Sf. Chao et al., MUTATIONAL SENSITIVITY PATTERNS DEFINE CRITICAL RESIDUES IN THE PALM SUBDOMAIN OF THE REVERSE-TRANSCRIPTASE OF HUMAN-IMMUNODEFICIENCY-VIRUSTYPE-1, Nucleic acids research, 23(5), 1995, pp. 803-810
We have analyzed 154 single amino acid replacement mutants within a 40
amino acid region (residues 164-203) of the reverse transcriptase (RT
) from human immunodeficiency virus type 1 (HIV-1). This region consis
ts of two antiparallel beta-strands (strands 9 and 10) flanked by two
alpha helices (E and F). The structure of this region of the 'palm' su
bdomain is conserved in a variety of DNA and RNA polymerases, indicati
ng a critical role in enzyme structure and function. Functional assays
were performed by screening RT activity of mutants expressed in E.col
i. A functionally important region corresponding closely to beta-stran
ds 9 and 10 and the loop joining them was revealed by its mutational s
ensitivity. Structural analysis of mutants was performed by using West
ern blots to assay correct folding, which is required for processing t
o produce the mature p66 and p51 RT species. This analysis indicates t
hat beta-strand 10 is a structurally important region. Combined analys
is of these two assays revealed diagnostic patterns of mutational sens
itivity which identify key positions in the RT sequence at which a spe
cific amino acid side chain is critical, either for structure or funct
ion, as well as residues which are external to the RT structure. This
work illustrates the utility of large-scale mutagenesis in relating pr
imary sequence to significant features of protein structure and functi
on.