Small RNAs and Hfq capture unfolded RNA target sites during transcription

Small ribonucleoproteins (sRNPs) target nascent precursor RNAs to guide folding, modification, and splicing during transcription. Yet, rapid co-transcriptional folding of the RNA can mask sRNP sites, impeding target recognition and regulation. To examine how sRNPs target nascent RNAs, we monitored binding of bacterial Hfq center dot DsrA sRNPs to rpoS transcripts using single-molecule co-localization co-transcriptional assembly (smCoCoA). We show that Hfq center dot DsrA recursively samples the mRNA before transcription of the target site to poise it for base pairing with DsrA. We adapted smCoCoA to precisely measure when the target site is syn-thesized and revealed that Hfq center dot DsrA often binds the mRNA during target site synthesis close to RNA poly-merase (RNAP). We suggest that targeting transcripts near RNAP allows an sRNP to capture a site before the transcript folds, providing a kinetic advantage over post-transcriptional targeting. We propose that other sRNPs may also use RNAP-proximal targeting to hasten recognition and regulation.

Automated summary

sRNPs play key roles in guiding the folding, modification, and splicing of nascent precursor RNAs during transcription. However, the rapid co-transcriptional folding of RNA can hinder the recognition and regulation of sRNP binding sites. In this study, the binding of Hfq•DsrA sRNPs to rpoS transcripts was examined using the single-molecule co-localization co-transcriptional assembly (smCoCoA) technique. It was found that Hfq•DsrA samples the mRNA before the transcription of the target site, preparing it for base pairing with DsrA. Furthermore, it was observed that Hfq•DsrA often binds to the mRNA close to RNA polymerase (RNAP) during the synthesis of the target site. This suggests that targeting transcripts near RNAP allows sRNPs to capture the site before the RNA folds, providing a kinetic advantage in recognition and regulation.