1. Co-translational regulation of gene expression
Our research endeavors in this area are aimed at gaining a thorough and detailed mechanistic understanding of the tmRNA mediated trans-translation process. We have identified a protein factor, small protein B (SmpB), that is essential for the initial binding and subsequent accommodation of tmRNA into the ribosomal A-site. We have identified key amino acid residues in SmpB that are essential for tmRNA binding. We are currently exploring the biochemical and mechanistic details of how these and additional SmpB residues participate in binding of tmRNA and the recognition of stalled ribosomes. Using mutational analysis and biochemical experiments, we have discovered a novel SmpB function associated with its C-terminal unstructured domain. This function is required for a step in the tagging process downstream of tmRNA binding and ribosome association but prior to transpeptidation and establishment of the tmRNA reading frame. Our studies have demonstrated that residues in the C-terminal tail of SmpB confer a previously unknown function that is essential for trans-translation. We are currently exploring the mechanistic detail of exactly how the SmpB C-terminal tail facilitates tmRNA accommodation into the ribosomal A-site, and what steps of the accommodation process are defective with the C-terminal tail variants.
2. Nonstop mRNA decay
We have discovered that defective mRNAs that promote ribosome stalling are selectively degraded by RNase R, a processive 3’-to-5’ exoribonuclease. This targeted RNase R activity is dependent on the presence of SmpB protein and tmRNA, suggesting a requirement for active trans-translation in facilitating RNase R engagement and nonstop mRNA decay. Interestingly, this RNase R activity targets aberrant (nonstop and multiple-rare-codon containing) mRNAs and does not affect the decay of related messages containing in-frame stop codons. Efforts are underway to elucidate the mechanism of how RNase R is targeted to nonstop mRNAs.
3. Directed proteolysis and the Lon Protease
We have recently demonstrated a direct role for the ATP-dependent protease Lon in the degradation of tmRNA-tagged proteins, in vivo and in vitro. We are interested in deciphering how tmRNA tagged proteins are recognized by Lon and what sequence determinants are required for the selective recognition and directed degradation by Lon.
4. Physiological Significance of the tmRNA System
We are investigating the role of the SmpB-SsrA system the in survival and virulence of two pathogenic bacterial species, Yersinia and Francisella. Our studies demonstrate that the SmpB-tmRNA tagging and ribosome rescue system is functional in both bacterial species. Furthermore, our investigations have shown that a ΔsmpB-ssrA mutant strain is avirulent and unable to cause mortality in a mouse infection model. Consistent with these observations, we have demonstrated that the expression of a key type three secretion system (TTSS) transcriptional activator, VirF, and a number of other Ysc-Yop system genes is reduced in the ΔsmpB-ssrA mutant. We are currently investigating the regulatory role of the SmpB-tmRNA system in controlling expression of the TTSS.
5. We are also interested in understanding how sequence and structure in RNA-binding proteins contributes to the formation of specific RNA-protein complexes and how these complexes promote specific biological functions.