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. 2005;6(8):R70.
doi: 10.1186/gb-2005-6-8-r70. Epub 2005 Aug 1.

Evidence for a Second Class of S-adenosylmethionine Riboswitches and Other Regulatory RNA Motifs in Alpha-Proteobacteria

Free PMC article

Evidence for a Second Class of S-adenosylmethionine Riboswitches and Other Regulatory RNA Motifs in Alpha-Proteobacteria

Keith A Corbino et al. Genome Biol. .
Free PMC article


Background: Riboswitches are RNA elements in the 5' untranslated leaders of bacterial mRNAs that directly sense the levels of specific metabolites with a structurally conserved aptamer domain to regulate expression of downstream genes. Riboswitches are most common in the genomes of low GC Gram-positive bacteria (for example, Bacillus subtilis contains examples of all known riboswitches), and some riboswitch classes seem to be restricted to this group.

Results: We used comparative sequence analysis and structural probing to identify five RNA elements (serC, speF, suhB, ybhL, and metA) that reside in the intergenic regions of Agrobacterium tumefaciens and many other alpha-proteobacteria. One of these, the metA motif, is found upstream of methionine biosynthesis genes and binds S-adenosylmethionine (SAM). This natural aptamer most likely functions as a SAM riboswitch (SAM-II) with a consensus sequence and structure that is distinct from the class of SAM riboswitches (SAM-I) predominantly found in Gram-positive bacteria. The minimal functional SAM-II aptamer consists of fewer than 70 nucleotides, which form a single stem and a pseudoknot. Despite its simple architecture and lower affinity for SAM, the SAM-II aptamer strongly discriminates against related compounds.

Conclusion: SAM-II is the only metabolite-binding riboswitch class identified so far that is not found in Gram-positive bacteria, and its existence demonstrates that biological systems can use multiple RNA structures to sense a single chemical compound. The two SAM riboswitches might be 'RNA World' relics that were selectively retained in certain bacterial lineages or new motifs that have emerged since the divergence of the major bacterial groups.


Figure 1
Figure 1
α-Proteobacterial RNA elements. (a) Consensus sequences and structures. Red and black positions for each RNA element indicate >95% and >80% conservation of a particular nucleotide, respectively. Purine (R) or pyrimidine (Y) designations are used when a single nucleotide is not >80% conserved. Solid black lines indicate variable regions, and solid grey lines are optional sequence insertions that are not present in all examples of an element. Circles represent single nucleotides whose presence (but not sequence) is conserved. Base pairs supported by strong (both bases in the pair vary) and weak (only one base in the pair varies) sequence covariation in a motif alignment have green and blue shaded backgrounds, respectively. (b) Phylogenetic distributions. Element names are chosen based on the proximity that representatives from A. tumefaciens have to genes.
Figure 2
Figure 2
The metA RNA element. (a) Sequence alignment of representative metA RNAs. Shaded nucleotides represent conserved base pairing regions. Lowercase and uppercase letters in the consensus line indicate 80% and 95% sequence conservation, respectively. A complete alignment is available in Additional data file 1. Organism abbreviations: Atu, Agrobacterium tumefaciens; Bja, Bradyrhizobium japonicum; Bme, Brucella melitensis; Mma, Magnetospirillum magnetotacticum; Mlo, Mesorhizobium loti; Rsp, Rhodobacter sphaeroides; Rpa, Rhodopseudomonas palustris; Sme, Sinorhizobium meliloti; Cbu, Coxiella burnetii; Bth, Bacteroides thetaiotaomicron; Bbr, Bordetella bronchiseptica. (b) Consensus sequence and structure of the SAM-I riboswitch aptamer found in Gram-positive bacteria. The consensus is updated from [17] and depicted using the same conventions as Figure 1a. The SAM-II aptamer structure is shown for comparison. (c) Comparison of genes in the methionine and SAM biosynthetic pathways found downstream of SAM-I and SAM-II riboswitches.
Figure 3
Figure 3
The metA element binds SAM. (a) In-line probing of 156 metA RNA from A. tumefaciens. 32P-labeled RNA (NR, no reaction) and products resulting from partial digestion with nuclease T1 (T1), partial digestion with alkali (-OH), and spontaneous cleavage during a 40 h incubation in the presence of varying of SAM concentrations (1 μM to 6 mM) were separated by polyacrylamide gel electrophoresis. Product bands corresponding to certain G residues (generated by T1 digestion) and full length 156 metA RNA (Pre) are labeled. (b) Sequence and secondary structure model for A. tumefaciens metA RNA. Sites of structural modulation for the 156 metA derived from in-line probing are circled with red, green and yellow representing reduced, increased, and constant scission in the presence of SAM, respectively. (c) Dependence of spontaneous cleavage in various regions of 156 metA on the concentration of SAM. Band intensities for the five regions (labeled 1-5) on the in-line probing gel in (a) were quantitated and normalized to the maximum modulation observed. Data from each of these sites corresponds to an apparent Kd of around 1 μM (producing half maximal modulation of cleavage) when plotted against the logarithm of the SAM concentration. Theoretical curves for single ligand binding at sites where cleavage increases (black) and decreases (gray) with a Kd of 1 μM are shown for comparison.
Figure 4
Figure 4
Molecular recognition characteristics of SAM-II aptamers. (a) In-line probing of A. tumefaciens 156 metA RNA in the presence of 1 mM SAM, SAH, SAC, and Met. See legend to Figure 3a for an explanation of the labels. (b) Chemical structures of SAM and a generalized SAM analogue. Arrows represent possible hydrogen bonds and electrostatic interactions that could serve as points of recognition by the aptamer. Circled interactions were determined to have strong (solid) or weak (dashed) contributions to binding affinity in singly substituted chemical analogues. Recognition of the N1 position of SAM was not tested. (c) Apparent Kd values of SAM analogues for binding to 156 metA. Columns (n, X, Y, Z, R1, R2, R3) correspond to groups on the core structure in (b). The S-methyl group (gray box) is not present for SAH and SAC.

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