Biochemical characterization of the p51 sub-unit of human immunodeficiency virus reverse transcriptase in homo- and heterodimeric recombinant forms of the enzyme.
The biochemical properties of the p51 subunit of HIV-1 reverse transcriptase (RT) were studied in order to understand its role in the heterodimeric form p66/p51 found in virions. A recombinant form of RT, p51/p51, expressed in yeast, was purified and characterized. The enzyme was affinity labeled using a 5' modified oligonucleotide primer, covalently linked, that was further elongated in the presence of a radioactive dNTP precursor. We found that the p51 subunit was labeled in the p51/p51 form, thus reflecting its activity, while this subunit was catalytically silent in the heterodimer, since only the p66 subunit was labeled in the latter recombinant form. Processivity studies showed long-sized products synthesized by p51/p51, as in the case of the other RT forms. The effect of primer tRNA(Lys) on the p51/p51 activity showed a strong inhibitory effect in the absence of KCl, similar to that observed with the p66/p51 form, while the same p51/p51 enzyme was strongly stimulated by tRNA(Lys), like RT p66/p66, when KCl was present in the incubation mixture.
D000345:Affinity Labels; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D012357:RNA, Transfer, Lys / Q000494:pharmacology; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D015003:Yeasts / Q000235:genetics
D000345:Affinity Labels; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D012357:RNA, Transfer, Lys; D011994:Recombinant Proteins; D011189:Potassium Chloride; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase