the presence or absence of SAM were sensitiveto cleavage directed by oligonucleotides complementary to residuesdownstream of the SD region (residues 98–108 or 106–114). Cleavagedirected by oligonucleotides complementary to either side of the coreelement required a 100-fold higher concentration as compared tooligonucleotides complementary to the SD region, consistent withlimited access to the region within the helical element even in theabsence of SAM. The SAM-dependent SD-ASD pairing appears, in

16151413121110987654321

*

*

*

*

**

*

*P

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+++++++++ + + +

–––

–––

––

––––

–

SAMChallenge oligo

Cleavage oligo 85–94 98–108 106–114

16151413121110987654321

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SAMOligo 106–11498–10890–9786–9385–9463–7225–32

85–94

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63–72

AS

D

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86–93

90–97

98–108

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SDASD

63–7225–32

a b

c

Figure 4 RNase H cleavage analysis of the E. faecalis SMK box RNA. (a) Positions of

oligonucleotide binding on the RNA. Arrows represent positions of binding of the antisense

oligonucleotides; numbers indicate the endpoints of the complementary residues. (b) Labeled

RNAs generated by T7 RNAP transcription in the presence (+) or absence (–) of SAM wereincubated with the indicated oligonucleotides (25–32 and 63–72 at 4.5 mM; all others at

45 nM), and RNA-DNA hybrids were then digested with RNase H. FL, full-length transcripts;

P, a pause transcript observed in all transcription reactions; asterisks, RNase H cleavage

products. (c) Effect of 3¢ cleavage on SAM-dependent protection. Labeled RNAs generated in the presence or absence of SAM were first incubated

with a 3¢-targeted cleavage oligonucleotide as shown, then digested with RNase H; for lanes indicated, the challenge oligonucleotide (25–32, which

is complementary to the 5¢ side of the internal loop) was then added (+) and incubation was continued to allow a second RNase H digestion step.

All oligonucleotides were used at 4.5 mM. Arrow marks the product of RNase H cleavage directed by the challenge oligonucleotide.

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contrast, to be susceptible to disruption by binding of a complemen-tary oligonucleotide at high concentration, suggesting that this pairingis not highly stable.

The RNase H assay was also used to further define the 3¢ border ofthe SMK element required for interaction with SAM. Oligonucleotidescomplementary to regions at the 3¢ ends of RNAs generated in thepresence or absence of SAM were used to direct RNase H–dependentremoval of specific downstream regions. The resulting RNA was thenincubated with a second oligonucleotide complementary to residues25–32, in a region protected by SAM binding in the full-length RNA(Fig. 4b). This assay therefore determined whether removal of 3¢regions of the RNA from the SAM–RNA complex can reverse theSAM-dependent protection of the 5¢ region of the RNA. Cleavagedirected by oligonucleotides 98–108 and 106–114 resulted in RNAproducts that were competent for SAM-dependent protection of the25–32 region, as cleavage by the challenge oligonucleotide occurred inthe absence of SAM (Fig. 4c, lanes 11 and 15) but not in the presenceof SAM (lanes 12 and 16). Cleavage directed by oligonucleotide98–108 results in an RNA product that includes the SD sequencebut not the AUG start codon (Fig. 4a). Retention of SAM-dependentprotection in an RNA extending past the SD sequence is consistentwith the results of the SAM-binding assays, where an RNA extendingto position 95 retained partial SAM-binding activity (Fig. 3b).Cleavage directed by oligonucleotide 85–94, which removes the SDsequence, resulted in loss of SAM-dependent protection from cleavagedirected by the 25–32 challenge oligonucleotide (Fig. 4c, lane 8),consistent with loss of SAM binding by RNAs extending toposition 90 (Fig. 3b). These results further support a model inwhich interaction of the SD region with the upstream element isrequired for SAM binding and a SAM-dependent RNA rearrangementthat sequesters the internal loop of the SMK box. Removal of the SDregion from a SAM-bound RNA is apparently sufficient to disrupt theSAM–RNA complex.

Structural mapping of the E. faecalis SMK box RNADigestion of 5¢ end–labeled E. faecalis SMK box RNA with RNases T1(which cleaves adjacent to unstructured G residues) and A (whichcleaves adjacent to unstructured U and C residues) was used tocharacterize the RNA structural arrangement and to identify SAM-induced structural alterations. Cleavage by RNases T1 and A was most

efficient at residues predicted to be unpaired in the structure shown inFigure 2a, with extensive cleavage by RNase T1 at G47 (in the terminalloop), G77 (downstream of the helical element) and G88 (firstposition of the SD) (Fig. 5a). Cleavage by RNase A was most efficientat U82 (downstream of the helical element), with additional productsrepresenting residues in the terminal loop and U65 (Fig. 5b). Productscorresponding to cleavage at residue A64 appeared in all assays,presumably owing to Mg+2-dependent cleavage. Comparison todenatured RNA revealed decreased cleavage by RNase T1 at positionsG50, G71 and G89–G92, suggesting that these residues are protectedin the folded RNA. Similarly, residues 35, 38, 42, 52, 56, 59 and72 showed reduced cleavage by RNase A in the folded RNA ascompared to the denatured RNA, consistent with the predictionthat the core element is structured even in the absence of SAM.Residues G47 and U48 (within the terminal loop) and U65 (within theinternal loop) showed increased cleavage in the folded RNA, suggest-ing that these residues become more exposed. The most prominentSAM-dependent change in the RNase T1 digestion pattern was theprotection of the SD region, particularly residue G88, in the presenceof SAM (Fig. 5a). Addition of SAM also resulted in protection of U86(2 nt upstream of the SD) and U95 (1 nt downstream of the regionpredicted to pair with the ASD) from digestion by RNase A (Fig. 5b).The protection of the SD region in the presence of SAM is consistentwith a model in which SAM binding results in sequestration of the SDregion, thereby inhibiting translation initiation. In contrast, residuesG104 and G108 (within and downstream of the AUG start codon) andU101 (immediately upstream of the start codon) showed increasedcleavage in the presence of SAM, which suggests that this regionbecomes less structured under these conditions.

The susceptibility of residue G88 to cleavage by RNase T1 was alsoused to quantitate the sensitivity to SAM. Incubation of the RNA withvarying concentrations of SAM, followed by RNase T1 digestion,resulted in a concentration-dependent pattern of protection, with50% protection of G88 at 0.9 mM SAM (data not shown). Bycomparison, the yitJ S box RNA shows half-maximal trans-cription termination in vitro at 0.15 mM SAM9. The yitJ assaymonitors a functional interaction with SAM, in the context of anascent transcript emerging from B. subtilis RNAP, whereas themetK assay monitors a structural change in T7 RNAP–generatedRNA that has been denatured and refolded. Despite these differences,it seems that S box and SMK box RNAs show a similar sensitivity toSAM concentrations.

Digestion with RNase V1 (which preferentially cleaves adjacent tostructured or stacked residues) resulted in increased cleavage in thepresence of SAM at residue U95 (immediately downstream of theresidues predicted to pair with the ASD sequence), consistent with aSAM-induced increase in structure in this region (Fig. 5c). An increase

NR10987654321Lane A T1–SAM +SAM–SAM +SAM–SAM +SAM

NR654321ALaneNR87654321T1Lane

37

41

47

50

63

66

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8286

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108106101

959288

77

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38

35

Time Time Time Time RNase V1 RNase V1a b c Figure 5 Structural mapping of the E. faecalis SMK box RNA. RNA

corresponding to positions 15–118 was generated by transcription with

T7 RNAP, gel-purified, 5¢ end–labeled, denatured and refolded in the

presence or absence of SAM, as indicated. (a) RNase T1 digestion. RNA

was incubated with RNase T1 for 10 min (lanes 1 and 5), 20 min (lanes 2

and 6), 30 min (lanes 3 and 7) or 45 min (lanes 4 and 8). (b) RNase A

digestion. RNA was incubated with RNase A for 10 min (lanes 1 and 4),

20 min (lanes 2 and 5) or 30 min (lanes 3 and 6). (c) RNase V1 digestion.

RNA was incubated for 10 min with RNase V1 (5 � 10–6 U ml–1, lanes 1

and 6; 8.3 � 10–6 U ml–1, lanes 2 and 7; 1.3 � 10–5 U ml-1, lanes 3 and 8;

2.5 � 10–5 U ml–1, lanes 4 and 9; 6.7 � 10–5 U ml–1, lanes 5 and 10).

In a–c, size standards (lanes T1 and A) were generated by digestion of

denatured RNA with RNases T1 and A, respectively; certain residues are

labeled to indicate key regions. NR, RNA that was not nuclease-digested.

