Human immunodeficiency virus reverse transcriptase expressed in transformed yeast cells. Biochemical properties and interactions with bovine tRNALys.
Human immunodeficiency virus (HIV) reverse transcriptase has been purified from yeast transformed by an autoreplicating plasmid containing the retroviral DNA polymerase gene. The previously described purification procedure for the yeast-expressed reverse transcriptase [Barr, P.J., Power, M.D., Chun Ting Lee-Ng, Gibson, H. & Luciw, P. (1987) Bio/Technology 5, 486-489] has been substantially modified, leading to an increased yield and a higher degree of purity. Several biochemical properties of the enzyme are described (template specificity, effect of DNA synthesis inhibitors); interestingly, HIV reverse transcriptase is highly resistant to N-ethylmaleimide. A complex between the human retroviral enzyme and the bovine tRNALys was shown, using a direct approach, by glycerol gradient centrifugation, as well as by the protective and specific effect of the tRNALys against enzyme inactivation by thermal denaturation and trypsin digestion. A competitive type of inhibition of HIV reverse transcriptase by tRNALys, but not by tRNAVal, is observed when viral RNA or activated DNA are used as templates.
Sallafranque-Andreola
M L
ML
Institut de Biochimie Cellulaire et de Neurochimie du Centre National de la Recherche Scientifique, Bordeaux, France.
Robert
D
D
Barr
P J
PJ
Fournier
M
M
Litvak
S
S
Sarih-Cottin
L
L
Tarrago-Litvak
L
L
D000818:Animals; D002417:Cattle; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification; D005814:Genes, Viral; D005822:Genetic Vectors; D006678:HIV / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008970:Molecular Weight; D010957:Plasmids; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012357:RNA, Transfer, Lys / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013698:Templates, Genetic; D014170:Transformation, Genetic; D015678:Viral Structural Proteins / Q000235:genetics*
D012345:RNA, Transfer, Amino Acid-Specific; D012357:RNA, Transfer, Lys; D015678:Viral Structural Proteins; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase