Atomic co-ordinates for yeast phenylalanine tRNA

Atomic coordinates are presented for yeast tRNA(Phe) derived from a wire skeletal model fitted to an electron density map at 2.5 Å resolution obtained by isomorphous replacement

Correlation between three-dimensional structure and chemical reactivity of transfer RNA

The bases of yeast tRNA(Phe) which react with carbodiimide and methoxyamine have been determined and this information has been combined with chemical modification studies of other workers to produce a composite picture of base accessibility in this tRNA. The results are compared with the three-dimensional structure which we have recently determined. The bases which react chemically lie in exposed positions in the three-dimensional model and those which do not are either in the double helical stem regions or else are involved in maintaining the tertiary structure through pairing or stacking interactions

Enzyme Engineering for Nonaqueous Solvents: Random Mutagenesis to Enhance Activity of Subtilisin E in Polar Organic Media

Enzyme activity is often dramatically reduced in polar organic solvents, even under conditions where the folded structures are stable. We have utilized random mutagenesis by polymerase chain reaction (PCR) techniques combined with screening for enhanced activity in the presence of dimethylformamide (DMF) to probe mechanisms by which improved enzymes for chemical synthesis in polar organic media might be obtained. Two amino acid substitutions which enhance subtilisin E activity in the presence of DMF, Q103R and D60N, were identified by screening on agar plates containing DMF and casein. The two substitutions are located near the substrate binding pocket or in the active site, and their effects on the catalytic efficiency k_(cat)/K_M for the hydrolysis of a peptide substrate are additive. The effects of D60N are apparent only in the presence of DMF, highlighting the importance of screening in the organic solvent. Protein engineering is an effective approach to enhancing enzyme activity in organic media: the triple mutant D60N+Q103R+N218S is 38 times more active than wild–type subtilisin E in 85% DMF. An evolutionary approach consisting of multiple steps of random muta–genesis and screening in continually higher concentrations of organic solvent should result in enzymes that are substantially more active in organic media