HIV-1 reverse transcriptase: polymerization properties of the p51 homodimer compared to the p66/p51 heterodimer.
The polymerase activity of the p51 homodimeric form of HIV reverse transcriptase was characterized by activity gel analysis, steady-state kinetic measurements, and processivity assays, and the activity was shown to be highly similar to that for the p66/p51 heterodimer. Recombinant 51- and 66-kDa reverse transcriptase proteins were individually expressed from an HIV-1 Pol gene having an accumulation of natural amino acid mutations compared to the BH10 clone (Ratner et al., 1985). The preparation of an active p51 homodimer critically depended on low temperature during its expression in bacterial cultures. Activity gel analysis demonstrates that refolded p51 protein derived from denatured p66/p51 heterodimer yields an active polymerase. The p51 homodimer has approximately one-half the activity and processivity of the heterodimer, while both enzymes have similar thermostability. Steady-state measurements reveal no significant differences in apparent affinities for substrate or homopolymeric template-primer, suggesting that the subunits in both enzyme forms have similar conformations. Template challenge experiments show that the off-rates for template-primer are lower, but as indicated by primer extension analyses, processivity is less for p51 homodimer. These results show that the RNase H domain is not essential for the assembly of the functional polymerase, but suggest that it enhances processivity.
Bavand
M R
MR
Institut für Molekularbiologie und Biophysik, ETH-Hönggerberg, Zürich, Switzerland.
Wagner
R
R
Richmond
T J
TJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D004279:DNA, Viral / Q000096:biosynthesis / Q000378:metabolism; D015751:Genes, pol; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping
D004279:DNA, Viral; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase