Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of Escherichia coli ProQ

A positive charge at conserved position 80 is not sufficient for RNA interaction in vivo or in vitro. (A) Design of B3H system to detect the interaction between ProQ and an RNA (malM 3′UTR). Interaction between the protein moiety and RNA moiety fused, respectively, to the NTD of the alpha subunit of RNA polymerase (RNAP) (α) and to one copy of the MS2 RNA hairpin (MS2^hp) activates transcription from test promoter, which directs transcription of a lacZ reporter gene (Berry and Hochschild 2018). Compatible plasmids direct the synthesis of the α-fusion protein (under the control of an IPTG-inducible promoter), the CI-MS2^CP adapter protein (under the control of a constitutive promoter; pCW17) and the hybrid RNA (under the control of an arabinose-inducible promoter). (B) Results of β-galactosidase (β-gal) assays performed in Δhfq plac-O_L2–62 reporter strain cells containing three compatible plasmids: one (α-ProQ) that encoded α (−) or the α-ProQ^ΔCTD (pKB955; residues 2–176) fusion protein (WT or an R80A or R80K mutant), another (CI-MS2^CP) that encoded λCI (−) or the λCI-MS2^CP fusion protein (+), and a third (Bait) that encoded a hybrid RNA with the 3′-UTR of malM (pKB1210) following one copy of an MS2^hp moiety (+) or an RNA that contained only the MS2^hp moiety (−). Cells were grown in the presence of 0.2% arabinose and 50 µM IPTG (see Materials and Methods). Bar graphs show the averages of three independent measurements and standard deviations. (C) (Left) Results of B3H assays detecting interactions between α-ProQ^ΔCTD and three RNA baits. β-gal assays were performed as in (B) but with three-hybrid RNA constructs (MS2^hp-malM-3′, MS2^hp-cspE-3′, MS2^hp-SibB). The bar graph shows the fold-stimulation over basal levels as averages and standard deviations of values collected from three independent experiments conducted in triplicate across multiple days. (Right) Western blot to compare steady-state expression levels of mutant α-ProQ^ΔCTD fusion proteins. Lysates were taken from the corresponding β-gal experiment containing MS2^hp-malM-3′ and all other hybrid components at 50 µM IPTG. Following electrophoresis and transfer, membranes were probed with anti-ProQ antibody. See Supplemental Figure S3 for loading controls and quantification of results.