Poliovirus RNA-Dependent RNA Polymerase (3D^pol):  Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mg²⁺

Abstract

We have solved the complete kinetic mechanism for correct nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3D pol . The phosphoryl-transfer step is flanked by two isomerization steps. The first conformational change may be related to reorientation of the triphosphate moiety of the bound nucleotide, and the second conformational change may be translocation of the enzyme into position for the next round of nucleotide incorporation. The observed rate constant for nucleotide incorporation by 3D pol (86 s -1 ) is dictated by the rate constants for both the first conformational change (300 s -1 ) and phosphoryl transfer (520 s -1 ). Changes in the stability of the "activated" ternary complex correlate best with changes in the observed rate constant for incorporation resulting from modification of the nucleotide. With the exception of UTP, the K d values for nucleotides are at least 10-fold lower than the cellular concentration of the corresponding nucleotide. Our data predict that transition mutations should occur at a frequency of 1/15000, transversion mutations should occur at a frequency of less than 1/150000, and incorporation of a 2′-deoxyribonucleotide with a correct base should occur at a frequency 1/7500. Together, these data support the conclusion that 3D pol is actually as faithful as an exonuclease-deficient, replicative DNA polymerase. We discuss the implications of this work on the development of RNA-dependent RNA polymerase inhibitors for use as antiviral agents.One positive aspect of the human immunodeficiency virus (HIV) 1 pandemic was the realization that, given the appropriate effort, antiviral agents targeting enzymes required for virus multiplication can be developed. The first HIV-encoded enzyme that emerged as a tractable target was the viral polymerase, reverse transcriptase (RT) (1-3). While most of the inhibitors of HIV RT currently in use are nucleoside analogues (1,4), many nonnucleoside analogue inhibitors of HIV RT have also been reported (1,4). In all cases, however, an understanding of the specificity and/or precise mechanism of action of these inhibitors has required evaluation of these compounds in the context of the complete kinetic mechanism for nucleotide incorporation catalyzed by this enzyme (1,4-7) and has benefited from structural information available for HIV RT in the absence and presence of substrates and/or inhibitors (8)(9)(10)(11)(12)(13)(14). Kinetic analysis of site-directed mutants of HIV RT has established some of the fundamental mechanisms employed by this enzyme to recognize its substrates, both nucleic acid and nucleotide, and to cooperate with other viral proteins during the process of converting the viral single-stranded RNA genome into double-stranded DNA (1,4,15,16). In addition, mechanistic studies of HIV RT have suggested alternative strategies to target regions of this enzyme distinct † This work was supported, in part, by a Howard Temin Award (CA75118) from the NCI, National Institutes of Health, and by a grant ...

Two proton transfers in the transition state for nucleotidyl transfer catalyzed by RNA- and DNA-dependent RNA and DNA polymerases

“…9A). The dependence of the magnitude of the isotope effect on the divalent cation used was consistent with previous observations that chemistry is partially rate limiting in Mg 2ϩ but solely rate limiting in Mn 2ϩ (1,11). However, to rule out conformational perturbations as the cause of the observed isotope effects, AMP incorporation in the steady state was evaluated.…”

“…2 A). Because a conformational change is partially rate limiting in Mg 2ϩ (1), these values may not reflect true pK a values (31), preventing unambiguous assignment of these pK a values to specific proton-transfer reactions. In Mn 2ϩ , however, chemistry is completely rate limiting so the pK a value of 8.2 observed here (Fig.…”

“…All classes of polymerases use the same five-step kinetic scheme for nucleotide incorporation (1)(2)(3)(4)(5)(6). The kinetic mechanism for the RNAdependent RNA polymerase (RdRp 1 ) from poliovirus (3D pol ) is shown in Scheme 1.…”

“…In one, chemistry (step three) is rate-limiting (10), and in some (e.g., T4 and RB69 DNA polymerases), the rate-limiting step has not been established. For 3D pol , both steps two and three are rate limiting when Mg 2ϩ is used as the divalent cation cofactor, and step three is rate limiting when Mn 2ϩ is used (1,11). Therefore, the 3D pol system provides a unique opportunity to interrogate the chemistry of polymerase catalyzed nucleotidyl transfer by using a nucleotide incorporation assay.…”

“…All pre-steady-state and steady-state experiments were performed as described (1,7,11,39,47). All details, including modifications, are provided in SI Text.…”