In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrC mRNA

Abstract

Small regulatory RNAs (sRNAs) from bacterial chromosomes became the focus of research over the past five years. However, relatively little is known in terms of structural requirements, kinetics of interaction with their targets and degradation in contrast to well-studied plasmid-encoded antisense RNAs. Here, we present a detailed in vitro analysis of SR1, a sRNA of Bacillus subtilis that is involved in regulation of arginine catabolism by basepairing with its target, ahrC mRNA. The secondary structures of SR1 species of different lengths and of the SR1/ahrC RNA complex were determined and functional segments required for complex formation narrowed down. The initial contact between SR1 and its target was shown to involve the 5′ part of the SR1 terminator stem and a region 100 bp downstream from the ahrC transcriptional start site. Toeprinting studies and secondary structure probing of the ahrC/SR1 complex indicated that SR1 inhibits translation initiation by inducing structural changes downstream from the ahrC RBS. Furthermore, it was demonstrated that Hfq, which binds both SR1 and ahrC RNA was not required to promote ahrC/SR1 complex formation but to enable the translation of ahrC mRNA. The intracellular concentrations of SR1 were calculated under different growth conditions.

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…131) This protein also represses sr1, 132) which encodes a small non-coding regulatory RNA that inhibits the translation of ahrC encoding a transcriptional regulator that activates the rocABC and rocDEF operons for arginine catabolism and represses the gene cluster for arginine biosynthesis. 108,[133][134][135] CcpN is active when cells are growing on a glycolytic substrate, even if the medium also contains a gluconeogenic substrate. 127) CcpN is inhibited on interaction with YqfL, which is active during gluconeogenesis, and pckA and gapB as well as sr1 are derepressed.…”

Clostridium difficile Hfq can replace Escherichia coli Hfq for most of its function

“…An interesting correlation has been emphasized between the number of positively charged amino acids (arginines or lysines) at the rim and the function of Hfq from Gram-positive bacteria in sRNA regulation (Panja et al 2013). Indeed, among the Hfq proteins studied in Gram-positive bacteria, L. monocytogenes Hfq carries the RKEK rim motif and stabilizes at least one sRNA-mRNA complex (Nielsen et al 2010(Nielsen et al , 2011, while B. subtilis Hfq having a RKEN motif associated with numerous RNAs was not required for sRNA regulation (Heidrich et al 2007;Gaballa et al 2008;Dambach et al 2013). The function of S. aureus Hfq carrying the KANQ rim motif remains unclear (Bohn et al 2007).…”

The role of mRNA structure in translational control in bacteria

“…The first class is the antisense RNAs that are transcribed in the strand opposite of the While many repressor RNAs mask directly the SD or the AUG initiation codon to sterically block the access of the 30S ribosomal subunit, recent studies also indicate that repressor RNAs regulate initiation of translation through interactions with regions of the target mRNAs located far upstream or downstream of the SD sequences. 48,49 In B. subtilis, SR1 RNA binds to the coding region 100 bp downstream the start site of ahrC mRNA. This binding induces a large conformational change of the mRNA that prevents the access of the 30S subunit to form the initiation complex.…”

“…This binding induces a large conformational change of the mRNA that prevents the access of the 30S subunit to form the initiation complex. 48 E. coli IstR-1 RNA represses the synthesis of tisAB mRNA encoding for an SOS-induced toxin. The repressor of toxin binds to an unpaired region 100 nt upstream of the tisAB ribosome binding site and prevents the binding of a stand-by ribosome.…”