Distinguishing between structural models for the Escherichia Coli RNA binding protein ProQ



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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 Neisseria meningitidis, and Lpp1663 in Legionella pneumophila. Previous work on ProQ has not supplied a satisfying answer on how ProQ binds to RNA, despite an available NMR structure. In July of 2021, the AlphaFold database was released, which included an alternate structure for ProQ. In order to critically evaluate both of these structures, I compared the structures of FinO domain proteins, examined highly conserved residues Y70 and R80 through the use of a forward genetic screen with our laboratory’s bacterial three-hybrid assay, and used the same assay to probe predicted interactions from the structural models with the use of site-directed mutagenesis. The available structures of FinO domains were found to vary from the NMR structure of ProQ in both quality and chemical properties. Two key residues on ProQ, Y70 and R80, were extremely sensitive to mutation. It is possible that these residues are directly involved in RNA binding by ProQ, a hypothesis supported by the structures and research on other FinO domain proteins. This work suggests that the NMR structure of ProQ should be examined more critically, and it is possible that the AlphaFold structure provides an alternate model for this protein. Through this, I hope to generate 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.



Biochemistry, Biology, Protein, Protein Structure, AlphaFold, Bacterial three-hybrid, ProQ, sRNA, mRNA