S-adenosylmethionine directly inhibits binding of 30S ribosomal subunits to the SMK box translational riboswitch RNA

Abstract

The S(MK) box is a conserved riboswitch motif found in the 5' untranslated region of metK genes [encoding S-adenosylmethionine (SAM) synthetase] in lactic acid bacteria, including Enterococcus, Streptococcus, and Lactococcus sp. Previous studies showed that this RNA element binds SAM in vitro, and SAM binding causes a structural rearrangement that sequesters the Shine-Dalgarno (SD) sequence by pairing with an anti-SD (ASD) element. A model was proposed in which SAM binding inhibits metK translation by preventing binding of the ribosome to the SD region of the mRNA. In the current work, the addition of SAM was shown to inhibit binding of 30S ribosomal subunits to S(MK) box RNA; in contrast, the addition of S-adenosylhomocysteine (SAH) had no effect. A mutant RNA, which has a disrupted SD-ASD pairing, was defective in SAM binding and showed no reduction of ribosome binding in the presence of SAM, whereas a compensatory mutation that restored SD-ASD pairing restored the response to SAM. Primer extension inhibition assays provided further evidence for SD-ASD pairing in the presence of SAM. These results strongly support the model that S(MK) box translational repression operates through occlusion of the ribosome binding site and that SAM binding requires the SD-ASD pairing.

Fig. 1.

Structural model of the S_MK box. Putative forms of E. faecalis metK RNA in the absence (a) or presence (b) of SAM (∗). Red, residues of the ASD sequence; blue, residues of the SD sequence; purple, AUG start codon; green arrows, location of stops observed in primer extension assays; black arrows, mutations in the E. faecalis S_MK box. The U14G substitution was introduced by including tandem G residues at the start-site for T7 RNAP transcription. Numbering of the sequence is relative to the predicted E. faecalis metK transcription start-site. The U22G and A93C substitutions were predicted to disrupt the SD-ASD pairing. Dotted lines represent potential pairings supported by phylogenetic and mutational analyses (ref. ; A. Smith, R.T.F., F.J.G., and T.M.H., unpublished results). The position of SAM within the RNA is not known.

Fig. 2.

Nitrocellulose filter binding assays. RNAs generated by T7 RNAP transcription were gel purified and end-labeled with [γ-³²P]ATP. Labeled RNAs were incubated with SAM or SAH followed by tRNA and 30S subunits (a) or tRNA and 30S subunits followed by SAM or SAH (b). Samples were passed through a nitrocellulose filter, and material retained by the filter was quantified by scintillation counting. Less than 0.03% of input RNA was retained by the filters in the absence of 30S subunits.

Fig. 3.

Effect of S_MK box mutations on binding of SAM and 30S ribosomal subunits. (a) SAM binding. RNAs generated by T7 RNAP transcription were gel-purified and then incubated in the presence of [methyl-¹⁴C]SAM. 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 residues 15–118 (Fig. 1). (b) Nitrocellulose filter binding assays. RNAs end-labeled with [γ-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 scintillation counting. Less than 0.03% of input RNA was retained by the filters in the absence of 30S subunits.

Fig. 4.

Primer extension inhibition analysis of S_MK box RNA. E. faecalis metK RNAs containing positions 15–208 were generated by T7 RNAP transcription, gel-purified, and then annealed to a [γ-³²P]ATP-labeled DNA primer complementary to positions 180–203. Annealed RNAs were incubated in the presence of SAM or SAH followed by tRNA and 30S subunits. dNTPs and reverse transcriptase were added, and reactions were quenched by the addition of gel-loading buffer. Radioactivity at the position of the U118 stop in lanes 7 and 8 was compared with the value for lane 6, which was normalized to 100. Lanes 10 and 11 were compared with lane 9, and lanes 13 and 14 were compared with lane 12 in a similar manner. RT, readthrough to 5' end.