In vivo, there are numerous mechanisms whereby RNA activities are precisely regulated by modulation or modification of RNA structures. Within the broad field of RNA modification, we are pursuing structural and biophysical studies on two systems. The first is a family of RNA chaperones known as the """"""""DEx(D/H)-box helicases"""""""" which catalyze rearrangement of RNA structures in ATP-dependent activities. These proteins are diverse with respect to size (ranging from approximately 400 to over 1200 residues) and function (ranging from participation in RNA splicing and ribosome assembly to RNA degradation); they share a conserved, approximately 400 residue helicase """"""""core fragment"""""""", and are distinguished by different peripheral domains that confer specificity. The crystallographic structure of the helicase core has been determined, revealing how this family of proteins fits into the broader picture of helicase architecture. The foci of future work in this area are (a) what are the global conformations of the helicases and their complexes with RNA that are relevant to helicase activity; (b) what is the structural basis for ATP-driven helicase activity and specific RNA substrate recognition; and (c) how general is the mechanism of target recruitment by a """"""""specificity domain""""""""? These questions are being addressed with a subfamily of the proteins that are involved in ribosome biogenesis, epitomized by E. coli DbpA, and B. subtilis YxiN, which bind specific RNA target sequences with high affinity. The long-term goals are to probe the solution conformations of the helicase using solution small-angle x-ray scattering, solve the structure of the helicase or subfragments, thereof, complexed to specific RNA oligonucleotides using x-ray crystallography, and to test the target recruiting mechanism by replacing the specificity domain with a U1A RNA binding module. The second system being studied is the endoribonuclease RNase E, which is both an essential enzymatic component of the prokaryotic RNA degradosome, and a processing enzyme for ribosomal and messenger RNA. RNase E is a large protein with both a catalytic domain that is responsible for the nuclease activity, and a scaffolding domain that recruits partner proteins to the degradosome. Through a systematic structural genomics approach, a catalytic fragment of RNase E has been defined and crystallized; the crystals diffract to ~3.2 A resolution; the structure will be solved using standard methods. The in vitro catalytic mechanism and substrate binding specificity of RNase E will be elucidated through a combination of mutagenesis, enzymatic assay and crystallographic studies. The effects of alteration of in vitro activity on in vivo activity and regulation of RNase E will be determined, in collaboration with the Stanley Cohen lab.
