Characterization of the interaction between the small RNA-encoded peptide SR1P and GapA from Bacillus subtilis

Abstract

Small regulatory RNAs (sRNAs) are the most prominent post-transcriptional regulators in all kingdoms of life. A few of them, e.g. SR1 from Bacillus subtilis, are dual-function sRNAs. SR1 acts as a base-pairing sRNA in arginine catabolism and as an mRNA encoding the small peptide SR1P in RNA degradation. Both functions of SR1 are highly conserved among 23 species of Bacillales. Here, we investigate the interaction between SR1P and GapA by a combination of in vivo and in vitro methods. De novo prediction of the structure of SR1P yielded five models, one of which was consistent with experimental circular dichroism spectroscopy data of a purified, synthetic peptide. Based on this model structure and a comparison between the 23 SR1P homologues, a series of SR1P mutants was constructed and analysed by Northern blotting and co-elution experiments. The known crystal structure of Geobacillus stearothermophilus GapA was used to model SR1P onto this structure. The hypothetical SR1P binding pocket, composed of two α-helices at both termini of GapA, was investigated by constructing and assaying a number of GapA mutants in the presence and absence of wild-type or mutated SR1P. Almost all residues of SR1P located in the two highly conserved motifs are implicated in the interaction with GapA. A critical lysine residue (K332) in the C-terminal α-helix 14 of GapA corroborated the predicted binding pocket.

A new role for CsrA: promotion of complex formation between an sRNA and its mRNA target in Bacillus subtilis

“…It base-pairs with ahrC mRNA encoding the transcriptional activator of the arginine catabolic operons rocABC and rocDEF and inhibits its translation [6][7][8]. In addition, SR1 codes for the small peptide SR1P that interacts with GapA [9][10][11]. Both functions of SR1 are remarkably conserved over one billion years of evolution [12].…”

Moonlighting in Bacillus subtilis: The Small Proteins SR1P and SR7P Regulate the Moonlighting Activity of Glyceraldehyde 3-Phosphate Dehydrogenase A (GapA) and Enolase in RNA Degradation

“…To gain an insight into the GapA–SR1P interaction surface, we predicted the SR1P structure and confirmed it by CD measurements with chemically synthesized SR1P [ 54 ]. Using the published 3D structure of B. stearothermophilus GapA we searched and found an SR1P binding pocket: it comprises both the N-terminal helix 1 and the C-terminal helix 14 of GapA and contains three adjacent lysine residues, of which Lys 332 was decisive for SR1P binding [ 54 ].…”

Small proteins in Gram-positive bacteria

“…To elucidate the SR1P–GapA interaction, comprehensive analyses were undertaken, employing peptide mutants in coelution experiments, complemented by functional assessments through northern blotting (Gimpel et al 2017 ). These investigations successfully unveiled the molecular interface between SR1P and GapA.…”