Abstract
<jats:p>Intrinsically disordered regions are widespread in RNA-processing machines. In Escherichia coli, RNase E uses its intrinsically disordered C-terminal domain (CTD) to recruit RNAs to the N-terminal catalytic domain, including mRNAs targeted by regulatory small RNAs (sRNAs), but the basis of substrate recognition and specificity is unclear. We engineered a protease-cleavable RNase E and used split-CRAC to isolate RNAs crosslinked to the AR2 sub-domain of the intrinsically disordered CTD fragment. AR2 preferentially engaged mRNAs and was depleted of sRNAs and sRNA-containing hybrids, supporting recognition of the mRNA. AR2 contacts concentrated on accessible A-rich motifs surrounding ribosome-binding sites and start codons, and purified AR2 recognised this motif in vitro. AR2 also contacted an AUAA motif in the rne translation-initiation region, and AR2 deletion increased RNase E abundance implicating this interaction in autoregulation. These findings define a relatively short AR2-binding motif and are consistent with CTD interactions with the 30S subunit that may provide additional specificity for a subset of mRNA translation initiation regions.</jats:p>