Poliovirus RNA-dependent RNA polymerase (3D(pol)). Assembly of stable, elongation-competent complexes by using a symmetrical primer-template substrate (sym/sub).
Detailed studies of the kinetics and mechanism of nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus, 3D(pol), have been limited by the inability to assemble elongation complexes that permit activity to be monitored by extension of end-labeled primers. We have solved this problem by employing a short, symmetrical, heteropolymeric RNA primer-template that we refer to as "sym/sub." Formation of 3D(pol)-sym/sub complexes is slow owing to a slow rate of association (0.1 microM(-1) s(-1)) of 3D(pol) and sym/sub and a slow isomerization (0. 076 s(-1)) of the 3D(pol)-sym/sub complex that is a prerequisite for catalytic competence of this complex. Complex assembly is stoichiometric under conditions in which competing reactions, such as enzyme inactivation, are eliminated. Inactivation of 3D(pol) occurs at a maximal rate of 0.051 s(-1) at 22 degrees C in reaction buffer lacking nucleotide. At this temperature, ATP protects 3D(pol) against inactivation with a K(0.5) of 37 microM. Once formed, 3D(pol)-sym/sub elongation complexes are stable (t((1)/(2)) = 2 h at 22 degrees C) and appear to contain only a single polymerase monomer. In the presence of Mg(2+), AMP, 2'-dAMP, and 3'-dAMP are incorporated into sym/sub by 3D(pol) at rates of 72, 0.6, and 1 s(-1), respectively. After incorporation of AMP, 3D(pol)-sym/sub product complexes have a half-life of 8 h at 22 degrees C. The stability of 3D(pol)-sym/sub complexes is temperature-dependent. At 30 degrees C, there is a 2-8-fold decrease in complex stability. Complex dissociation is the rate-limiting step for primer utilization. 3D(pol) dissociates from the end of template at a rate 10-fold faster than from internal positions. The sym/sub system will facilitate mechanistic analysis of 3D(pol) and permit a direct kinetic and thermodynamic comparison of the RNA-dependent RNA polymerase to the other classes of nucleic acid polymerases.
Arnold
J J
JJ
Department of Biochemistry, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Cameron
C E
CE
D000255:Adenosine Triphosphate / Q000378:metabolism; D020137:Base Pair Mismatch; D002712:Chlorides / Q000494:pharmacology; D017931:DNA Primers / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D007700:Kinetics; D017895:Manganese Compounds / Q000494:pharmacology; D008954:Models, Biological; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase; D013696:Temperature; D013698:Templates, Genetic; D013997:Time Factors; D014158:Transcription, Genetic; D014542:Uridine Monophosphate / Q000378:metabolism
D002712:Chlorides; D017931:DNA Primers; D017895:Manganese Compounds; D012367:RNA, Viral; D000255:Adenosine Triphosphate; D014542:Uridine Monophosphate; C090443:polymerase 3Dpol, poliovirus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; C025340:manganese chloride