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We measured kinetic parameters in vitro and directly analyzed aminoacylaton and formylation levels in vivo to study recognition ofEscherichia coli initiator tRNA mutants by E. coli Met-tRNA synthetase and Met-tRNA transformylase. We show that, in addition to the anticodon sequence, mutations in the "discriminator" base A73 also affect aminoacylation. An A73 -+ U change has a small effect, but a change to G73 or C73 significantly lowers VI.X/Kr" for in vitro aminoacylation and leads to appreciable accumulation of uncharged tRNA in vivo. S cantly, coupling of the G73 mutation with G72, a neighboring-base mutation, results in a tRNA essentially uncharged in vivo. Coupling of C73 and U73 mutations with G72 does not have such an effect. Elements crucial for Met-tRNA tansformylase recognition of tRNAs are located at the end of the acceptor stem. These elements include a weak base pair or a mismatch between nucleotides (nt) 1 and 72 and base pairs 2-71 and 3'70. The natures of nt 1 and 72 are les important than the fact that they do not form a strong Watson-Crick base par. Interestingly, the negative effect of a C-G base pair between nt 1 and 72 Is suppressed by mutation of the neighboring nucleotide A73 to either C73 or U73. The presence of C73 or U73 could destabilize the C1lG72 base pair at the end of an RNA helix. Thus, in some tRNAs, the discriminator base could affect stability of the base pair between nt 1 and 72 and thereby the structure of tRNA at the end of the acceptor stem.As part of structure-function relationship studies of Escherichia coli initiator methionine tRNA (Fig. 1), we previously described mutagenesis of the tRNAfMet gene and presented functional studies of the mutant tRNAs in vitro and in vivo. These studies showed that (i) the (GGG)-(CCC) sequence conserved in the anticodon stem of initiator tRNAs was important in targeting the tRNA to the ribosomal P site (1), (ii) the C1 x A72 mismatch at the end ofthe acceptor stem was important in preventing the tRNA from binding to the elongation factor EF-Tu and, thereby, from functioning in the elongation step of protein synthesis (2, 3), and (iii) the sequence and/or structural features important for recognition of tRNA by Met-tRNA transformylase were mostly clustered at the end of the acceptor stem (4).We are also studying recognition of the initiator tRNA by E. coli Met-tRNA synthetase (MetRS). Schulman and coworkers (5) have concluded that the anticodon sequence of methionine tRNA contains the major determinants for recognition by E. coli MetRS. This conclusion was based on the findings that (i) mutations in the anticodon nucleotides greatly affected the kinetic parameters in aminoacylation (6, 7) and (ii) transfer of methionine anticodon CAU to E. coli AOH C C A.

FT-IR difference spectroscopy can provide detailed information about structure and conformational changes in proteins.* 1 This approach is greatly enhanced by site-directed isotope labeling (SDIL).2-4 In contrast to earlier methods employing site-directed mutagenesis, SDIL allows vibrational band assignments to be made in an essentially unaltered system through the replacement of single amino acid residues with their isotopic analogs.

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