Challenges of ligand identification for riboswitch candidates

Abstract

Expanding DNA sequence databases and improving methods for comparative analysis are being exploited to identify numerous noncoding RNA elements including riboswitches. Ligands for many riboswitch classes usually can be inferred based on the genomic contexts of representative RNAs, and complex formation or genetic regulation subsequently demonstrated experimentally. However, there are several candidate riboswitches for which ligands have not been identified. In this report, we discuss three of the most compelling riboswitch candidates: the ykkC/ykkD, yybP/ykoY and pfl RNAs. Each of these RNAs is numerous, phylogenetically widespread, and carries features that are hallmarks of metabolite-binding riboswitches, such as a well-conserved aptamer-like structure and apparent interactions with gene regulation elements such as ribosome binding sites or intrinsic transcription termination stems. These RNAs likely represent only a small sampling of the challenging motifs that researchers will encounter as new noncoding RNAs are identified.

Figure 1

Consensus sequences and structure models for widespread candidate riboswitches. (A) ykkC/yxkD RNA. (B) yybP/ykoY RNA. (C) pfl RNA. Diagrams were constructed using data from representatives identified in RefSeq38, and metagenomic data sets as describes in references and . Y = U or C, R = G or A.

Figure 2

Sequence, secondary structure and activity of a ykkC RNA representative. (A) Bacillus subtilis ykkC RNA construct used for reporter assays with integration vector pDG1661. The reporter plasmid contains a ribosome binding site and start codon that follow ∼50 bp after the inserted riboswitch candidate. Mutants M1 and M3 were designed to disrupt base-pairing in P1 and M2 disrupts a highly-conserved region of the RNA. M4 deletes the right shoulder of the putative expression platform terminator stem. (B) β-galactosidase expression levels measured in Miller units using methods described previously in reference , for wild-type and the various mutants described in (A) under minimal and rich media (M4 only) as indicated. When present, error bars represent standard deviation among three independent replicates.

Figure 3

Sequence, secondary structure and activity of a pfl RNA representative. (A) C. acetobutylicum pfl RNA construct used in reporter assays with pDG1661 in B. subtilis. Mutants M1, M3, and M5 disrupt P1, pseudoknot and P2 formation, respectively. M4 and M6 restore the disruptions caused by M3 and M5, respectively. M2 disrupts formation of the transcription terminator stem. (B) β-galactosidase expression measured in Miller units as described previously in reference , for wild-type and mutant constructs under both minimal and rich media conditions. Error bars represent standard deviation among three independent replicates.