Nucleic Acids Research, 1995, Vol. 23, No. 5 803-810
© 1995
MOLECULAR BIOLOGY |
Mutational sensitivity patterns define critical residues in the palm subdomain of the reverse transcriptase of human immunodeficiency virus type 1
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA 1Department of Microbiology, University of Toronto Toronto M5S 1A8, Canada 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA 3Department of Medicine, University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA 4Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill Chapel Hill, NC 27599, USA
*To whom correspondence should be addressed
Received November 10, 1994. Revised January 27, 1995. Accepted January 27, 1995.
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 consists of two antiparallel ß-strands (strands 9 and 10) flanked by two 
helices (E and F). The structure of this region of the ‘palm’ subdomain is conserved in a variety of DNA and RNA polymerases, indicating a critical role in enzyme structure and function. Functional assays were performed by screening RT activity of mutants expressed in E.coli.A functionally important region corresponding closely to ß-strands 9 and 10 and the loop joining them was revealed by its mutational sensitivity. Structural analysis of mutants was performed by using Western blots to assay correct folding, which is required for processing to produce the mature p66 and p51 RT species. This analysis indicates that ß-strand 10 is a structurally important region. Combined analysis of these two assays revealed diagnostic patterns of mutational sensitivity which identify key positions in the RT sequence at which a specific amino acid side chain is critical, either for structure or function, as well as residues which are external to the RT structure. This work illustrates the utility of large-scale mutagenesis in relating primary sequence to significant features of protein structure and function.
*Present addresses: Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Present addresses: 37-121 CHS, UCLA School of Medicine, 10833 Leconte, Los Angeles, CA 90024, USA
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