Biophysical Chemistry of a DEAD/H Protein

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DESCRIPTION (provided by applicant): The long term goal of this project is to obtain a mechanistic understanding of a large, ubiquitous class of proteins, termed DEXD/H proteins, which participate as essential factors in many cellular processes involving RNA. DExD/H proteins are believed to act as RNA helicases to catalyze conformational changes in large RNAs, however, other functions have been proposed. The intent is to perform biochemical and biophysical experiments on purified proteins that will complement extensive efforts by many other groups applying molecular genetic and molecular biological methods to the same proteins in their more complex physiological setting. This project initially chose E. coli DbpA as a model for detailed study, because of its exceptional experimental tractability. Not only is it biochemically well-behaved, but, in contrast with all other DExD/H proteins, it shows very tight binding and high specificity for its target RNA, which simplifies structural and biochemical experiments. We have established that DbpA interacts with RNA in a unique manner, shown that it has helicase activity and understood how the high affinity and specificity is achieved. Current aims include (1) mechanistic experiments to understand how DbpA acts as a helicase and whether it is designed to only open a few base pairs. (2) biochemical experiments defining how DbpA interacts with 23S rRNA and (3) molecular microbiological experiments designed to determine the step in the bacterial ribosome assembly pathway where DbpA acts. Finally, high throughput RNA binding, ATPase and RNA helicase assays in microtiter plates will be developed, in order to assay many other DExD/H proteins, including the 18 family members involved in yeast ribosome assembly.