Linh Nguyen, Biochemistry
Optimization of RNA Display Using GC-Clamp Modifications to Improve Genetic Detection of Bacterial RNA-Protein Interactions
Project advisor: Katie Berry

Non-coding small regulatory RNAs (sRNAs) have an important role in bacterial stress responses.1 The binding of sRNAs to their target mRNAs is facilitated by RNA-binding proteins such as Hfq or ProQ.2,3 The Berry Lab has developed a bacterial three-hybrid (B3H) assay to detect the binding of RNA with both of these RNA chaperones in vivo, by connecting the strength of an RNA-protein interaction to the expression of a reporter gene. The interaction between a “prey” protein fused to the α-subunit of RNA polymerase (RNAP) and a “bait” RNA tethered upstream of a test promoter stabilizes the binding of RNAP and increases transcription of the reporter gene lacZ. 4 Despite the promise of the B3H system and its success in detecting many high-affinity interactions, low signal-to-noise for other RNA-protein interactions currently limits the broader utility of the assay. Computational structure predictions suggested that certain RNAs of interest could misfold when hybridized with other components of the hybrid RNA construct, e.g., an MS2 hairpin (MS2hp) or an exogenous intrinsic terminator. Such misfolding would likely disrupt a bait RNA’s interaction with the prey protein. To avoid this limitation, this study aims to optimize the hybrid RNA construct to improve the breadth of detectable interactions in the B3H assay. To this end, we designed new hybrid RNA constructs with the addition of a GC-clamp – a short insert of guanines (G) and cytosines (C) flanking a region of interest – to promote proper folding and optimal display of RNA. Several sRNAs and 5’ untranslated regions (5’UTRs) were cloned into GC-clamp designs and their in vivo interactions with Hfq were tested in the B3H assay. Preliminary results demonstrate the promise of a short GC clamp in improving the B3H signal for many sRNA-Hfq interactions, and we are currently working to test these GC constructs with additional RNAs. Increasing the sensitivity and generalizability of the B3H assay to study bacterial RNA-protein interactions will help shed light on the molecular mechanisms of RNA-chaperone proteins and the important processes in bacteria they regulate, such as adaptation to stress, biofilm formation and virulence.
1 Storz, G., Vogel, J., & Wassarman, K. M. (2011). Regulation by small RNAs in bacteria: expanding frontiers. Molecular cell, 43(6), 880–891. https://doi.org/10.1016/j.molcel.2011.08.022
2 Gottesman, S., & Storz, G. (2011). Bacterial small RNA regulators: versatile roles and rapidly evolving variations. Cold Spring Harbor perspectives in biology, 3(12), a003798. https://doi.org/10.1101/cshperspect.a003798
3 Olejniczak, M., & Storz, G. (2017). ProQ/FinO-domain proteins: another ubiquitous family of RNA matchmakers?. Molecular microbiology, 104(6), 905–915. https://doi.org/10.1111/mmi.13679
4 Berry, K. E., & Hochschild, A. (2018). A bacterial three-hybrid assay detects Escherichia coli Hfq-sRNA interactions in vivo. Nucleic acids research, 46(2), e12. https://doi.org/10.1093/nar/gkx1086
 

Amy Wang, Biochemistry
A Multicopy Reporter System for in vivo Detection of Bacterial RNA-Protein Interactions
Project advisor: Katie Berry

RNA-binding proteins are critical in regulating gene expression. Being able to identify and quantify those interactions can help us understand their mechanisms of action and evolutionary significance. One strategy to characterize RNA-protein interactions is an in vivo bacterial three-hybrid (B3H) assay that is able to detect Escherichia coli Hfq and ProQ interactions with their cognate RNAs. In the B3H system, the binding of RNA polymerase (RNAP) to a lacZ reporter gene is weak, but when an RNA-of-interest (“bait”) interacts with the protein-of-interest (“prey”) fused to a subunit of RNAP, binding is stabilized at a test promoter (OL2-62) and lacZ is expressed. Thus, the strength of an RNA-protein interaction can be measured by the amount of β-galactosidase generated. The current B3H reporter system utilizes a test promoter driving lacZ expression on a single-copy F’ episome, which also carries the lacI repressor gene, while each of the hybrid components needed for the assay (DNA-RNA adapter, bait RNA, prey protein) is expressed from a compatible multicopy plasmid.

The status quo B3H can successfully detect many protein-RNA interactions consistently, including Hfq-ChiX, ProQ-sibB, and ProQ-cspE interactions, but it fails to pick up weaker but known interactions. Computational steady-state modeling of the assembly of B3H assay components suggests that having only a single copy of the test promoter per cell may be a limiting factor in signal. We therefore hypothesize that a multicopy reporter would increase B3H signal and sensitivity, thereby broadening the utility of the assay. In order to test this hypothesis, three candidate multicopy reporter plasmids have been generated using Gibson assembly. These introduce OL2-62-lacZ and/or lacI into either pBait or pAdapter in various orientations.

Preliminary results with an Hfq-ChiX interaction indicate that a multicopy reporter system can successfully detect protein-RNA interactions, and that the specific location of the lacZ reporter gene strongly impacts the behavior of the system. Currently, a pAdapter-lacI-OL2-lacZ provides the best signal of our multicopy reporters, and we are comparing this construct to single-copy reporters across multiple RNA-protein interactions. In addition to further modifications to reduce background signal and improve the fold-stimulation of lacZ in the presence of an RNA-protein interaction, we are excited for the possibility of easily changing out the reporter gene in the system and plan to create constructs with fluorescent-protein reporters soon. In summary, we have validated that the B3H assay can be successfully implemented with either a multicopy or single-copy lacZ reporter, and we are optimizing the multicopy system to increase signal-to-noise across a wide range of interactions and expand the reporter genes available for detecting bacterial RNA-protein interactions in vivo.

Katherine Dailey, Biochemistry
Distinguishing Between Structural Models for the E. coli RNA Binding Protein ProQ
Project advisor: Katie Berry

Evolving research on small RNAs (sRNAs) in bacteria implicates sRNAs as a key effector of gene regulation, influencing expression for genes involved in processes from basic bacterial biology to serious public health issues such as virulence and antibiotic resistance. While some sRNAs are able to act independently, many are dependent on an RNA-binding protein, such as the well-established Hfq in Escherichia coli. Another family of RNA-binding proteins is the FinO family, including ProQ and FinO in E. coli, NMB1681 in N. meningitidis, and Lpp1663 in L. pneumophila. Structures for these proteins have been solved through both NMR and X-ray diffraction, in addition to computational predictions1 . While many structural elements are common across all structures, there are interesting differences in regions that have been implicated by genetic experiments to be important for RNA binding. In order to investigate the structure and function relationships of these proteins, we have analyzed the available models for FinO family proteins to compare intriguing structural features, including the position and predicted contacts of a universally conserved arginine that plays a critical role in RNA binding. Finally, we have probed predicted interactions from structural models with the use of site-directed mutagenesis and our laboratory’s bacterial three-hybrid (B3H) assay2 . Together, this work is generating insights into the most relevant structural conformations for in vivo RNA binding by FinO proteins and the ways in which the structure of E. coli ProQ is both similar and distinct from orthologous FinO domain proteins.
1 Alexandru F. Ghetu et al., “Crystal Structure of the Bacterial Conjugation Repressor FinO,” Nature
Structural Biology 7, no. 7 (July 1, 2000): 565–69, https://doi.org/10.1038/76790; Grecia M. Gonzalez et
al., “Structure of the Escherichia Coli ProQ RNA-Binding Protein,” RNA 23, no. 5 (May 2017): 696–711,
https://doi.org/10.1261/rna.060343.116; Carina Immer, Carolin Hacker, and Jens Wöhnert, “Solution
Structure and RNA-Binding of a Minimal ProQ-Homolog from Legionella Pneumophila (Lpp1663),” RNA
26, no. 12 (December 2020): 2031–43, https://doi.org/10.1261/rna.077354.120; Steven G Chaulk et al., “N.
Meningitidis 1681 Is a Member of the FinO Family of RNA Chaperones,” RNA Biology 7, no. 6
(November 2010): 812–19, https://doi.org/10.4161/rna.7.6.13688; John Jumper et al., “Highly Accurate
Protein Structure Prediction with AlphaFold,” Nature 596, no. 7873 (August 26, 2021): 583–89,
https://doi.org/10.1038/s41586-021-03819-2.
2 Katherine E Berry and Ann Hochschild, “A Bacterial Three-Hybrid Assay Detects Escherichia Coli
Hfq–SRNA Interactions in Vivo,” Nucleic Acids Research 46, no. 2 (January 25, 2018): e12–e12,
https://doi.org/10.1093/nar/gkx1086.
 

Sungeun Jo, Biochemistry
Searching for Novel sRNA-Binding Proteins in Chlamydia trachomatis
Project advisor: Katie Berry

Non-coding small RNAs (sRNAs) in prokaryotes work as regulators of mRNA expression, and sRNA-mediated gene expression plays an important role in bacterial processes such as stress responses and virulence, providing a promising field of study to understand bacterial behavior. In E. coli, well-studied global sRNA-binding proteins such as Hfq and ProQ are encoded and known to be important for supporting the stability and function of sRNAs. However, many bacterial species that produce sRNAs do not encode either of these sRNA-binding proteins. This raises the intriguing possibility that this subset of bacteria may have as-of-yet-undiscovered RNA-binding proteins that possess important roles in bacterial behavior. Chlamydia trachomatis, a human pathogen that causes a sexually transmitted infection, is one of such bacteria with numerous identified sRNAs1,2 and the most common cause of blindness worldwide, adding significance to the study of this strain.

In this study, we are employing a bacterial three-hybrid (B3H) assay to search for new RNA-binding proteins encoded in this bacterial genome. This B3H assay, developed to assess RNA-protein interactions3 , was derived from a bacterial two-hybrid (B2H) assay, which was previously used to identify a new protein-protein interaction in the same strain4 . To screen for new sRNA-protein interactions in C. trachomatis, previously identified four sRNAs (ctrR1, ctrR2, ctrR0332, and IhtA)5 were cloned into the B3H system’s pBait (RNA moiety) and transformed into reporter cells with a C. trachomatis genomic-fragment library as a pPrey (protein moiety). Some candidate sRNA-binding peptides and proteins have been found through this screening process. And, β-galactosidase assays were conducted to quantify the interactions of these candidates with sRNAs, along with computational analysis to map the candidate fragments back to Chlamydial genomic/proteomic context. In the future, a complementary candidate-based approach can be used to more specifically explore RNA-binding activity of recently identified putative RNA-binding proteins in C. trachomatis. Furthermore, the expansion of the screening method to other bacterial genomes such as Mycobacterium tuberculosis would provide a better understanding of diverse RNA-based translational regulations.
1 Elwell, C., Mirrashidi, K., & Engel, J. (2016). Chlamydia cell biology and pathogenesis. Nature reviews. Microbiology, 14(6), 385–400. https://doi.org/10.1038/nrmicro.2016.30
2 Klepsch, M. (2020). Small RNA-binding complexes in Chlamydia trachomatis identified by Next-Generation Sequencing techniques. Doctoral Thesis
3 Berry, K. E., & Hochschild, A. (2018). A bacterial three-hybrid assay detects Escherichia coli Hfq-sRNA interactions in vivo. Nucleic acids research, 46(2), e12. https://doi.org/10.1093/nar/gkx1086
4 Rao, X., Deighan, P., Hua, Z., Hu, X., Wang, J., Luo, M., Wang, J., Liang, Y., Zhong, G., Hochschild, A., & Shen, L. (2009). A regulator from Chlamydia trachomatis modulates the activity of RNA polymerase through direct interaction with the beta subunit and the primary sigma subunit. Genes & development, 23(15), 1818–1829. https://doi.org/10.1101/gad.1784009
5 Albrecht, M., Sharma, C. M., Reinhardt, R., Vogel, J., & Rudel, T. (2010). Deep sequencing-based discovery of the Chlamydia trachomatis transcriptome. Nucleic acids research, 38(3), 868–877. https://doi.org/10.1093/nar/gkp1032

Morgan Tanguay, Biochemistry
The Study of RNA-Binding Protein Interactions in a B3H Assay
Project Advisor: Kathryn McMenimen

Hfq is a vital, RNA-binding protein that helps sRNAs regulate mRNAs through base-pairing and was first studied in E. coli, but is now being observed in many other bacteria, such as B. subtilis. Through genetic and biochemical approaches, both protein-protein and RNA-protein interactions can be studied to better understand the critical, regulatory functions that Hfq has in species-specific models. In Biochemistry 314 lab, full-length Hfq from B. subtilis was cloned into viable vectors using PCR and used in two different assays, B2H and B3H. B2H, or bacterial two-hybrid, and B3H, bacterial three-hybrid, take advantage of the expression of the lacZ gene, which encodes for beta-galactosidase, through the regulation of RNA polymerase. In both assays, the Hfq protein was used as prey and was linked to the alpha subunit of RNA pol. In contrast, the baits for either assay differed in that for B2H, another Hfq protein from a different organism was used while the B3H assay used RNA for the bait, which also differed in species origin. Interactions between bait and prey in both assays were evaluated through the detection of beta-galactosidase, with high expression correlating to a high degree of interaction. The goal was to identify new binding partners for full-length Hfq in B. subtilis and compare the degrees of binding with each partner. By observing these interactions between BsHfq and other organisms's Hfq and RNAs, future research can be geared towards exploring the functions of other RNA-binding proteins homologous to BsHfq.