Post-transcriptional regulation of gene expression in bacteria

Our lab focuses on understanding the coordination between RNA targeting, degradation, and translation in bacteria.

Most species of bacteria are harmless and are often beneficial, but others can cause infectious diseases. The expression of virulence genes is often regulated through RNA chaperone protein Hfq and small RNAs (sRNAs). Hfq mediates the base pairing between sRNAs and target mRNAs which leads to up- or downregulation of the target. Down- or upregulation of a target occurs mainly via altering the translation initiation or target RNA degradation. RNA degradation in bacteria is facilitated by a degradosome, a complex of ribonuclease E (RNase E), enolase, RhlB helicase, and PNPase.

Fig. 1. Posttranscriptional regulation of gene expression in bacteria mediated via Hfq protein and sRNAs.

The degradosome can also preassemble with sRNA and Hfq. This complex could be potentially more efficient in regulating target mRNA in comparison to sequential targeting by sRNA and degradosome. Since complexes assemble via alternative pathways, we will also investigate if the formation mechanism changes the regulatory outcome.

Moreover, these complexes in cells attempt to bind mRNA that is likely undergoing translation. Timely coordination between targeting, degradation and translation is crucial for efficient mRNA regulation. However, how it occurs in real-time is not known. To test this, we will reconstitute active translation in vitro and test the assembly of targeting and degrading complexes.

Single-molecule TIRF microscopy

Significant effects of sRNAs in gene expression are observed as quickly as 1-2 minutes from signal induction, yet we do not fully understand how the coordination between these processes occurs so efficiently on this time scale. To address this issue, we need to monitor the assembly of RNA-protein complexes as it evolves in real-time on time scales relevant in vivo. Moreover, the fate of individual sRNAs can vary, e. g. some sRNAs might fail to bind the protein, or to find a cognate mRNA.  

With this in mind, to really dive deep into the molecular mechanism of sRNA action, we need a method that:

• provides a high temporal resolution (scale of milliseconds to minutes)
• provides spatial resolution to resolve changes in the conformation of RNA-protein or RNA-RNA complexes.
• allows the visualization of various pathways of the reaction

Single-molecule TIRF microscopy allows the visualization of hundreds of biomolecules immobilized on the microscopic slide. However, each molecule is analyzed separately. Molecules are fluorescently labeled, and the interactions are detected by colocalization of fluorophores or FRET.