The SAM-responsive S(MK) box is a reversible riboswitch

Abstract

The S(MK) (SAM-III) box is an S-adenosylmethionine (SAM)-responsive riboswitch found in the 5' untranslated region of metK genes, encoding SAM synthetase, in many members of the Lactobacillales. SAM binding causes a structural rearrangement in the RNA that sequesters the Shine-Dalgarno (SD) sequence by pairing with a complementary anti-SD (ASD) sequence; sequestration of the SD sequence inhibits binding of the 30S ribosomal subunit and prevents translation initiation. We observed a slight increase in the half-life of the metK transcript in vivo when Enterococcus faecalis cells were depleted for SAM, but no significant change in overall transcript abundance, consistent with the model that this riboswitch regulates at the level of translation initiation. The half-life of the SAM-S(MK) box RNA complex in vitro is shorter than that of the metK transcript in vivo, raising the possibility of reversible binding of SAM. We used a fluorescence assay to directly visualize reversible switching between the SAM-free and SAM-bound conformations. We propose that the S(MK) box riboswitch can make multiple SAM-dependent regulatory decisions during the lifetime of the transcript in vivo, acting as a reversible switch that allows the cell to respond rapidly to fluctuations in SAM pools by modulating expression of the SAM synthetase gene.

Figure 1. The E. faecalis S_MK box riboswitch

(A) In the absence of SAM (left), the ASD sequence (red) is predicted to be single stranded and helix P0 is formed. In this conformation, the SD sequence (green) is accessible for translation initiation. SAM binding (right) causes disruption of helix P0 and stabilization of helices P1, P2, and P4. In this conformation, the SD sequence is sequestered and translation initiation is inhibited. Helix P3 is predicted to be present in both conformations. The AUG start codon of metK is indicated in yellow. (B) The SAM-binding pocket is at the center of a 3-way junction between helices P1, P2, and P4. The three-dimensional cartoon is rotated relative to (A) to facilitate visualization of the ligand-binding pocket. SAM makes direct contacts with the mRNA via its adenosine moiety, while its methionine tail extends into a solvent-filled cavity (Lu et al., 2008). This image was generated from coordinates deposited in the RCSB Protein Data Bank under accession number 3E5C.

Figure 2. Measurement of SAM pools in E. faecalis

E. faecalis cells were grown in minimal medium containing high methionine (230 μM) until mid-log phase. Cells were collected by centrifugation and resuspended in fresh media containing low methionine (1.8 μM; open circles). Samples were collected over time and the concentration of SAM in formic acid extracts of the cells was determined in an in vitro transcription assay with a SAM-responsive S box RNA, by comparison to a standard curve of transcription reactions carried out with known concentrations of SAM. Reversibility of the response was determined by addition of methionine (to 230 μM) after the 60 min sample (closed circles).

Figure 3. Dissociation of the SAM-S_MK box riboswitch complex

(A) A bipartite RNA corresponding to the E. faecalis metK leader (with a nick at the top of the terminal stem-loop) was used for fluorescence assays. A 2-AP fluorescent probe (arrow) was incorporated in the upstream RNA at a region predicted to undergo a conformational rearrangement in response to SAM binding. (B) Dissociation of SAM from the S_MK box riboswitch was monitored at 22 °C in a stopped-flow spectrometer. A 5-fold molar excess of a SAM-sequestering competitor RNA (B. subtilis yitJ leader) was added to the E. faecalis S_MK box RNA-SAM complex, resulting in an increase in 2-AP fluorescence. Data points (gray spheres) represent the averages from six individual traces. The residuals plot (bottom panel) shows the difference between the actual (gray spheres) and calculated (black trace) data sets.

Figure 4. Conformational switching of the S_MK box riboswitch

Fluorescence of an E. faecalis metK leader RNA (1 μM) containing a 2-AP fluorescent probe was monitored over time. Addition of SAM (2 μM) at 60 sec resulted in fluorescence quenching, while addition of yitJ competitor RNA (4 μM) at 180 sec caused an increase in fluorescence. Addition of more SAM (20 μM) at 300 sec reversed the phenotype again, leading to an even greater decrease in fluorescence. Gaps in the data represent the time required for addition of sequential reaction components. Values plotted represent the averages from four experiments.

Figure 5. Repression of E. faecalis metK-lacZ translational fusions in B. subtilis by addition of methionine or SAM

Cells were grown in Spizizen medium (Anagnostopoulos and Spizizen, 1961) in the presence of methionine, collected by centrifugation, and resuspended in fresh medium with or without methionine. After 120 min, methionine or SAM was added to cultures as indicated. Values represent the averages from at least two experiments. MU, Miller units (Miller, 1972).