Document 0891
 DOCN  M9540891
 TI    Construction of an enzymatically active ribonuclease H domain of human
       immunodeficiency virus type 1 reverse transcriptase.
 DT    9504
 AU    Stahl SJ; Kaufman JD; Vikic-Topic S; Crouch RJ; Wingfield PT; Protein
       Expression Laboratory, National Institute of Child Health; and Human
       Development, National Institutes of Health, Bethesda,; MD 20892.
 SO    Protein Eng. 1994 Sep;7(9):1103-8. Unique Identifier : AIDSLINE
       MED/95132587
 AB    The isolated ribonuclease (RNase) H domain of human immunodeficiency
       virus type 1 (HIV-1) is enzymatically inactive. The incorporation of the
       putative substrate binding site of Escherichia coli RNase HI (amino acid
       residues 76-102, the alpha c-helix and adjacent loop region) into the
       equivalent position of the RNase H domain of HIV-1 resulted in a highly
       active hybrid protein dependent on Mn2+. Similar restoration of RNase H
       activity has been observed when histidine residues are added to either
       the N- or C-terminus of the HIV-1 RNase H domain. The hybrid HIV-1/E.
       coli RNase H protein is approximately 10-fold more active than HIV-1
       reverse transcriptase and 30-fold more active than the histidine-tagged
       proteins, indicating that the alpha c-helix and adjacent loop region of
       E. coli RNase HI is an excellent substrate binding region because of its
       sequence and/or location. The RNase H hybrid produced the same specific
       cleavage in the model tRNA(Lys3) primer removal assay as HIV-1 reverse
       transcriptase, showing that substrate binding and specificity are
       separable and that the specificity determinants are at least partially,
       if not totally, contained in the amino acid sequence of the hybrid
       protein derived from HIV-1 reverse transcriptase.
 DE    Amino Acid Sequence  Base Sequence  Binding Sites/GENETICS  DNA
       Primers/GENETICS  Escherichia coli/ENZYMOLOGY/GENETICS  Human
       HIV-1/*ENZYMOLOGY/GENETICS  Molecular Sequence Data  Molecular Structure
       Protein Engineering  Recombinant Proteins/CHEMISTRY/GENETICS/METABOLISM
       Reverse Transcriptase/*CHEMISTRY/GENETICS/METABOLISM  Ribonuclease H,
       Calf Thymus/*CHEMISTRY/GENETICS/METABOLISM  Sequence Homology, Amino
       Acid  Sequence Tagged Sites  Support, U.S. Gov't, P.H.S.  JOURNAL
       ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).