title: Photoaffinity labeling of the primer binding domain in murine leukemia virus reverse transcriptase.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103.
authors.forename: R S
authors.initials: RS
authors.lastname: Tirumalai
authors.forename: M J
authors.initials: MJ
authors.lastname: Modak
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D007700:Kinetics; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic; D014357:Trypsin; D014466:Ultraviolet Rays
chemical_list: D000345:Affinity Labels; D000596:Amino Acids; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C027903:oligo (dT); D012194:RNA-Directed DNA Polymerase; D014357:Trypsin