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Deciphering the effects of small RNA interactions in individual bacterial cells

Bacteria use many different strategies to regulate gene expression in response to the volatile, and often stressful, conditions in their environments. One type of organization includes non-coding RNA molecules called small RNAs (sRNAs), which are found in all areas of life. A new study led by researchers at the University of Illinois describes, for the first time, the effects of RNA interactions on individual bacterial cells. Their findings were reported in the journal Nature Communications, With the paper identified as that of the most prominent editors.

Bacterial RNAs are often involved in regulating stress responses using mechanisms that include base-coupling interactions with a target mRNA and enhancing or suppressing its stability or the amount of protein produced from mRNA. Hfq, a hexagonal RNA accompaniment protein, facilitates binding between RNAs and enhances RNA stability. Although the kinetics of the mRNA-mRNA interactions has been studied in vitro, the mutagenic effects on the base pairing reactions in vivo remain largely unknown.

“We wanted to understand how individual interactions between base pairs between small RNA and one of its targets contributed to the overall regulatory outcome, and thus the amount of protein that is produced from mRNA under these conditions,” said Illinois professor of microbiology Carey Carey. Vanderpool, who is also a faculty member at the Carl R. Woese Institute for Genome Biology (IGB), who co-led the study. “We took an approach that allowed us to visualize and enumerate individual small RNA and mRNA molecules within bacterial cells, giving us insight into what is happening at the molecular level.”

The research team collaborated with professor of biophysics Taekjip Ha (Johns Hopkins University) and professor of chemistry at Illinois and IGB Zaida Luthey-Schulten College. The researchers used mathematical modeling and ultra-high-resolution quantitative imaging to examine the consequences of changing individual base pair interactions on downregulation kinematic parameters such as the time required for sRNA to find a target mRNA.

The study focused on sRNA SgrS, which is made in bacterial cells when sugar transport exceeds what the cell can handle through metabolism. Previous work by the Vanderpool group showed that under glucose stress conditions, SgrS binds to the primary target mRNA, ptsG, which encodes part of the glucose transporter mechanism. Together with Hfq, SgrS inhibits ptsG translation and thus prevents the production of new glucose transporters to slow down transport so the metabolism can catch up.

To identify key SgrS regions important for ptsG regulation, the researchers used high-throughput sequencing to analyze thousands of mutants in parallel and find those with the strongest influences on regulatory interactions.

“What we found is that the interactions of Al Qaeda’s lone husband had some effect on the organization, but were relatively minor effects,” Vanderbull said. The biggest effects we saw were when we saw mutations in sRNA that disrupted its ability to interact with Hfq. If sRNA cannot bind effectively to the utility, it will be much slower to find its mRNA target, and once found, it becomes unlinked more quickly.

Anustup Poddar, postdoctoral researcher and first author of the paper, said. “The fact that these values ​​are much smaller than the dynamically predicted values ​​tells us that there is much to learn about the role of the flap proteins in target search mediated by base conjugation.”

Even with base pairing disrupted, SgrS regulation of ptsG is still observed as long as Hfq is present. The study clearly demonstrated the important role of Hfq in promoting and stabilizing interactions between SgrS and ptsG. Vanderpol said these results were surprising as the greatest effects were thought to come from disrupting the base coupling interactions.

“I learned a different way of thinking from the collaborators and realized how important and powerful mathematical modeling is,” said Mohamed Azzam, a former graduate student who worked on the study.

The Vanderpool Group has an ongoing collaboration with Professor of Biochemistry and Molecular Biology Jingyei Fei (University of Chicago) to investigate the kinetic parameters of other SgrS targets, with the overall goal of understanding the hierarchy of regulation.

“We had the questions that we wanted to ask but we didn’t have the high-precision methods of looking at this in a quantitative manner,” Vanderbull said. “The best kind of collaboration is when each person brings some type of experience and different ways of looking at a problem on the table that make the result both exciting and enjoyable.”

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Vanderbull was recently elected a Fellow of the American Academy of Microbiology in recognition of her scientific achievement and original contributions to the advancement of the field of microbiology. The National Institutes of Health and the National Science Foundation supported this work.

https: //www.igb.Illinois.Edo /Article /Deciphering small effects of individual bacterial cell interactions

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