Kinetic analysis of the catalysis of strand transfer from internal regions of heteropolymeric RNA templates by human immunodeficiency virus reverse transcriptase.
The kinetic mechanism of HIV reverse transcriptase catalyzed strand transfer synthesis (i.e. switching of the primer to a new template) from internal regions of natural sequence RNA was investigated. The system consisted of a 142 nucleotide RNA template (donor), primed with a specific 20 nucleotide DNA oligonucleotide that was used to initiate synthesis. An RNA with homology to an internal region of the donor was used as acceptor template. Using 32P-labeled DNA oligonucleotide, the primer-extension products made from full-length synthesis on the donor (108 bases in length) or homologous transfer to and extension on the acceptor (155 bases) were monitored. Results indicated that the maximum efficiency of transfer (the ratio of transfer products to donor-directed+transfer products x 100) in this particular system was about 25% while the theoretical Vmax for the rate of appearance of transfer products at infinite acceptor concentration was about 20-fold lower than the measured rate for full-length donor-directed products. The Km for acceptor template in the transfer reaction was about 8 nM. Experiments using the above donor template hybridized to a specific DNA that has been shown to transfer to the acceptor indicated that RNase H-mediated rapid release of this DNA from the donor while subsequent association with the acceptor was relatively slow.
DeStefano
J J
JJ
University of Maryland, College Park 20742.
D002384:Catalysis; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018014:Gene Transfer Techniques; D054303:HIV Reverse Transcriptase; D006801:Humans; D007700:Kinetics; D016350:Leucine Zippers; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011108:Polymers; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D011995:Recombination, Genetic; D013379:Substrate Specificity; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000235:genetics
D017931:DNA Primers; D009841:Oligonucleotides; D011108:Polymers; D012367:RNA, Viral; C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase