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PreprintCited by 9Bacterial Genetics and BiotechnologyRNA and protein synthesis mechanismsRNA Research and Splicing

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

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Stein, Ewa M., Wang, Suxuan, Dailey, Katherine, Gravel, Chandra M, Wang, Shiying, Olejniczak, Mikołaj, Berry, Katherine E·2023·RNA
DOI 10.1101/2023.04.25.538249PMC PMC10578477

Preprint — published as 10.1261/rna.079697.123 · Paper page

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Abstract

RNA-binding proteins play important roles in bacterial gene regulation through interactions with both coding and non-coding RNAs. ProQ is a FinO-domain protein that binds a large set of RNAs in Escherichia coli , though the details of how ProQ binds these RNAs remain unclear. In this study, we used a combination of in vivo and in vitro binding assays to confirm key structural features of E. coli ProQ’s FinO domain and explore its mechanism of RNA interactions. Using a bacterial three-hybrid assay, we performed forward genetic screens to confirm the importance of the concave face of ProQ in RNA binding. Using gel shift assays, we directly probed the contributions of ten amino acids on ProQ binding to seven RNA targets. Certain residues (R58, Y70, and R80) were found to be essential for binding of all seven RNAs, while substitutions of other residues (K54 and R62) caused more moderate binding defects. Interestingly, substitutions of two amino acids (K35, R69), which are evolutionarily variable but adjacent to conserved residues, showed varied effects on the binding of different RNAs; these may arise from the differing sequence context around each RNA’s terminator hairpin. Together, this work confirms many of the essential RNA-binding residues in ProQ initially identified in vivo and supports a model in which residues on the conserved concave face of the FinO domain such as R58, Y70 and R80 form the main RNA-binding site of E. coli ProQ, while additional contacts contribute to the binding of certain RNAs.

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FIGURE 1. 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 (MS2hp) 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-MS2CP 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-OL2–62 reporter strain cells containing three compatible plasmids: one (α-ProQ) that encoded α (−) or the α-ProQΔCTD (pKB955; residues 2–176) fusion protein (WT or an R80A or R80K mutant), another (CI-MS2CP) that encoded λCI (−) or the λCI-MS2CP fusion protein (+), and a third (Bait) that encoded a hybrid RNA with the 3′-UTR of malM (pKB1210) following one copy of an MS2hp moiety (+) or an RNA that contained only the MS2hp 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-ProQΔCTD and three RNA baits. β-gal assays were performed as in (B) but with three-hybrid RNA constructs (MS2hp-malM-3′, MS2hp-cspE-3′, MS2hp-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-ProQΔCTD fusion proteins. Lysates were taken from the corresponding β-gal experiment containing MS2hp-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.
FIGURE 1. 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 (MS2hp) 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-MS2CP 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-OL2–62 reporter strain cells containing three compatible plasmids: one (α-ProQ) that encoded α (−) or the α-ProQΔCTD (pKB955; residues 2–176) fusion protein (WT or an R80A or R80K mutant), another (CI-MS2CP) that encoded λCI (−) or the λCI-MS2CP fusion protein (+), and a third (Bait) that encoded a hybrid RNA with the 3′-UTR of malM (pKB1210) following one copy of an MS2hp moiety (+) or an RNA that contained only the MS2hp 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-ProQΔCTD and three RNA baits. β-gal assays were performed as in (B) but with three-hybrid RNA constructs (MS2hp-malM-3′, MS2hp-cspE-3′, MS2hp-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-ProQΔCTD fusion proteins. Lysates were taken from the corresponding β-gal experiment containing MS2hp-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.CC BY · PubMed Central
FIGURE 2. Saturation mutagenesis screen to identify compensatory mutants that rescue RNA-binding activity of ProQR80K. (A) Residues selected for saturation mutagenesis are highlighted in yellow, green, and cyan on the NMR structure of ProQNTD (Gonzalez et al. 2017). Arginine 80 is shown in orange sticks in the convex-face position found in the NMR structure and in pink sticks in a hypothetical concave-face position analogous to its position in other orthologs. (B) Compensatory mutations found in pPrey-ProQR80K plasmids that produced blue colonies on X-gal-indicator plates with pBait-malM-3′ are listed. Codons identified in multiple colonies are indicated by 2× and 3×. (C) Results confirming the effects of compensatory mutants in a plate-based B3H assay, detecting interactions between variants of ProQΔCTD with malM-3′ RNA. β-gal assays were performed with Δhfq reporter strain cells containing three compatible plasmids: one that encoded the CI-MS2CP fusion protein, another that encoded α or an α-ProQΔCTD fusion protein (wild-type, WT, or the indicated mutant), and a third that encoded a hybrid RNA (MS2hp-malM-3′) (see Materials and Methods). Quantification of color intensity shown in Supplemental Figure S4B. (D,E) The two strongest compensatory substitutions (V74K and L91V) are shown on (D) the NMR structure and (E) the AlphaFold Model (Varadi et al. 2022) of ProQNTD. Side chains were mutated in PyMol to the lowest-energy rotamer to show predicted structures of the compensatory substitutions. Distances between the terminal atom of each side chain (R80K, V74K, L71V) are shown in Angstroms and visualized with dashed lines.
FIGURE 2. Saturation mutagenesis screen to identify compensatory mutants that rescue RNA-binding activity of ProQR80K. (A) Residues selected for saturation mutagenesis are highlighted in yellow, green, and cyan on the NMR structure of ProQNTD (Gonzalez et al. 2017). Arginine 80 is shown in orange sticks in the convex-face position found in the NMR structure and in pink sticks in a hypothetical concave-face position analogous to its position in other orthologs. (B) Compensatory mutations found in pPrey-ProQR80K plasmids that produced blue colonies on X-gal-indicator plates with pBait-malM-3′ are listed. Codons identified in multiple colonies are indicated by 2× and 3×. (C) Results confirming the effects of compensatory mutants in a plate-based B3H assay, detecting interactions between variants of ProQΔCTD with malM-3′ RNA. β-gal assays were performed with Δhfq reporter strain cells containing three compatible plasmids: one that encoded the CI-MS2CP fusion protein, another that encoded α or an α-ProQΔCTD fusion protein (wild-type, WT, or the indicated mutant), and a third that encoded a hybrid RNA (MS2hp-malM-3′) (see Materials and Methods). Quantification of color intensity shown in Supplemental Figure S4B. (D,E) The two strongest compensatory substitutions (V74K and L91V) are shown on (D) the NMR structure and (E) the AlphaFold Model (Varadi et al. 2022) of ProQNTD. Side chains were mutated in PyMol to the lowest-energy rotamer to show predicted structures of the compensatory substitutions. Distances between the terminal atom of each side chain (R80K, V74K, L71V) are shown in Angstroms and visualized with dashed lines.CC BY · PubMed Central
FIGURE 3. Mutations in the central pocket have strong detrimental effects on the binding of seven RNAs. (A) The location of substitutions on the AlphaFold model of ProQNTD (Varadi et al. 2022). (B) The raw gel data for 1 nM 32P-labeled malM-3′ binding to the eight proteins. (C) The plots of fraction bound versus protein concentration for the binding of cspE-3′, cspE81-3′, gapA-3′, malM-3′, RybB, SibA, and SibB RNAs to WT ProQNTD, and T65A, K54A, R62A, R58A, Y70F, R80A, and R80K mutants measured using the gel shift assay. The data sets in which the maximum fraction bound was above 40% were analyzed by fitting to the quadratic equation. Raw gel data for all RNAs are presented in Supplemental Figures S7–S13. The obtained Kd values are shown in Table 1.
FIGURE 3. Mutations in the central pocket have strong detrimental effects on the binding of seven RNAs. (A) The location of substitutions on the AlphaFold model of ProQNTD (Varadi et al. 2022). (B) The raw gel data for 1 nM 32P-labeled malM-3′ binding to the eight proteins. (C) The plots of fraction bound versus protein concentration for the binding of cspE-3′, cspE81-3′, gapA-3′, malM-3′, RybB, SibA, and SibB RNAs to WT ProQNTD, and T65A, K54A, R62A, R58A, Y70F, R80A, and R80K mutants measured using the gel shift assay. The data sets in which the maximum fraction bound was above 40% were analyzed by fitting to the quadratic equation. Raw gel data for all RNAs are presented in Supplemental Figures S7–S13. The obtained Kd values are shown in Table 1.CC BY · PubMed Central
FIGURE 4. Mutations on the periphery of the central pocket have differential effects on the binding of seven RNAs. (A) The location of substitutions on the AlphaFold model of ProQNTD (Varadi et al. 2022). (B) The raw gel data for 1 nM 32P-labeled malM-3′ binding to the five proteins. (C) The plots of fraction bound versus protein concentration for the binding of cspE-3′, cspE81-3′, gapA-3′, malM-3′, RybB, SibA, and SibB RNAs to WT ProQNTD, and R32A, D41A, K35A, and R69A mutants measured using the gel shift assay. The data sets in which the maximum fraction bound was above 40% were analyzed by fitting to the quadratic equation. Raw gel data for all RNAs are presented in Supplemental Figures S7–S13. The obtained Kd values are shown in Table 1.
FIGURE 4. Mutations on the periphery of the central pocket have differential effects on the binding of seven RNAs. (A) The location of substitutions on the AlphaFold model of ProQNTD (Varadi et al. 2022). (B) The raw gel data for 1 nM 32P-labeled malM-3′ binding to the five proteins. (C) The plots of fraction bound versus protein concentration for the binding of cspE-3′, cspE81-3′, gapA-3′, malM-3′, RybB, SibA, and SibB RNAs to WT ProQNTD, and R32A, D41A, K35A, and R69A mutants measured using the gel shift assay. The data sets in which the maximum fraction bound was above 40% were analyzed by fitting to the quadratic equation. Raw gel data for all RNAs are presented in Supplemental Figures S7–S13. The obtained Kd values are shown in Table 1.CC BY · PubMed Central
FIGURE 5. The sequence and structure of the lower part of the terminator hairpins explain differences in the binding of ProQNTD and its mutants by cspE-3′ and SibB RNAs. (A–D), The secondary structures of RNA molecules are shown above the corresponding plots of fraction-bound data for WT ProQNTD (NTD) or its R69A mutant. The RNA secondary structures were predicted using RNAStructure software (Reuter and Mathews 2010). The changes introduced into the sequence of SibB are marked in red font. The data sets in which the maximum fraction bound was above 40% were analyzed by fitting to the quadratic equation. Data for SibB and cspE-3′ are the same as in Figure 3. The obtained Kd values are shown in Table 2, and raw gel data are presented in Supplemental Figure S15.
FIGURE 5. The sequence and structure of the lower part of the terminator hairpins explain differences in the binding of ProQNTD and its mutants by cspE-3′ and SibB RNAs. (A–D), The secondary structures of RNA molecules are shown above the corresponding plots of fraction-bound data for WT ProQNTD (NTD) or its R69A mutant. The RNA secondary structures were predicted using RNAStructure software (Reuter and Mathews 2010). The changes introduced into the sequence of SibB are marked in red font. The data sets in which the maximum fraction bound was above 40% were analyzed by fitting to the quadratic equation. Data for SibB and cspE-3′ are the same as in Figure 3. The obtained Kd values are shown in Table 2, and raw gel data are presented in Supplemental Figure S15.CC BY · PubMed Central
FIGURE 6. The substitutions of peripheral residues K35 and R69 have indirect effects on the RNA binding in the central pocket of the ProQNTD. The RNA secondary structures were predicted using RNAstructure software (Reuter and Mathews 2010). The data sets in which the maximum fraction bound was above 40% were analyzed by fitting to the quadratic equation. Data for cspE-3′ are the same as in Figures 3 and 4. The average equilibrium dissociation constant (Kd) values are shown in Table 3, and raw gel data are presented in Supplemental Figure S16.
FIGURE 6. The substitutions of peripheral residues K35 and R69 have indirect effects on the RNA binding in the central pocket of the ProQNTD. The RNA secondary structures were predicted using RNAstructure software (Reuter and Mathews 2010). The data sets in which the maximum fraction bound was above 40% were analyzed by fitting to the quadratic equation. Data for cspE-3′ are the same as in Figures 3 and 4. The average equilibrium dissociation constant (Kd) values are shown in Table 3, and raw gel data are presented in Supplemental Figure S16.CC BY · PubMed Central
FIGURE 7. Model for ProQ–RNA interaction. (A) Alignment of AlphaFold model of FinO domain of ProQ (Jumper et al. 2021; Varadi et al. 2022) and Lp RocC/RocR cocrystal structure (PDB ID: 7RGU; Kim et al. 2022). (B) Summary of in vitro biochemical results. Residues which were modified for gel shift experiments are colored based on the size of the detrimental effects on RNA binding when modified: strong (R80, Y70, R58; magenta), moderate (K35, K54, R62; cyan), effects that varied across RNA ligands (K35, R69; green), and those with only weak effects (D41, T65; orange). (C,D) Surface representation of the concave-face binding pocket of ProQ, with residues colored as in (B). The terminal two pyrimidine nucleotides from RocR are shown in stick representation and are colored by atomic identity. The two panels compare (C) ProQ residues identified as important for RNA interactions in vitro in the current work and (D) residues found in previous B3H experiments to contribute to cspE-3′ and SibB binding in vivo (Pandey et al. 2020).
FIGURE 7. Model for ProQ–RNA interaction. (A) Alignment of AlphaFold model of FinO domain of ProQ (Jumper et al. 2021; Varadi et al. 2022) and Lp RocC/RocR cocrystal structure (PDB ID: 7RGU; Kim et al. 2022). (B) Summary of in vitro biochemical results. Residues which were modified for gel shift experiments are colored based on the size of the detrimental effects on RNA binding when modified: strong (R80, Y70, R58; magenta), moderate (K35, K54, R62; cyan), effects that varied across RNA ligands (K35, R69; green), and those with only weak effects (D41, T65; orange). (C,D) Surface representation of the concave-face binding pocket of ProQ, with residues colored as in (B). The terminal two pyrimidine nucleotides from RocR are shown in stick representation and are colored by atomic identity. The two panels compare (C) ProQ residues identified as important for RNA interactions in vitro in the current work and (D) residues found in previous B3H experiments to contribute to cspE-3′ and SibB binding in vivo (Pandey et al. 2020).CC BY · PubMed Central
Ewa Stein
Ewa Stein CC BY · PubMed Central

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Metadata

Authors
Stein, E. M., Wang, S., Dailey, K., Gravel, C. M., Wang, S., Olejniczak, M., & Berry, K. E.
Affiliations
  • Adam Mickiewicz University in Poznań (PL)
  • Mount Holyoke College (US)
Corresponding author
  • Olejniczak, Mikołaj
  • Berry, Katherine E
ORCID
Venue
RNA
Publisher
openRxiv
Published
2023-04-25
Volume
29
Issue
11
Pages
1772-1791
ISSN
1355-8382 (ISSN-L)
DOI
10.1101/2023.04.25.538249
PMC
PMC10578477
OpenAlex
W4386070208
Cited by
9
References
1
Citations by year
2025: 6 · 2024: 3
FWCI
1.49
Type
journal article
Language
en
Open access
Licence
other-oa
Locations
  • RNA (publishedVersion, other-oa)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
  • HighWire Press Open Archive (submittedVersion)
Funders
  • Camille and Henry Dreyfus Foundation
  • Narodowym Centrum Nauki (2018/31/B/NZ1/02612)
  • National Institutes of Health
  • National Institute of General Medical Sciences (R15GM135878)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
ProQ, RNA-binding proteins, RNA binding sites, Escherichia coli, bacterial gene regulation, RNA-binding residues, forward genetic screen

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Stein, E. M., Wang, S., Dailey, K., Gravel, C. M., Wang, S., Olejniczak, M., & Berry, K. E. (2023). Biochemical and genetic dissection of the RNA-binding surface of the FinO domain of Escherichia coli ProQ. RNA. https://doi.org/10.1101/2023.04.25.538249

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