Identification of 22 candidate structured RNAs in bacteria using the CMfinder comparative genomics pipeline

Abstract

We applied a computational pipeline based on comparative genomics to bacteria, and identified 22 novel candidate RNA motifs. We predicted six to be riboswitches, which are mRNA elements that regulate gene expression on binding a specific metabolite. In separate studies, we confirmed that two of these are novel riboswitches. Three other riboswitch candidates are upstream of either a putative transporter gene in the order Lactobacillales, citric acid cycle genes in Burkholderiales or molybdenum cofactor biosynthesis genes in several phyla. The remaining riboswitch candidate, the widespread Genes for the Environment, for Membranes and for Motility (GEMM) motif, is associated with genes important for natural competence in Vibrio cholerae and the use of metal ions as electron acceptors in Geobacter sulfurreducens. Among the other motifs, one has a genetic distribution similar to a previously published candidate riboswitch, ykkC/yxkD, but has a different structure. We identified possible non-coding RNAs in five phyla, and several additional cis-regulatory RNAs, including one in epsilon-proteobacteria (upstream of purD, involved in purine biosynthesis), and one in Cyanobacteria (within an ATP synthase operon). These candidate RNAs add to the growing list of RNA motifs involved in multiple cellular processes, and suggest that many additional RNAs remain to be discovered.

Figure 1

Consensus sequences and structures are depicted for seven of the 22 motifs identified. Other motifs are presented as Supplementary Data, as are the alignments on which these diagrams are based. Calculations for conservation of nucleotide identity/presence and evidence of covariation are described in the ‘Materials and methods’ section. Proposed base pairs with more than 5% non-canonical or missing nucleotides are not classified as covarying. Note that the levels of nucleotide conservation are affected both by biochemical constraints on the motif and by phylogenetic diversity; motifs with limited range (e.g. the COG4708 motif) will appear more conserved. Some covarying positions in variable-length stems are not shown.

Figure 2.

Common features of GEMM motifs. Two GEMM instances were selected to illustrate common features, although these two examples do not represent the full 322 GEMMs (see Supplementary Data). (A) This putative RNA contains a canonical GNRA tetraloop and receptor (gray regions). Almost 50% of GEMM instances contain a likely tetraloop receptor. Only the first gene in the downstream operon is shown. (B) Some GEMM RNAs lack the tetraloop receptor, but there are two extra bulged A residues (gray shading) that are found in roughly half of the sequences lacking a receptor. Gray overlined nucleotides can fold to form a stem of a rho-independent transcription terminator (followed by 3′-trailing Us). This terminator appears to compete with the 3′ part of the P2 stem (right-most hairpin). 78 of 322 GEMM instances have predicted transcription terminators overlapping P2.