A GENETIC APPROACH FOR IDENTIFYING CRITICAL RESIDUES IN THE FINGERS AND PALM SUBDOMAINS OF HIV-1 REVERSE-TRANSCRIPTASE

By using oligonucleotide-directed saturation mutagenesis, we collected 366 different single amino acid substitutions in a 109-aa segment (re sidues 95-203) in the fingers and palm subdomains of the HIV-1 reverse transcriptase (RT), the enzyme that replicates the viral genome, Afte r expression in Escherichia coli, two phenotypic assays were performed . The first assay tested for RNA-dependent DNA polymerase activity, Th e other assay used Western blot analysis to estimate the stability of each mutant protein by measuring the processing of the RT into its mat ure heterodimeric form, consisting of a 66-kDa subunit and a 51-kDa su bunit, The resulting phenotypic data provided a ''genetic'' means to i dentify amino acid side chains that are important for protein function or stability, as well as side chains located on the protein surface, Several HIV-1 RT crystal structures were used to evaluate the mutation al analysis. Our genetic map correlates well with the crystal structur es, Combining our phenotype data with crystallographic data allowed us to study the genetically defined critical residues, The important fun ctional residues are found near the enzyme active site, Many residues important for the stability of the RT participate in potential hydroge n bonding or hydrophobic interactions in the protein interior, In addi tion to providing a better understanding of the HIV-1 RT, this work de monstrates the utility of saturation mutagenesis to study the function , structure, and stability of proteins in general. This strategy shoul d be useful for studying proteins for which no crystallographic data a re available.