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in cleavage in the region surrounding the AUG codon was alsoobserved. Increased cleavage in this region by all three RNasesprecludes a clear understanding of how the structure is affected inthe presence of SAM. Residue C75 showed higher cleavage by RNaseV1 in the absence of SAM, suggesting that pairing in this region (forexample, as shown in Fig. 2a) occurs in the absence of SAM; a similarpattern was observed for residues U69 and U70, although cleavage wasless efficient. The most efficient cleavage by RNase V1 occurred atresidue U42, within the upper helix; in conjunction with efficientRNase T1 cleavage of G47 (in the terminal loop) in the presence orabsence of SAM, this observation suggests that the structural arrange-ment of the upper domain of the SMK box is not strongly affected bySAM. Overall, these results support a model in which at least a portionof the internal loop of the SMK box and the ASD sequence areunpaired in the absence of SAM, and binding of SAM alters thepairing in the bottom portion of the helical element and promotesprotection of the SD region, presumably through pairing with theASD sequence (Fig. 6).

DISCUSSIONIn this study we report the identification of the SMK box, a new SAM-binding riboswitch RNA that regulates SAM synthetase genes in theLactobacillales group of lactic acid bacteria. Binding of SAM topurified SMK box RNA in vitro resulted in a rearrangement of theRNA structure that sequestered the SD sequence. No factors otherthan SAM were necessary for SAM binding or the SAM-dependentstructural change. The E. faecalis SMK box was sufficient to confertranslational repression of a lacZ reporter in vivo under conditionswhere SAM pools are elevated. These data support a model in whichSAM binding to the SMK box represses metK expression by inhibitingtranslation initiation.

The SMK box shows no similarity to the previously identified S boxSAM-binding riboswitch. A major difference between the SMK box andS box RNAs is that the SAM-binding domain of the SMK box RNAsincludes the SD sequence, which serves as the target for regulation(Fig. 6). In contrast, the SAM-binding domain of S box RNAs doesnot include the target of regulation (the terminator helix) and includesonly the 5¢ portion of the antiterminator element. Certain S box RNAsare predicted to regulate gene expression at the level of translation

initiation rather than transcription termination1. In genes of this class,the terminator helix is replaced by a helix that sequesters the SD bypairing it with a complementary ASD sequence, and the antitermi-nator helix is replaced by a helix that sequesters the ASD sequence bypairing it with another competing sequence, the anti-ASD or AASD(Fig. 6). Although no S box RNAs of this type have been characterizedbiochemically, it seems, by analogy with other S box RNAs, that therole of SAM binding is to prevent formation of the ASD-sequesteringhelix (AASD), without having a direct effect on the SD sequence itself.Other metabolite-binding RNA riboswitches that operate at the levelof translation are arranged similarly to the S box RNAs1–3, so thatdirect participation of the regulatory target in the metabolite-bindingelement is an unusual feature of SMK box RNAs.

Although the affinity of the SMK box RNAs for SAM has not yetbeen determined, the concentration of SAM required for protection ofthe SD sequence from RNase digestion (o1 mM) is similar to thatrequired for SAM-dependent transcription termination in vitro forS box RNAs5,9. SAM concentrations have been reported at 80–400 mMfor B. subtilis in vivo, depending on growth conditions12, but it is notknown what fraction of the SAM pool is in complexes with othercellular components. SMK box and S box RNAs also show a similarability to discriminate between SAM and related compounds such asSAH, which differs from SAM by a single methyl group. As SAH is thebyproduct of use of SAM as a methyl donor, discrimination betweenSAM and SAH is of crucial biological importance. Although the S boxand SMK box show little overall sequence or structural similarity, wenote that the pairing between the residues corresponding to E. faecalismetK 29–31 and 67–69 has a pattern of sequence conservation similarto that of the tertiary interaction in S box leaders (Fig. 6)9. In bothcases, this pairing is both stimulated by and required for SAM binding.The major differences between the S box and SMK box RNA motifsdemonstrate that there are multiple ways to build RNA elementswith similar biochemical and regulatory properties. A third classof SAM-binding RNA elements has recently been found upstreamof methionine-related genes, including metK, primarily ina-proteobacteria13; this element differs from the S box and SMK boxin sequence and structural arrangement, and it is often foundupstream of transcriptional terminators, although SAM-dependenttranscription termination has not been demonstrated. These results

Figure 6 SMK box and S box mechanisms. In the SMK box model (top), the

ASD and SD regions (blue) are unpaired and available for interaction in the

absence of SAM (left). The covarying residues (green) are also unpaired.

In the presence of SAM (right), binding of SAM (asterisk) results in a

rearrangement of the RNA that includes pairing of the covarying residues

(green) and of the ASD and SD regions (blue) to form a pseudoknot.

