Binding and kinetic properties of HIV-1 reverse transcriptase markedly differ during initiation and elongation of reverse transcription.
We recently showed that primer tRNA3Lys, human immunodeficiency virus type 1 (HIV-1) RNA and HIV-1 reverse transcriptase (RT) form a specific complex of initiation of reverse transcription that can be functionally distinguished from the elongation complex, which can be obtained by substituting an 18mer oligodeoxyribonucleotide (ODN) for the natural primer (Isel et al., 1996). Here, we compared the binding properties and the single and multiple turnover kinetics of HIV-1 RT in the initiation and elongation complexes. Even though the equilibrium dissociation constants of HIV-1 RT are not very different for the two complexes, RT dissociates approximately 200-fold faster from the initiation complex. Furthermore, nucleotide incorporation by the pre-formed primer-template-RT complexes is reduced by a approximately 50-fold factor during initiation of reverse transcription, compared with elongation. As a consequence, processivity of HIV-1 RT in the initiation complex is close to unity, while it increases by four orders of magnitude during elongation, as expected for a replication enzyme. This processivity change is reminiscent of the transition from initiation to elongation of transcription. Furthermore, our results indicate that the post-transcriptional modifications of tRNA3Lys play a role similar to that of the sigma factor in transcription by the Escherichia coli RNA polymerase: they favour the formation of the specific initiation complex but do not affect the polymerization rate of the bound enzyme.
Lanchy
J M
JM
Unité Propre de Recherche No. 9002 du Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
Ehresmann
C
C
Le Grice
S F
SF
Ehresmann
B
B
Marquet
R
R
D017931:DNA Primers; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D007700:Kinetics; D011485:Protein Binding; D013698:Templates, Genetic; D014158:Transcription, Genetic
D017931:DNA Primers; D054303:HIV Reverse Transcriptase