Revealing Hidden Biology by Identifying Ligands for the ykkC Orphan Riboswitche

Riboswitches are non-coding RNAs that bind a small molecule or ion ligand to regulate gene expression. The cognate ligands have been identified for dozens of riboswitch classes, but the ligand identities are unknown for many promising structured RNA motifs, which are known as orphan riboswitches. The ykkC RNA motif was discovered over a decade ago by using bioinformatics and, until recently, was the longest standing orphan riboswitch candidate. The cognate ligand of ykkC riboswitches remained unsolved mostly due to the seemingly disparate set of genes under its regulatory control. These genes commonly code for proteins annotated as urea carboxylases, multi-drug efflux transporters, sulfonate/nitrate/carbonate transporters, arginases, de novo purine biosynthesis enzymes, and branched-chain amino acid enzymes.;My colleagues found that guanidine induces expression of a riboswitch- lacZ reporter gene fusion in a large screen of various growth conditions. They collected additional evidence implicating guanidine as the elusive ligand for ykkC riboswitches by demonstrating that reporter gene expression is activated at a lower concentration of guanidine when the ykkCD operon is knocked out in B. subtilis. These gene products, which likely function as a selective guanidine transporter, are commonly associated with this riboswitch class. I used in-line probing as an in vitro biochemical approach to further evaluate guanidine as the cognate ligand and found that some ykkC motif RNAs from distantly related bacteria become more structured at specific sites in a dose-dependent manner upon the addition of guanidine.;I subsequently determined that representatives of two other RNA motifs, called mini-ykkC and ykkC-III, also bind guanidine in vitro. All three guanidine-binding RNA motifs have similar downstream gene associations, and therefore these three distinct classes of RNA represent the guanidine-I, -II and -III riboswitch classes. I performed experiments on the protein family whose expression is most commonly regulated by guanidine riboswitches and demonstrated that these proteins are involved in breaking down guanidine into non-toxic molecules. Although the biological relevance of guanidine is still poorly understood, the wide distribution of these riboswitches and the genes they control suggest that free guanidine is a greatly underappreciated metabolite.;While the majority of ykkC RNAs selectively bind guanidine, I found that the remaining 30% of ykkC RNAs do not bind guanidine and carry nucleotide changes in their binding pockets to allow recognition of a different ligand. I analyzed the consensus sequence and structure models as well as the downstream gene associations for these variant ykkC RNAs, which revealed at least four distinct candidate riboswitch classes in addition to the guanidine-I riboswitch class. I have validated the cognate ligands for two of these variant ykkC riboswitch classes, both of which are RNA nucleotide derivatives.;I discovered that the first variant class selectively binds guanosine tetraphosphate (ppGpp), a bacterial alarmone derived from the ribonucleotide GTP. The ppGpp riboswitch class primarily regulates transcription of genes involved in branched-chain amino acid biosynthesis. Shortly thereafter, I determined that the second of these variant ykkC riboswitch classes binds a similar molecule called phosphoribosyl pyrophosphate (PRPP), the initial substrate for the biosynthesis of RNA monomers, to regulate the expression of genes for purine nucleotide biosynthesis. Members of these newly validated riboswitch classes frequently form tandem arrangements between and other RNA-based regulatory elements. I demonstrated that these tandem arrangements accomplish more complex gene control systems than a typical singlet riboswitch system. The existence of sophisticated RNA regulatory systems that respond to a widespread RNA-derived signaling molecule and a fundamental RNA biosynthetic precursor supports the hypothesis that RNA World organisms could manage a complex metabolic state without the assistance of protein factors.