Site-specific crosslinking of 4-thiouridine-modified human tRNA(3Lys) to reverse transcriptase from human immunodeficiency virus type I.
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.
Mishima
Y
Y
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.
Steitz
J A
JA
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D004247:DNA / Q000235:genetics; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012357:RNA, Transfer, Lys / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D013891:Thiouridine / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D012357:RNA, Transfer, Lys; D013891:Thiouridine; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D003488:Cyanogen Bromide