Poliovirus RNA-dependent RNA polymerase (3Dpol): pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of Mg2+.
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.
Arnold
Jamie J
JJ
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Cameron
Craig E
CE
D000249:Adenosine Monophosphate / Q000737:chemistry; D002384:Catalysis; D002413:Cations, Divalent; D004256:DNA Polymerase I / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D011756:Diphosphates / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry*; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D017955:Poliovirus / Q000201:enzymology* / Q000502:physiology; D012323:RNA Processing, Post-Transcriptional; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D012265:Ribonucleotides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013698:Templates, Genetic; D013816:Thermodynamics; D019065:Virus Assembly
D002413:Cations, Divalent; D017931:DNA Primers; D011756:Diphosphates; D012265:Ribonucleotides; D000249:Adenosine Monophosphate; C034970:bacteriophage T7 induced DNA polymerase; C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium