I. Gene Regulation by Bacterial small RNAs 

Hfq image

We are studying how Hfq and small RNAs work together to regulate gene expression in bacteria. The figure above, is a superposition from a pair of X-ray crystal structures showing Hfq bound to 2 RNAs. In blue is a short RNA with the sequence AUUUUUG, a classic model for bacterial sRNAs. In red, is an AAAAAA oligo representing the (ARN)x motif coming from an mRNA that would be the target of sRNA regulation. We use the tools of biochemistry, biophysics and molecular biology to probe how target recognition is achieved, how the RNAs undergo refolding in response to sRNA:mRNA pairing and how that pairing interaction leads to down-stream regulatory events including up- or down-regulation of translation or targeted RNA degradation. 

A more complete list of papers can be found on the publications page, but examples of our work on Hfq and sRNA systems can be found in the following papers:

     Salim et al. (2012) Nucl. Acids Res. (in press)

     Salim and Feig (2010) PLOS One.

     Mikulecky et al. (2004) NSMB.

     Brescia et al. (2003) RNA. 

Discovery of new sRNAs and new Hfq homologs in pathogenic organisms

     Lybecker et al. (2010) Mol. Microbiology

     Tsui et al. (2010) J. Bact. 

II. Biochemistry of Clostridium difficile Toxins

cdiff image

Clostridium difficile is a pathogenic bacteria that causes antibiotic associated diarrhea. This is one of the most common hospital-acquired infection and causes increased mortality and prolonged hospitalization. Current therapies for treating C. diff are only modestly effective and recurrence is a serious problem as many patients who appear to cover find themselves back in the hospital several days after release due to either reinfection or incomplete irradication of the organism from the GI tract. Of particular concern is an epidemic strain that is both more virulent and more resistant to current antibiotics. Our approach to C. difficile associated diarrhea has been to study the toxins responsible for virulence and cellular damage. These are two large protein toxins called Toxin A and Toxin B. We have been studying how these proteins work, how they enter cells and more importantly how to deactivate/inhibit the toxins to prevent damage to the GI tract. We have developed small molecules and peptides that inhibit toxin function and are studying how we can convert these into agents that can transition from the laboratory to the clinic. 

We are also looking at ways to repurpose these toxins for biotechnology applications.  These proteins hijack the endocytotic pathway of normal cells, translocating large proteins into the cytosol. We have used reporter systems fused in place of the catalytic domain to show that we can take advantage of this machinery to translocate proteins of our design into eukaryotic cells. This has potential to perform gene therapy type applications without the gene as these proteins required to replace defective genes can be taken up by cells in a manner not accessible in most cases. 

A more complete list of papers can be found on the publications page, but examples of our work on bacterial toxins includes:

     Abdeen, Swett and Feig (2010) ACS Chem. Biol. 

     Kern and Feig (2011) Biochem. Biophys. Res. Commun.

III. Kinetics and Thermodynamics of RNA Structural Rearrangements 

energy landscape

RNA folding is often studied using thermal denaturation methods. These techniques taught us a lot about global aspects of RNA fold and low energy structures, and the rugged energy landscapes these RNAs encounter. In biology however, except during co-transcriptional folding, RNAs are more likely to encounter refolding transitions, conversion from one stable fold to another in response to a regulatory signal. Such refolding events are important in RNA-mediated gene regulation by sRNAs, viral translation initiation and packaging of dimeric genomic RNAs into virus particles such as HIV. We are studying the kinetics and thermodynamics of these transitions using a variety of biophysical techniques such as ITC, SPR and single-molecule FRET. The energy landscape above represents the transitions between kissing hairpins and extended duplex conformations and the energy landscape such transitions encounter in the absence of the proteins that help chaperone such rearrangements. Note the large energy barrier encountered between wells C and D representing a kissing complex and the corresponding rearranged form with an extended duplex structure. We are now studying how proteins change these landscapes to enhance the conversion rate between transient intermediates liked C and stable regulatory states like D. This also leads us to better understand how transient interactsion like seed pairing between sRNAs and mRNAs assists in develop selectivity for cognate partners while selecting against non-cogante pairings that would lead to off-target effects in regulatory circuits.

A more complete list of papers can be found on the publications page, but examples of our work on RNA structural rearrangements can be found in the following papers:

     Salim et al. (2012) Biophys. J.

     Salim and Feig (2009) Methods.

     Zhao et al. (2010) Biophys J.

     Mikulecky and Feig (2006) Biopolymers

     Mikulecky, Takach and Feig (2004) Biochemistry

© Andrew Feig 2013