Olejniczak, M. | Search bioRxiv

bioRxiv2021

Bacterial chaperone protein Hfq facilitates the annealing of sponge RNAs to small regulatory RNAs

Bacterial small RNAs (sRNAs) in association with the chaperone protein Hfq regulate the expression of many target mRNAs. Since sRNAs action is crucial to engender a response to changing environmental conditions, their activity needs to be regulated. One such mechanism occurs at posttranscriptional level and involves sponge RNAs (or anti-sRNAs) which sequester sRNAs affecting their regulatory output. Both types of RNAs were identified on Hfq, but it is not known how Hfq interacts with RNA sponges and stimulates their base-pairing with sRNAs. Here, we used biochemical methods to demonstrate that anti-sRNAs resemble sRNAs by their structure and their modes of Hfq binding. Hfq facilitates sponge RNA annealing to sRNA, and each surface of the protein plays a particular role in the process. Moreover, we found that the efficiency of sponge RNA interactions with sRNAs can be improved, therefore, we propose that natural RNA sponges might not sequester sRNAs optimally.

molecular biology↗

bioRxiv2020

Determinants of RNA recognition by the FinO domain of the Escherichia coli ProQ protein

The regulation of gene expression by small RNAs in Escherichia coli depends on RNA binding proteins Hfq and ProQ, which bind mostly distinct RNA pools. To understand how ProQ discriminates between RNA substrates, we compared its binding to six different RNA molecules. Full-length ProQ bound all six RNAs similarly, while the isolated N-terminal FinO domain (NTD) of ProQ specifically recognized RNAs with Rho-independent terminators. Analysis of malM 3-UTR mutants showed that tight RNA binding by the ProQ NTD required a terminator hairpin of at least two base pairs preceding an 3 oligoU tail of at least four uridine residues. Substitution of an A-rich sequence on the 5 side of the terminator to uridines strengthened the binding of several ProQ-specific RNAs to the Hfq protein, but not to the ProQ NTD. Substitution of the motif in the malM-3 and cspE-3 RNAs also conferred the ability to bind Hfq in E. coli cells, as measured using a three-hybrid assay. In summary, these data suggest that the ProQ NTD specifically recognizes 3 intrinsic terminators of RNA substrates, and that the discrimination between RNA ligands by E. coli ProQ and Hfq depends both on positive determinants for binding to ProQ and negative determinants against binding to Hfq.

molecular biology↗