Sequestration of the SD within the pseudoknot is predicted to prevent

translation initiation. In the S box model (bottom), the anti-antiterminator

helix is unpaired in the absence of SAM (left), allowing formation of the

antiterminator helix (AT, magenta and blue) which prevents formation of the

terminator helix. In the presence of SAM (right), the anti-antiterminator helix

is stabilized (AAT, black and magenta) and the tertiary interaction between

the covarying residues is stabilized (green); the antiterminator is unpaired,

allowing formation of the terminator helix (T, blue) which results in

premature termination of transcription. A similar RNA rearrangement inS box RNAs can result in inhibition of translation initiation if the terminator

helix is replaced by a helix that sequesters the SD sequence by pairing

with the ASD sequence, the antiterminator is replaced with a helix that

sequesters the ASD by pairing with the AASD sequence, and SAM promotes

sequestration of the AASD. The most highly represented sequence is shown

for the SAM-dependent tertiary interaction9 (green), but covariation of the paired residues is found in both the S box and SMK box sequences. The position of

SAM within the structures is not known. For the SMK box, SAM binding requires sequences extending past the SD region and the SD-ASD pairing. For the

S box, SAM binding requires the complete AAT structure but not the terminator helix (or SD-ASD pairing).

T3′

5′

5′5′

3′

3′

5′3′

AATAT

(AA

SD

)

(AA

SD

)

(AS

D)

(AS

D) (S

D)

*

*

+SAM–SAM *

SD

(SD)

SD

ASD AS

D

SMK box

S box

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therefore suggest that there are at least three different classes of SAM-dependent riboswitches in bacteria.

A variety of mechanisms for regulation of methionine metabolismgenes have been uncovered in bacteria. Most Gram-positive bacteriawith low G + C content, including certain members of the Lactoba-cillales, use the S box system to control expression of both methioninebiosynthesis and metK genes at the level of transcription terminationin response to SAM4,5,10,11. A T box motif predicted to monitorcharging of tRNAMet has been identified upstream of methionine-related genes in Enterococcus, Lactobacillus and Staphylococcus spp., butmetK genes are not included in this group (F.J.G. and T.M.H.,unpublished data)5. In Bacillus spp., the cysE cysteine biosynthesisgene is also a member of the T box family, responding to tRNACys

(ref. 14), whereas other cysteine-related genes are regulated at the levelof transcription initiation15,16. Cysteine and methionine biosynthesisgenes in L. lactis are regulated at the level of transcription initiation bya LysR-type activator, FhuR (also called CmbR), which responds toO-acetyl serine17,18, and a similar mechanism using the MtaR proteinhas been suggested for Streptococcus spp.10,19. Regulation at the level oftranscription initiation is also used in Escherichia coli, with separateregulators for cysteine and methionine genes; metK is coordinatelyregulated with methionine biosynthesis genes, using SAM as theeffector20. It therefore seems that at least some of the lactic acidbacteria differ from other organisms both in coordinating regulationof cysteine and methionine biosynthesis genes and in separatelyregulating metK expression. Although the generality of these patternsneeds further investigation, it is notable that many lactic acid bacteriarequire an exogenous source of methionine for optimal growth andreadily interconvert methionine and cysteine. SAM synthetase activityis essential for cell viability under all growth conditions, and inde-pendent regulation of metK in response to SAM may ensure thatappropriate levels of SAM are maintained regardless of fluctuations inmethionine availability. A further potential advantage of a transla-tional control mechanism is that the switch could be reversible,allowing rapid response to variations in SAM concentration by releaseof the SD when SAM pools decline. It will be necessary to test whetherthe regulatory pattern demonstrated here in a heterologous host canalso be demonstrated in a system in which the SMK box is found.

This study demonstrates that the SMK box serves as a previouslyundiscovered SAM-binding cis-acting regulatory RNA. The structuralmapping and mutational analyses suggest that in the absence of SAMthe SD and ASD regions are unpaired and that binding of SAMstimulates pairing within the core element and between the SD andASD elements. The data also indicate that the SD sequence and pairingbetween the SD and ASD are required for SAM binding. Theobservation that the SMK box RNA confers translational repressionin vivo under conditions where SAM pools are elevated supports amodel in which SAM-dependent pairing of the SD to the ASDprevents translation initiation by blocking access of the ribosome tothe ribosome binding site. Direct testing of this model represents thenext major goal of our analysis.

METHODSDNA constructs. DNA templates for construction of lacZ fusions and

T7 RNAP transcription were generated by combining pairs of complementary

oligonucleotides (Supplementary Table 1 online) as previously described9,21.

