NUCLEOTIDES IN PRECURSOR TRANSFER-RNAS THAT ARE REQUIRED INTACT FOR CATALYSIS BY RNASE-P RNAS

Precursor tRNA(Asp) molecules, containing a 26-base 5' leader, were treated with diethylpyrocarbonate, 50% hydrazine or anhydrous hydrazine/3M NaCI and then subjected to processing by RNase P RNAs from Escherchia coli or Bacillus subtilis. Fully processed tRNAs and material not successfully cleaved by the catalytic RNAs were analyzed for their content of chemically altered nucleotides. Several bases were identified as being required intact for optimal activity as substrate as judged by exclusion of chemically modified residues from processed molecules, and simultaneous enhancement in material that was not recognized as substrate. Such nucleotides cluster near the site of cleavage at the mature 5' end and in the T stem and loop. Purines at residues 1 and 2 adjacent to the site of cleavage, position 57 in the T loop, and site 64 in the T stem exhibited the most pronounced effects. These results suggest a model of recognition of substrate by RNase P RNAs in which the ribozyme interacts with the corner of the precursor tRNA's three dimensional structure, where the T- and D-loops are juxtaposed, and extends along the top of the molecule back towards the site of catalysis.