Genetic identification of the functional surface for RNA binding by Escherichia coli ProQ

Abstract

The FinO-domain-protein ProQ is an RNA-binding protein that has been known to play a role in osmoregulation in proteobacteria. Recently, ProQ has been shown to act as a global RNA-binding protein in Salmonella and E. coli, binding to dozens of small RNAs (sRNAs) and messenger RNAs (mRNAs) to regulate mRNA-expression levels through interactions with both 5' and 3' untranslated regions (UTRs). Despite excitement around ProQ as a novel global RNA-binding protein interacting with many sRNAs and mRNAs, and its potential to serve as a matchmaking RNA chaperone, significant gaps remain in our understanding of the molecular mechanisms ProQ uses to interact with RNA. In order to apply the tools of molecular genetics to this question, we have adapted a bacterial three-hybrid (B3H) assay to detect ProQ's interactions with target RNAs.Using domain truncations, site-directed mutagenesis and an unbiased forward genetic screen, we have identified a group of highly conserved residues on ProQ's NTD as the primary face for in vivo recognition of two RNAs, and propose that the NTD structure serves as an electrostatic scaffold to recognize the shape of an A-form RNA duplex. INTRODUCTION Regulatory, small RNAs (sRNAs) are found in nearly all bacterial species and implicated in important processes such as virulence, biofilm formation, host interactions and antibiotic resistance.(1-3) These sRNAs typically regulate messenger RNA (mRNA) translation through imperfect base pairing near an mRNA's ribosomal binding site.(2, 4-6) In many bacterial species, the stability and function of sRNAs are supported by global RNA-binding proteins, such as the protein Hfq.(1, 4, 7-9) Given that Hfq is not present in all bacterial species and that not all sRNAs depend on Hfq for their function, there is increasing interest in other RNA-binding proteins that may play a role in global gene-regulation in bacteria,(2, 10-13) including a class of proteins that contain FinO domains.(14-17) The Escherichia coli protein FinO is the founding member of the FinO structural class of RNA-binding proteins. In E. coli, FinO binds the FinP sRNA and regulates the 5´ untranslated region (UTR) of traJ.(18, 19) Similarly, Legionella pneumophila RocC contains a FinO-domain and binds the sRNA RocR along with at least four 5' UTRs of mRNAs involved in competence.(20) In E. coli, another FinO-domain-containing protein called ProQ was initially characterized as an RNA-binding protein contributing to osmoregulation through expression of proP.(21) ProQ was recently identified through Grad-Seq experiments to bind to dozens of cellular RNAs,(17) including a large number of sRNAs and mRNA 3'UTRs in Samonella and E. coli.(22) ProQ binding has been shown to regulate mRNA-expression levels through interactions with both 5' and 3' UTRs. It has been shown to form a ternary complex with an sRNA (RaiZ) and an mRNA (hupA), to support RaiZ's repression of hupA,(23) and to protect mRNAs from exonucleolytic degradation by binding to 3' ends.(22) Further, ProQ supports the sRNA SraL in preventin...

Determinants of RNA recognition by the FinO domain of theEscherichia coliProQ protein

“…The HDX study suggested that the sRNA SraB contacts the linker and CTD of E. coli ProQ more extensively than the cspE 3ʹ-UTR (19). In agreement with this observation, the linker and the CTD regions of ProQ contributed to the binding of SibB sRNA, but not the cspE 3ʹ-UTR, when measured by the three-hybrid assay (30).…”

“…Given that previous studies showed contributions of ProQ's FinO domain to RNA binding (19,29,30), the binding of this isolated domain to the six RNAs was also examined ( Fig. 3, Table 1, Suppl.…”

“…An important question is whether these adenosines play a similar role in controlling Hfq-and ProQ-interactions in vivo. To investigate this, we utilized a bacterial three-hybrid (B3H) system for RNA-protein interactions that has been demonstrated to detect both Hfq-and ProQ-RNA interactions in vivo (30,51). In this assay, expression of three hybrid components make reporter-gene (lacZ) transcription from a test promoter dependent on a productive interaction between an RNA 'bait' and a protein 'prey' (Fig.…”

“…We began our B3H analysis with a fragment of cspE that has been previously used in ProQ B3H assays, and with Δhfq reporter cells, which have yielded optimal signal-to-noise for B3H interactions with both ProQ and Hfq (30,51). Patches of Δhfq reporter cells grown on X-gal-indicator medium appeared much more blue when all three hybrid components were present than in each of the three negative-control conditions when half of each component was absent (-Adapter, -Prey or -Bait; Fig.…”

“…In agreement with that suggestion mapping of the ProQ surfaces affected by the binding of two natural ProQ ligands, the 3ʹ-UTR of cspE mRNA and the sRNA SraB, using hydrogen-deuterium exchange (HDX) experiments, showed the strongest protection by each RNA on partly overlapping surfaces of the FinO domain (19). In further support, a study using a bacterial three-hybrid assay showed that mutations in the FinO domain of ProQ were detrimental for the binding of the cspE 3ʹ-UTR and SibB sRNA in E. coli cells, and that this domain alone was sufficient for the binding of each RNA (30).…”