Catalytic features of the recombinant reverse transcriptase of bovine leukemia virus expressed in bacteria.
We have expressed the recombinant reverse transcriptase (RT) of bovine leukemia virus (BLV) in bacteria. The gene encoding the RT was designed to start at its 5' end next to the last codon of the mature viral protease, namely the amino terminus of the RT matches the last 26 codons of the pro gene and is coded for by the pro reading frame. The RT sequence extends into the pol gene, utilizing the pol reading frame after overcoming the stop codon by adding an extra nucleotide (thus imitating the naturally occurring frameshift event). Hence we have generated a transframe polypeptide that is a 584-residues-long protein (see Rice, Stephens, Burny, and Gilden (1985) Virology 142, 357-377). This protein was partially purified after adding a six-histidine tag and studied biochemically testing a variety of parameters. The enzyme exhibits all activities typical of RTs, i.e., both RNA- and DNA-dependent DNA polymerase as well as a ribonuclease H (RNase H) activity. Unlike most RTs, the BLV RT is enzymatically active as a monomer even after binding a DNA substrate. The enzyme shows a preference for Mg2+ over Mn2+ in both its DNA polymerase and RNase H activities. BLV RT is relatively resistant to nucleoside triphosphate analogues, which are known to be potent inhibitors of other RTs such as that of HIV.
Perach
M
M
Sackler School of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel.
Hizi
A
A
D000818:Animals; D002384:Catalysis; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D001909:Leukemia Virus, Bovine / Q000201:enzymology / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase