Distinguishing between structural models for the Escherichia Coli RNA binding protein ProQ
Date
2022-07-01
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Abstract
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.
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Keywords
Biochemistry, Biology, Protein, Protein Structure, AlphaFold, Bacterial three-hybrid, ProQ, sRNA, mRNA