A Search for Ribonucleic Antiterminator Sites in Bacterial Genomes: Not Only Antitermination

Abstract

BglG/LicT-like proteins are transcriptional antiterminators that prevent termination of transcription at intrinsic terminators by binding to ribonucleic antiterminator (RAT) sites and stabilizing an RNA conformation which is mutually exclusive with the terminator structure. The known RAT sites, which are located in intergenic regions of sugar utilization operons, show low sequence conservation but significant structural analogy. To assess the prevalence of RATs in bacterial genomes, we employed bioinformatic tools that describe RNA motifs based on both sequence and structural constraints. Using descriptors with different stringency, we searched the genomes of Escherichiacoli K12, uropathogenic E. coli and Bacillus subtilis for putative RATs. Our search identified all known RATs and additional putative RAT elements. Surprisingly, most putative RATs do not overlap an intrinsic terminator and many reside within open reading frames (ORFs). The ability of one of the putative RATs, which is located within an antiterminator-encoding ORF and does not overlap a terminator, to bind to its cognate antiterminator protein in vitro and in vivo was confirmed experimentally. Our results suggest that the capacity of RAT elements has been exploited during evolution to mediate activities other than antitermination, for example control of transcription elongation or of RNA stability.

Competitive folding of RNA structures at a termination–antitermination site

“…Although transcription antitermination by BglG/SacY-like proteins was first described over 30 yr ago (Steinmetz and Aymerich 1986;Mahadevan and Wright 1987) and has been the subject of intensive studies since then, there are still many aspects of the molecular mechanism by which these proteins perform their regulatory task that remain elusive in the absence of experimental evidence. Although noncanonical RAT sequences that are not necessarily associated with terminators and whose function remains unclear have been identified (Tortosa and Le Coq 1995;Daguer et al 2004;Gordon et al 2015), the general mechanism of regulation mediated by BglG/SacY proteins relies on the competition between mutually exclusive RNA terminatorantiterminator structures. The development of single-molecule fluorescence-based methods have recently opened the way to new fields of investigation, in particular regarding critical aspects of the structure and dynamics of the short RNA hairpins targeted by these antitermination proteins, on which little is still known.…”