Document 0108
 DOCN  M9590108
 TI    Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to
       reverse transcriptase from human immunodeficiency virus type I.
 DT    9509
 AU    Mishima Y; Steitz JA; Department of Molecular Biophysics and
       Biochemistry, Howard; Hughes Medical Institute, Boyer Center for
       Molecular Medicine,; Yale University School of Medicine, New Haven, CT
       06536-0812,; USA.
 SO    EMBO J. 1995 Jun 1;14(11):2679-87. Unique Identifier : AIDSLINE
       MED/95300801
 AB    We have mapped specific RNA-protein contacts between human
       immunodeficiency virus (HIV) type I reverse transcriptase (RT) and its
       natural primer, human tRNA(3Lys), using a site-specific crosslinking
       strategy. Four different tRNA(3Lys) constructs with a single 32P-labeled
       4-thiouridine (4-thioU) residue at positions 1, 16, 36 or 41 were
       synthesized. After incubation with RT followed by irradiation,
       crosslinks were localized to either the p66 or p51 subunit of RT by
       digestion with nuclease and SDS gel fractionation. 4-thioU at position
       -1 or 16 transferred label to the p66 subunit almost exclusively (>
       90%), whereas position 36 labeled both p66 and p51 (3:1). Position 41
       yielded no detectable crosslinks. The region of p66 contacted by
       position -1 of tRNA(3Lys) was localized to the 203 C-terminal amino
       acids of RT by CNBr cleavage, whereas a 127 amino acid-CNBr peptide
       (residues 230-357) from both p66 and p51 was labeled by position 36.
       Functionality of the 4-thioU-modified tRNA(3Lys)(-1) crosslinked to RT
       in the presence of an RNA but not a DNA template was demonstrated by the
       ability of the tRNA to be extended. These results localize the 5' half
       of the tRNA on the interface between the two RT subunits, closer to the
       RNase H domain than to the polymerase active site, in accord with
       previous suggestions. They argue further that a specific binding site
       for the 5' end of the primer tRNA(3Lys) may exist within the C-terminal
       portion of the p66 subunit, which could be important for the initiation
       of reverse transcription.
 DE    Base Sequence  Binding Sites  Cross-Linking Reagents  Cyanogen Bromide
       DNA/GENETICS  Human  HIV-1/*ENZYMOLOGY  In Vitro  Models, Molecular
       Molecular Sequence Data  Protein Conformation  Reverse
       Transcriptase/CHEMISTRY/*METABOLISM  RNA, Transfer,
       Lys/CHEMISTRY/GENETICS/*METABOLISM  Support, U.S. Gov't, P.H.S.
       Thiouridine/CHEMISTRY/METABOLISM  JOURNAL ARTICLE

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