Sequence variations were verified by DNA sequencing. For templates for

T7 RNAP transcription, the upstream pair of oligonucleotides included a

promoter recognized by T7 RNAP. DNA constructs for lacZ fusions replaced

the E. faecalis metK promoter with the B. subtilis glyQS promoter. Sequence

changes were introduced by replacement of a pair of oligonucleotides with a

pair containing the desired changes. The metK constructs matched the

sequences for E. faecalis strain V583 (NCBI AE016830), L. plantarum strain

WCFS1 (NCBI AL935263) and S. gordonii Challis (The Institute for Genomic

Research, http://www.tigr.org).

b-galactosidase assays. The E. faecalis metK leader region, including 15 nt

downstream of the translational start site, was positioned downstream of the

B. subtilis glyQS promoter. The resulting DNA fragment was inserted into a

lacZ fusion vector (pFG328)22 to generate either a metK-lacZ transcriptional

fusion (using a variant of lacZ in which the E. coli trpA translational start region

is fused to the third codon of lacZ to allow efficient translation initiation in

B. subtilis)23 or an in-frame metK-lacZ translational fusion (containing the first

5 codons of metK fused to codon 18 of lacZ, generated by deletion of the

trpA-lacZ junction). Constructs were introduced into the chromosome of

B. subtilis strain BR151 (metB10 lys-3 trpC2) by integration into an SPbprophage. B. subtilis strains containing lacZ fusions were grown at 37 1C in

Spizizen minimal medium24 containing methionine (50 mg ml–1) until early

exponential growth. The cells were harvested by centrifugation at 8,000g and

resuspended in Spizizen medium either with or without methionine, and

samples were collected at 1-h intervals and assayed for b-galactosidase activity

after toluene permeabilization of the cells25. Growth experiments were repeated

at least twice, and reproducibility was ±10%.

T7 RNA polymerase transcription reactions. RNA was generated by using a

MEGA-shortscript T7 RNAP high-yield transcription kit (Ambion). The RNA

products were gel-purified as described previously21.

SAM-binding assays. T7 RNAP–transcribed RNA (3 mM) in 1� transcription

buffer26 was heated to 65 1C and slow-cooled to 40 1C. Competitor compounds

(unlabeled SAM or SAH) were added at 400 mM as indicated in Figure 3 and

[methyl-14C]SAM (ICN; 52 mCi mmol–1 (1.92 GBq mmol–1)) was added to a

final concentration of 8 mM. After incubation for 10 min at room temperature,

samples were passed through a Nanosep 10K Omega filter and washed four

times with 150 ml 1� transcription buffer. Material retained by the filter was

mixed with Packard BioScience Ultima Gold scintillation fluid and counted in a

Packard Tri-Carb 2100TR liquid scintillation counter.

RNase H probing of E. faecalis metK transcripts. Labeled RNAs were

generated by T7 RNAP transcription in the presence of [a-32P]UTP. RNAs

generated in the presence or absence of SAM (125 mM) were incubated for

5 min with antisense DNA oligonucleotides (as indicated in Figure 4), then

digested with RNase H (0.45 U ml–1; Ambion) for 10 min; incubation

temperatures (24–37 1C) were optimized for each oligonucleotide. The RNA

products were resolved by denaturing PAGE and visualized by PhosphorImager

analysis (Molecular Dynamics).

Structural mapping of the E. faecalis metK leader RNA. RNAs synthesized by

T7 RNAP transcription were 5¢ end–labeled with [g-32P]ATP (7,000 Ci mmol–1)

using a KinaseMax kit (Ambion) and passed through a MicroSpin G-50 column

(Amersham Biosciences). Labeled RNAs (600 nM) in 1� transcription buffer in

the presence or absence of SAM (140 mM) were heated to 65 1C and

slow-cooled to 40 1C. RNase T1 (0.01 U ml–1), RNase A (0.67 ng ml–1) or

RNase V1 (5 � 10–6 to 6.7 � 10–5 U ml–1) was added and samples were

incubated at room temperature for 5–30 min. Reactions were halted by addition

of Precipitation Inactivation Buffer (Ambion) and digestion products were

resuspended in gel-loading buffer, resolved using 10% denaturing PAGE and

visualized by PhosphorImager analysis (Molecular Dynamics). Size standards

were generated by RNase digestion of denatured RNAs.

Note: Supplementary information is available on the Nature Structural & MolecularBiology website.

ACKNOWLEDGMENTSWe thank S. Tigert for technical assistance. Preliminary sequence data wereobtained from The Institute for Genomic Research website at http://www.tigr.org.This work was supported by the US National Institutes of Health (grantGM63615).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

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Published online at http://www.nature.com/nsmb/

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions/

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