Photoaffinity labeling of the primer binding domain in murine leukemia virus reverse transcriptase

Biochemistry. 1991 Jul 2;30(26):6436-43. doi: 10.1021/bi00240a014.


We have labeled the primer binding domain of murine leukemia virus reverse transcriptase (MuLV RT) by covalently cross-linking 5' end labeled d(T)8 to MuLV RT, using ultraviolet light energy. The specificity and the functional significance of the primer cross-linking reaction were demonstrated by the fact that (i) other oligomeric primers, tRNAs, and also template-primers readily compete with radiolabeled d(T)8 for the cross-linking reaction, (ii) under similar conditions, the competing primers and template-primer also inhibit the DNA polymerase activity of MuLV RT to a similar extent, (iii) substrate deoxynucleotides have no effect, and (iv) the reaction is sensitive to high ionic strength. In order to identify the primer binding domains/sites in MuLV RT; tryptic digests prepared from the covalently cross-linked MuLV RT and [32P]d(T)8 complexes were resolved on C-18 columns by reverse-phase HPLC. Three distinct radiolabeled peptides were found to contain the majority of the bound primer. Of these, peptide I contained approximately 65% radioactivity, while the remainder was associated with peptides II and III. Amino acid composition and sequence analyses of the individual peptides revealed that peptide I spans amino acid residues 72-80 in the primary amino acid sequence of MuLV RT and is located in the polymerase domain. The primer cross-linking site appears to be at or near Pro-76. Peptides II and III span amino acid residues 602-609 and 615-622, respectively, and are located in the RNase H domain. The probable cross-linking sites in peptides II and III are suggested to be at or near Leu-604 and Leu-618, respectively.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Affinity Labels / metabolism*
  • Amino Acid Sequence
  • Amino Acids / analysis
  • Avian Myeloblastosis Virus / enzymology
  • Binding Sites
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • Kinetics
  • Molecular Sequence Data
  • Moloney murine leukemia virus / enzymology*
  • Oligodeoxyribonucleotides / metabolism
  • Peptide Fragments / isolation & purification
  • RNA-Directed DNA Polymerase / metabolism*
  • Recombinant Proteins / metabolism
  • Templates, Genetic
  • Trypsin
  • Ultraviolet Rays


  • Affinity Labels
  • Amino Acids
  • Oligodeoxyribonucleotides
  • Peptide Fragments
  • Recombinant Proteins
  • oligo (dT)
  • RNA-Directed DNA Polymerase
  • Trypsin