We are happy to announce that Dr. Ewelina Małecka-Grajek, the Head of the Laboratory of Single-Molecule Biophysics, has received funding for a research project under the SONATA BIS 12 competition! The title of this project is "Dynamics of RNA-degrading complexes in bacteria" and the funding is more than PLN 3 million! Congratulations! Please find below a description of the winning project.

All biological processes, starting from bacteria fighting against viruses, to heart development in humans, are controlled by changes in the expression of genetic material and dynamic interactions between protein and RNA molecules. We already know a lot about how these proteins look structurally, what RNA elements they recognize, and what effect or lack of it such interactions have on the cell. In recent years, more and more emphasis has been placed on learning about the molecular mechanisms that govern biological processes. Through such research, not only can we understand how the mechanisms evolve, but we can also manipulate them better, for example, in the development of vaccines.

The goal of this project is to study the molecular mechanisms that govern communication between specific processes such as RNA silencing, RNA degradation, and translation in bacteria. Post-transcriptional regulation of genes is responsible for the bacterial response to stress, such as switching metabolism to available nutrients or adapting to host conditions in the case of pathogenic strains. This regulation is enabled by small RNAs (sRNAs) interacting with informational RNAs (mRNAs) on a complementary basis. The chaperone protein Hfq is involved in pairing sRNA and mRNA. The binding of mRNA by sRNA often leads to the degradation of both molecules, thus it lowers the expression of the target mRNA. In contrast, the degradation of most bacterial RNAs, including those paired by Hfq, is the responsibility of a protein complex called the degradosome containing the enzyme that catalyzes RNA cutting: ribonuclease E (RNase E). Interestingly, sRNAs are found in cells in complexes with Hfq, but also Hfq and the degradosome. It is not known what functional differences divide these complexes. The degradosome often also includes the RhlB helicase that unravels structured RNAs, so we hypothesize that the presence of the degradosome allows sRNAs to access previously inaccessible sites in target mRNAs. In addition, it is not known whether the formation of a stable mRNA-degrading complex depends on the order in which the individual components are attached. The previous studies have shown that most, but not all, sRNAs are degraded together with the target mRNA which would also lead to regulatory silencing. However, it is not known what factors determine sRNA degradation or whether Hfq is present in this process. If so, it is possible that short fragments remaining after RNA degradation remain bound to Hfq affecting the subsequent fate of target mRNAs. We are planning to reconstitute a catalytically active degradosome that also has the ability to interact with proteins. Using fluorescently labeled components, we will be able to track the folding of complexes in parallel with RNA catalysis.

The additional complexity of coordinating these processes is the fact that RNA undergoes continuous translation. It is known that sRNAs often target the ribosome's binding region on mRNA, but it is unclear whether there is a
direct competition between the ribosome and the sRNA-Hfq complex. In addition, degradosomes carrying out cutting in mRNA coding regions must coordinate directly with ribosomes.
We want to visualize how dynamic these processes are. The research conducted in this project will provide insight into the communication between key cellular processes involved in the expression of genetic information. We will learn about the dynamics of these mechanisms on the millisecond scale, and thus at the real time resolution at which these processes occur in the cell. By monitoring single molecules, we will also gain insight into the heterogeneity of these processes. All this information will be valuable in designing artificial regulators of gene expression and simulating their effects.