Holmes 9408813 This proposal describes one of the first extensive efforts to understand how a specific RNA molecule is recognized by a protein and modified at a specific position. This protein, M1G Methyltransferase which is encoded by the trmD gene, introduces a methyl group from S-adenosyl-methionine into the 1 position of quanine at position 37 for a number of tRNA species. Here we propose to first, determine what tRNA structures are required enzyme recognition. This will be accomplished by the synthesis then in vitro expression of synthetic tRNA genes to produce unmodified variant molecules. Methylation of these substrates should indicate which structures are essential for maximum binding and/or catalytic activity. Binding will be assessed by a zonal electrophoretic method. A major question to be addressed here will be whether specific base residues or general tRNA structure be important for protein-RNA interaction. We will explore the nature of complexes formed using chemical, physical, and enzymatic methodologies which may determine the rough conformation of RNA-protein complexes. For example will protein interact with a large or small portion of the tRNA molecules and will enzyme interact with only one face of the tRNA? We propose to determine if M1G Transferase can bind tRNA like structures in mRNA encoding that enzyme. It appears that enzyme production in vivo is somehow limited suggesting an autoregulatory mechanism. In order to test this, synthetic transcripts from various sites in the trmD operon will be prepared in vitro and utilized as substrates for enzyme using radiolabeled AdoMet. Finally, we will identify regions of M1G Transferase involved in tRNA binding and recognition. This will be accomplished by the analysis of selected mutant enzymes which we believe cannot interact properly with tRNA. %%% This research is aimed at determining how cellular enzymes very specific RNA molecules in cells than introduce changes in the structure of th ose molecules. We have focused on one specific system which involves the methylation of a specific tRNA molecules found in the bacterium E. coli. The enzyme (M1G Transferase) places a methyl group at a very specific position on that tRNA which is required for the correct function of that RNA molecules in protein synthesis. Most of the RNA molecules in all cells are modified in various ways in order that they can function correctly in normal cell growth. Ours is one of the first proposals of this kind aimed at determining how RNA is modified and it is likely that all cells will use mechanisms which may be elucidated in this study. In order to study this problem we must prepare synthetic tRNA which can be acted upon by the enzyme. This can be done in the test tube using new technologies for the synthesis of totally synthetic RNA molecules. We can and will prepare modified forms of tRNA in order to determine what structures in the RNA are important for how the enzyme recognizes the tRNA molecule among many different kinds of cellular tRNA. In addition we will use chemical and enzymatic methods to get an idea about what the complex between RNA and protein may look like. Such knowledge permits us to speculate about the exact mechanisms required for recognition and may provide information of great general value helping us to study how enzymes from many other cells may carry out similar modifications. Finally, we will begin making structural changes in the enzyme in order to determine what protein structures are required for the recognition and binding of RNA. The proposal outlined here impinges on important questions of biological specificity. For example, how does RNA correctly participate in accurate protein synthesis which is essential for life in all cells? We believe this work impinges on many broader problems about RNA structure and function and will be useful in understanding many cellular events involving RNA. ***
