Rate-limiting Pyrophosphate Release by HIV Reverse Transcriptase Improves Fidelity

Abstract

Previous measurements of the rates of polymerization and pyrophosphate release with DNA templates showed that pyrophosphate (PP) dissociation was fast after nucleotide incorporation so that it did not contribute to enzyme specificity (k/K). Here, kinetic parameters governing nucleotide incorporation and PP release were determined using an RNA template. Compared with a DNA template of the same sequence, the rate of chemistry increased by up to 10-fold (250 versus 24 s), whereas the rate of PP release decreased to approximately 58 s so that PP release became the rate-limiting step. During processive nucleotide incorporation, the first nucleotide (TTP) was incorporated at a fast rate (152 s), whereas the rates of incorporation of remaining nucleotides (CGTCG) were much slower with an average rate of 24 s, suggesting that sequential incorporation events were limited by the relatively slow PP release step. The accompanying paper shows that slow PP release allows polymerization and RNase H to occur at comparable rates. Although PP release is the rate-determining step, it is not the specificity-determining step for correct incorporation based on our current estimates of the rate of reversal of the chemistry step (3 s). In contrast, during misincorporation, PP release became extremely slow, which we estimated to be ∼0.002 s These studies establish the mechanistic basis for DNA polymerase fidelity during reverse transcription and provide a free energy profile. We correct previous underestimates of discrimination by including the slow PP release step. Our current estimate of 2.4 × 10 is >20-fold greater than estimated previously.

Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance

“…Presumably, the altered conformation of the dNTPbinding pocket in Q151Mc is able to discriminate the flexible sugar ring of a ddNTP from the 3=-endo dNTP during the process of catalytic incorporation. Recent kinetic studies revealed that the lower rate of pyrophosphate (PPi) release by RT could decrease the rate of polymerization (43). Our recent structure of the drug foscarnet (PFA), a pyrophosphate analog, in complex with RT/DNA showed that R72 directly interacts with PFA (32), and the interaction likely plays an important role in PPi release.…”

Architecture of an HIV-1 reverse transcriptase initiation complex

“…The global structure here is represented in surface mode to highlight potential steric clashes. c, Reaction coordinate diagram and corresponding reaction scheme of HIV-1 reverse transcription on an RNA template using published values 29 . The +1 RTIC appears to be trapped in a pre-translocation state (green box).…”

“…The fingers domain of RT adopts a semi-open conformation similar to that of RT structures bound to nucleic acids that lack an incoming nucleotide 2,22 (Figure 4a, Extended Data Figure 9). Based on prior mechanistic studies of RT enzymology 8,29 , the RTIC here is blocked in a pre-translocation conformation for the primer-template complex (Figure 4c). Unlike NNRTI-bound complexes, the RTIC is functional and can incorporate the next dNTP, suggesting conformational plasticity within the RT active site.…”

“…Although RT contacts the PBS in the cleft, the disrupted palm subdomain contacts between the PBS and RT explain the poor affinity of RT for the vRNA/tRNA complex. Within the framework of the standard dNTP incorporation mechanism 29 , RT in this +1 initiation complex adopts a pre-translocation conformation with an open active site and improper positioning of nucleic acid for catalysis (Figure 4c). The vRNA helices, whose orientation hinders productive binding to RT, must be displaced and/or unfolded for the tRNA primer terminus to reposition within the active site such that the RT fingers can clamp down on an incoming nucleotide (Figure 4b).…”

Pyrophosphate Release in the Protein HIV Reverse Transcriptase

“…Moreover, if RNA template (instead of DNA) is considered, step (4) was shown experimentally to be slower than step (3). 6 The time scales of the chemical and the escape steps are therefore not very different and changes in templates (for example) can modify the rate-determining event.…”