Interaction of primer tRNA^Lys3 with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation

Abstract

In the interaction between HIV-1 RT and tRNA Lys3 each subunit of the heterodimer interacts with tRNA showing a different affinity: Kd (p66) = 23 nM, Kd (pS1) = 140 nM. Preincubation of heterodimeric RT with tRNA, at concentrations similar to that of the Kd value for p51, leads to an increase of the catalytic activity on poly(A)-oligo(dT). These results were compared to those using different tRNA analogs: oxidized tRNA, tRNAs lacking one, two or three nucleotides from the 3'-end, or ribo-and deoxyribonucleotides mimicking the anticodon loop sequence. In all cases, tRNA analogs were weaker activators of HIV-1 liT than natural tRNA. A possible mechanism of RT p661p51 activation by tRNA and its analogs, mediated through the p5I subunit, is discussed.

DNA synthesis primed by mononucleotides (de novo synthesis) catalyzed by HIV‐1 reverse transcriptase: tRNALys,3 activation

“…We have also shown that the preincubation of HIV-1 RT (either the homodimer p66/p66 [16] or the heterodimer p66/p51 [10]) with tRNA Ly~' 3 led to conformational changes of the enzyme that increases its activity when the assay was performed with the classical poly(A)-oligo(dT) primer-template duplex. [3H]dTTP was used both as primer and as nucleotide precursor.…”

“…We have already described that the maximal activation of HIV-1 RT by tRNA LyS' 3 takes place at concentrations oftRNA allowing each subunit of the p66/p51 HIVol RT to form a complex with tRNA Lys' 3 [10]. The next step was to determine, in the presence of tRNA, the concentration of each compound allowing optimal activation.…”

“…Complex formation with primer tRNA ,nduces significant structural changes in HIV-1 RT, as followed ~y fluorescence emission or by the accessibility of the RT p66/ ~51.tRNA complex to chymotrypsin [9]. Preincubation of ~IIV-1 RT with either tRNA Lys'3 or oligonucleotides mimicking :he anticodon loop sequence of tRNA Lye'3 leads to an increase 9f the catalytic activity on poly(A)-oligo(dT) [10]. Each subunit )fthe heterodimeric form of HIV-I RT (p66/p51) interacts with *Corresponding author.…”

“…Our previous results indicate that the activation of DNA polymerase activity associated to the p66 subunit would result from the interaction of primer tRNA with the p51 subunit of p66/p51 HIV-1 RT [10]. Most DNA polymerases initiates DNA synthesis from a short oligonucleotide.…”

Interaction of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with Primer tRNALys3 and Affinity Modification of The Enzyme by tRNALys3 Derivatives

“…The stoichiometry and Kd values of the reverse transcriptase and tRNA'2y'' complex formation have been estimated from a Scatchard plot using spectrofluorometric titration data. The p66/p51 heterodimer binds two molecules of tRNA with different affinities (Zakharova et al, 1995a).…”