SAM-VI RNAs selectively bind S-adenosylmethionine and exhibit similarities to SAM-III riboswitches

Abstract

Five distinct riboswitch classes that regulate gene expression in response to the cofactor S-adenosylmethionine (SAM) or its metabolic breakdown product S-adenosylhomocysteine (SAH) have been reported previously. Collectively, these SAM- or SAH-sensing RNAs constitute the most abundant collection of riboswitches, and are found in nearly every major bacterial lineage. Here, we report a potential sixth member of this pervasive riboswitch family, called SAM-VI, which is predominantly found in Bifidobacterium species. SAM-VI aptamers selectively bind the cofactor SAM and strongly discriminate against SAH. The consensus sequence and structural model for SAM-VI share some features with the consensus model for the SAM-III riboswitch class, whose members are mainly found in lactic acid bacteria. However, there are sufficient differences between the two classes such that current bioinformatics methods separately cluster representatives of the two motifs. These findings highlight the abundance of RNA structures that can form to selectively recognize SAM, and showcase the ability of RNA to utilize diverse strategies to perform similar biological functions.

Keywords: SAM; aptamer; cofactor; gene regulation; metA; metK; noncoding RNA.

Figure 1.

The SAM-VI riboswitch class (Bifido-metK) shares architectural and sequence similarities with the SAM-III riboswitch class. (A) Consensus sequence and secondary structure model of 17 unique examples of the SAM-VI riboswitch. The Shine-Dalgarno (SD) sequence and the AUG start codon (start) are embedded within the SAM-VI aptamer. (B) Consensus sequence and secondary structure model of 936 unique sequences of SAM-III (S_MK box) RNAs. Annotations are as described for A. (C) Some nucleotides in the three-stem junction of SAM-VI (top, shaded in cyan) appear to have similar identity and location to ligand-contacting nucleotides of the SAM-III aptamer. The highlighted nucleotides for SAM-VI are similar to those of a representative SAM-III RNA that directly contact the ligand (bottom, shaded in cyan), as presented in an atomic-resolution model generated from x-ray crystallography data [21].

Figure 2.

A representative SAM-VI riboswitch aptamer directly binds SAM. (A) Sequence and secondary structure model of the 66 RNA derived from the metK gene of Bifidobacterium bifidum. Guanosine nucleotides at the 5′ end denoted in lowercase letters were added to enhance in vitro transcription efficiency. Colored circles are based on data derived from B. (B) PAGE analysis of 5ˊ ³²P-radiolabeled 66 metK RNA that was subjected to in-line probing assays in the absence (-) or presence of SAM ranging in concentration from 1 nM to 100 µM. NR, T1 and ^‒ OH indicate no reaction, RNase T1 partial digestion (cleavage after G nucleotides), and partial alkaline-mediated digestion (cleaves after each nucleotide), respectively. Bands corresponding to precursor RNA (Pre) and enzymatic cleavage after certain G residues are annotated. Notable regions of ligand-mediated structural modulation are numbered 1 through 4, and the regions corresponding to the base-paired stems annotated. (C) Plot depicting the fraction of RNA bound to ligand versus the logarithm of the concentration of SAM (see inset for the chemical structure). The plot was constructed as described in the methods by quantifying the band intensities in regions that were most robustly modulated (regions 1 and 2).

Figure 3.

SAM-VI RNA binds SAH with a weaker affinity compared to SAM. (A) Sequence and secondary structure model of the B. bifidum 66 metK RNA depicting the sites of modulation by SAH. (B) PAGE analysis of in-line probing reactions in the absence (‒) or presence of SAH ranging in concentration from 1 nM to 3 mM. Additional annotations are described in the legend to Fig. 2B. (C) The plot of the fraction of RNA bound to SAH versus the logarithm of the molar concentration of SAH (see inset for the chemical structure). Additional details are as described in the legend to Fig. 2C.

Figure 4.

Mutational analysis of a SAM-VI aptamer. (A) Locations of nucleotide changes made to generate 66 metK mutant RNA constructs M1 through M4. Nucleotides in red are the highly conserved (>97%) positions as determined by comparative sequence analysis (Fig. 1A). Single nucleotide positions that were mutated in each RNA construct are boxed and annotated with the mutant nucleotide identity. (B) PAGE analysis of in-line probing assays performed with wild-type (WT) or each mutant RNA construct as indicated in the presence of 0, 1 µM, 10 µM, or 100 µM SAM (from left to right). Additional annotations are as described in the legend to Fig. 2B.