Human immunodeficiency virus reverse transcriptase. Expression in Escherichia coli, purification, and characterization of a functionally and structurally asymmetric dimeric polymerase.
Human immunodeficiency virus (HIV) reverse transcriptase isolated from viral particles contains two subunits, p51 and p66. We have produced both subunits in separate Escherichia coli strains using expression vectors. Stop codons were placed immediately after the codon for the carboxyl-terminal residue of the mature processed p51 and p66 subunits found in viral particles. Insertion of a methionine in front of the HIV protease cleavage site in the recombinant protein enabled synthesis of both subunits with the natural amino-terminal proline, since E. coli methionine aminopeptidase cleaves a Met-Pro amino-terminal linkage. That this occurred to an extent greater than 95% was confirmed by sequencing the purified subunits. Examination of the activities of the individual p51 and p66 subunits on a variety of templates and under solution conditions optimized for each subunit revealed a significant catalytic activity for the natural p51 subunit. This result contrasts to results reported earlier for many recombinant forms without the natural amino and/or carboxyl termini. As expected from earlier work, the optimal homopolymeric template for the p66 subunit was poly(rA). For the p51 subunit, poly(dC) was found to be the optimal template; its activity is 2- to 4-fold greater than p66 on poly(dC). The p51 subunit is 13- to 50-fold less active on poly(rC). These findings are discussed in the context of our earlier hypothesis (McHenry, C. S. (1989) in Molecular Biology of Chromosome Function (Adolph, K., ed) Chap. 5, Springer-Verlag, New York) that the HIV reverse transcriptase might be functionally asymmetric with distinct plus- and minus-strand polymerases.
Thimmig
R L
RL
Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver 80262.
McHenry
C S
CS
D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004279:DNA, Viral; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D054303:HIV Reverse Transcriptase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
D004279:DNA, Viral; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase