MUTATIONAL SENSITIVITY PATTERNS DEFINE CRITICAL RESIDUES IN THE PALM SUBDOMAIN OF THE REVERSE-TRANSCRIPTASE OF HUMAN-IMMUNODEFICIENCY-VIRUSTYPE-1

Citation
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
Citations number
36
Categorie Soggetti
Biology
Journal title
ISSN journal
03051048
Volume
23
Issue
5
Year of publication
1995
Pages
803 - 810
Database
ISI
SICI code
0305-1048(1995)23:5<803:MSPDCR>2.0.ZU;2-B
Abstract
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.