Variant Bacterial Riboswitches Associated with Nucleotide Hydrolase Genes Sense Nucleoside Diphosphates

Abstract

The ykkC RNA motif was a long-standing orphan riboswitch candidate that has recently been proposed to encompass at least five distinct bacterial riboswitch classes. Most ykkC RNAs belong to the subtype 1 group, which are guanidine-I riboswitches that regulate the expression of guanidine-specific carboxylase and transporter proteins. The remaining ykkC RNAs have been organized into at least four major categories called subtypes 2a-2d. Subtype 2a RNAs are riboswitches that sense the bacterial alarmone ppGpp and typically regulate amino acid biosynthesis genes. Subtype 2b riboswitches sense the purine biosynthetic intermediate PRPP and frequently partner with guanine riboswitches to regulate purine biosynthesis genes. In this study, we examined ykkC subtype 2c RNAs, which are found upstream of genes encoding hydrolase enzymes that cleave the phosphoanhydride linkages of nucleotide substrates. Subtype 2c representatives mostly recognize adenosine and cytidine 5'-diphosphate molecules in either their ribose or deoxyribose forms (ADP, dADP, CDP, and dCDP). Other nucleotide-containing compounds, especially nucleoside 5'-triphosphates, are strongly rejected by some members of this putative riboswitch class. High ligand concentrations in vivo are predicted to turn on expression of hydrolase enzymes, which presumably function to balance cellular nucleotide pools. These results further showcase the striking functional diversity derived from the structural scaffold shared among all ykkC motif RNAs, which has been adapted to sense at least five different types of natural ligands. Moreover, riboswitches for nucleoside diphosphates provide additional examples of the numerous partnerships observed between natural RNA aptamers and nucleotide-derived ligands, including metabolites, coenzymes, and signaling molecules.

Former orphan riboswitches reveal unexplored areas of bacterial metabolism, signaling, and gene control processes

“…For example, numerous representatives of eight novel RNA motifs were identified by comparing all intergenic regions from Bacillus subtilis with those from 90 additional bacterial species ). Seven of these eight motifs have since proven to represent distinct riboswitch classes (McCown et al 2017;Sherlock et al 2018a, Sherlock et al 2018b, Sherlock et al 2019).…”

“…Each of these riboswitch candidate classes, termed subtypes 2a through 2d, has distinct differences in the nucleotide positions corresponding to the binding pocket of guanidine-I aptamers that were predicted to alter ligand specificity. At this time, the ligands for subtypes 2a (Sherlock et al 2018a), 2b (Sherlock et al 2018b), and 2c (Sherlock et al 2019) have been established as noted below. The ligand for 2d has yet to be experimentally determined.…”

“…Subtype 2c ykkC RNAs regulate the expression of genes encoding NUDIX and HAD-like hydrolases in response to nucleoside diphosphates (Sherlock et al 2019). Representatives of this riboswitch class selectively recognizes adenosine and cytidine diphosphate in both the ribose and Cold Spring Harbor Laboratory Press on July 31, 2020 -Published by rnajournal.cshlp.org Downloaded from deoxyribose forms.…”

“…EamA (originally called phn_DUF6) and TauE (also known as SafE) Sherlock et al 2019) in response to a ligand that remains undetermined at this time. Thus, at least one more orphan riboswitch class is present among the original ykkC motif RNAs.…”

“…For example, the initial consensus models for c-di-GMP (Weinberg et al 2007), SAM-VI (Mirihana Arachchilage et al 2018), and ykkC subtype 2 riboswitches were missing important structural features. In these cases, either the RNA constructs tested by in-line probing were fortuitously long enough to encompass the entire aptamer (Sudarsan et al 2008;Mirihana Arachchilage et al 2018), or additional conservation was found by comparative sequence analysis at a later time and the RNA constructs used for evaluation were adjusted accordingly (Sherlock et al 2018a(Sherlock et al , 2018b(Sherlock et al , 2019.…”