HIV-1 reverse transcriptase shows no specificity for the binding of primer tRNA(Lys3).
The transcription initiation primer for HIV-1 is a specific cellular tRNA species, tRNA(Lys3). We used several methods to assess the binding of tRNA by recombinant HIV-1 p51/p66 reverse transcriptase (RT), gel retardation analysis, intrinsic RT protein fluorescence quenching, and nitrocellulose filter binding assays. The binding of tRNA to RT was saturable, implying a distinct site or sites on the enzyme for tRNA interaction. However, this binding was non-selective, with all tRNA isoacceptors and total unfractionated tRNA binding with similar affinity as primer tRNA(Lys3). In contrast, no significant binding of tRNA by RT was noted. Our results show that HIV-1 RT has no specificity for the binding of primer tRNA(Lys3), and imply that factors other than RT sequences may be important for the selective incorporation of primer tRNA into the virion particle.
Arion
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D
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis, Jewish General Hospital, Montreal, Quebec, Canada.
Harada
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R
Li
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X
Wainberg
M A
MA
Parniak
M A
MA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
C034477:RNA primers; D012329:RNA, Bacterial; D012357:RNA, Transfer, Lys; D011994:Recombinant Proteins; D012313:RNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase