A second riboswitch class for the enzyme cofactor NAD⁺

Abstract

A bacterial noncoding RNA motif almost exclusively associated with pnuC genes was uncovered using comparative sequence analysis. Some PnuC proteins are known to transport nicotinamide riboside (NR), which is a component of the ubiquitous and abundant enzyme cofactor nicotinamide adenine dinucleotide (NAD +). Thus, we speculated that the newly found 'pnuC motif' RNAs might function as aptamers for a novel class of NAD +sensing riboswitches. RNA constructs that encompass the conserved nucleotides and secondary structure features that define the motif indeed selectively bind NAD + , nicotinamide mononucleotide (NMN), and NR. Mutations that disrupt strictly conserved nucleotides of the aptamer also disrupt ligand binding. These bioinformatic and biochemical findings indicate that pnuC motif RNAs are likely members of a second riboswitch class that regulates gene expression in response to NAD + binding.

Probing and Perturbing Riboswitch Folding Using a Fluorescent Base Analogue

“…We identified key interactions, such as the Hoogsteen base-pair between A31 and U8 and tertiary interactions between A28 and P1, that are critical for ligand recognition, observations that are supported by the high level of sequence conservation in L3 (Roth et al 2007). Other riboswitches that adopt pseudoknot structures, particularly ones that contain A-rich domains like L3 (Panchapakesan et al 2021), are likely to take advantage of similar interactions.…”

Comprehensive discovery of novel structured noncoding RNAs in 26 bacterial genomes

“…Additional bioinformatic and biochemical analyses [ 23 ] identified a central core of the motif that is required for its function. These efforts trimmed the collection of non-redundant representatives to 43, which were used to create a consensus sequence and structural model ( Fig.…”

“…These characteristics solidified our preliminary classification of the pnuC motif as a strong riboswitch candidate. Indeed, binding assays confirmed that pnuC motif RNAs selectively bind various nicotinamide derivatives, including NR and NAD + [ 23 ]. Thus, each pnuC motif RNA serves as the aptamer for a second riboswitch class for this coenzyme, which we have named the NAD + -II riboswitch as published elsewhere [ 23 ].…”

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Figure 1. Consensus sequence and structural models for the (A) pnuC (NAD+-II riboswitch) [ 23 ], (B) icd -II, (C) carA , (D) gltD , (E) ldh2 and (F) proX motif RNAs, which are among the collection of strong riboswitch candidates identified in this study. Annotations are as defined in the key depicted in A

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“…A focus of our research team in recent years has been the discovery of riboswitches that sense specific metabolites or inorganic ions to regulate the expression of genes whose protein products synthesize, transport, utilize, or respond to the ligands being monitored. Over 50 distinct classes of riboswitches have been experimentally validated to date [11][12][13][14][15][16][17][18][19][20][21][22][23], and many of these sense nucleotide-derived compounds proposed to have been present during an era of biology before the evolutionary emergence of proteins [24][25][26][27][28]. Thus, uncovering additional classes of riboswitches promises to reveal more about how modern cells regulate critical biochemical processes, and perhaps also to provide insight into the functions of ancient RNA-based regulatory systems [29,30].…”