; R o o t E n t r y F mhm C o m p O b j b W o r d D o c u m e n t u O b j e c t P o o l i i A B C D E F G H I J F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; ' , R 9 @ D ` ' G c 3 q G - ; v ' b N c 9420416 Winkler PRIVATE The overall goal of this proposal is to investigate the molecular basis for RNA:protein recognition by an important class of tRNA modification enzymes, the tRNA prenyl transferases. As an experimental model system, the E. coli K 12 MiaA tRNA prenyl transferase will be analyzed. The MiaA enzyme catalyzes the addition of a (2 isopentenyl group from dimethylallyl diphosphate (DMADP) to the N6 nitrogen of adenosine adjacent to the anticodon at position 37 of 8 of 46 E. coli tRNA species (i.e., i6A 37 formation). The MiaA prenyl transferase will be overexpressed and purified to homogeneity, possibly with the aid of removable affinity tags. The combined tRNA sequence and secondary structure determinants required by the MiaA enzyme for i6A 37 formation will be established. Finally, the roles of four highly conserved amino acid motifs in the MiaA enzyme will be ascertained for substrate binding and i6A 37 formation. These combined data should lead to a more complete picture of how cells regulate tRNA modification levels and coordinate modification with changes in tRNA levels unde r different physiological conditions. Finally, and perhaps most importantly, the proposed experiments may lead to new concepts in the general problem of RNA:protein recognition. None of the conserved protein motifs in MiaA strongly matches known RNA or DMADP binding sites, and one motif corresponds to an ATP/GTP binding site, even though nucleotide triphosphates are not known to be required for i6A 37 formation. Several hypotheses will be tested for the role of these conserved motifs in MiaA enzyme function. %%% RNA is a large nucleic acid molecule that plays numerous essential biological roles in the cells of all organisms. To function, different kinds of RNA interact specifically with proteins, which act as enzyme catalysts and structural components in cells. Thus, it is of fundamental biological importance to learn the principles by which different kinds of RNA recognize specific proteins. To date, these principles are known for only a small number of RNA:protein interactions, and no consensus rules have emerged. The overall goal of this NSF grant is to determine the principles of RNA:protein recognition for an important model bacterial protein, called the MiaA prenyl transferase, that specifically binds to and changes bases of (i.e., modifies) certain transfer RNA (tRNA) molecules.. By correlating structural changes with functional properties, it should be possible to work out the rules for tRNA:protein recognition for these important kinds of macromolecules. *** ; @ ....()()))()() Z : phoenix W CH o ; S u m m a r y I n f o r m a t i o n ( @ Oh +' 0 $ H l D h R:WWUSERTEMPLATENORMAL.DOT marcia steinberg marcia steinberg @ @ @ UZ @ Microsoft Word 6.0 3 e 3 e > u 1 C L N N N l
