This study is aimed at understanding how a specific tRNA is recognized and enzymatically modified at specific sites. One such enzyme to be studied here is M1G methyltransferase (1MGT) from Escherichia coli, which methylates position G37 (1 position of the guanine ring) using S-Adenosyl Methionine as the substrate. This important modification is required for the correct "in frame" reading of codons which begin with C. 1MGT is the product of the trmD gene, which has been isolated from both E. coli and Salmonella typhimurium. Relatively little is known about how the many modifications are introduced into cellular RNA in any system, and only a few of the possibly hundreds of specific modification enzymes have been isolated and characterized. It is likely that each enzyme has evolved to recognize and modify a unique RNA "determinant", and it will be important to know how this recognition is achieved in so specific a manner, and to ultimately understand the cellular function of these modifications. In a general sense, the remarkable specificity of modification enzymes, both in selection for the tRNA substrate and particular residue to be modified, raises important questions: What are the structural features of cellular RNA and scores of specific enzymes which permit recognition. What are the catalytic mechanisms for these many interesting reactions? What do these structures look like? How have these genes evolved? Can we modify or otherwise use these enzymes to engineer RNA with expanded function? Using information obtained from solution probing and genetic experiments (and eventually form structural information gotten via NMR or X-ray crystallography), detailed structure function analyses will be carried out on protein and RNA. With these general ideas in mind, this study has the following specific aims: a. define the minimal domain of 1MGT that exhibits activity and tRNA binding. Enzyme deleted for residues at the amino or carboxyl terminus will be tested for catalytic efficiency and ability to bind tRNA, with key amino acid residues and protein domains required for RNA binding and subsequent, catalytic events identified. Amino acid residues that are important for GpG and AdoMet interactions will also be determined. In the case of AdoMet, specific AdoMet consensus sequence will be elucidated by a mutagenesis approach. The GpG interaction site will be labeled using analogues of GpG that might interact covalently with residues in or near the GpG catalytic site. tRNA domains of tRNA, which have been shown to make contact with enzyme, will also be examined by the mutagenesis approach, with a focus on the anticodon stem-loop and variable loop. b. examine the nature of enzyme and RNA structural perturbations, which accompany occupancy of the GpG binding site by either tRNA or GpG. Changes in tRNA structure in normal and relevant mutant tRNA will be examined as well in complex. This will be accomplished initially using CD spectroscopy. Ultimately, NMR approaches will be employed to identify specific domains or residues involved in protein structural perturbations. Specific structural variants of 1MGT affected in binding and or catalysis for the ability to undergo structural changes will be tested. c. continue the ongoing collaborations with Drs. Ng, Giege and Scarsdale on structural determination of 1MGT, using the information so obtained as a basis for a more direct approach to enzyme structure function studies employing site directed mutagenesis.
2. non-techincal
RNA has been shown to be made up of many unusual building blocks, which consist of modified nucleotides. At present over 100 of these unusual bases have been found in naturally occurring RNA. The function of these many nucleotides has not been fully examined. This study seeks to understand how one such modified base in tRNA is synthesized. This modification which involves methylation of the 1 position of Guanine 37, is carried out by the Escherichia coli enzyme tRNA (guanosine-1) methyltransferase (1MGT). This question being examined concerns how this enzyme binds the correct tRNA structure and inserts a methyl group at just the correct site in the highly structured tRNA molecule. First, what portions of the protein bind RNA and are required for recognition will be identified. Next how changes in protein and RNA structure are involved in binding and or RNA recognition will be examined. Finally, attempts will be made to determine the structure of the enzyme using techniques of X-ray crystallography, and Nuclear Magnetic Resonance. This work raises important questions of general interest. What are the structural features of RNA and scores of specific enzymes, which permit recognition? What are the exact catalytic mechanisms for RNA modification? How have genes evolved to produce enzymes that modify RNA? Can we modify or otherwise use these enzymes to engineer RNA with expanded function?
