TY - JOUR
T1 - Poliovirus RNA-dependent RNA polymerase (3D(pol)) is sufficient for template switching in vitro
AU - Arnold, Jamie J.
AU - Cameron, Craig E.
PY - 1999/1/29
Y1 - 1999/1/29
N2 - We have performed a systematic, quantitative analysis of the kinetics of nucleotide incorporation catalyzed by poliovirus RNA-dependent RNA polymerase, 3D(pol). Homopolymeric primer/templates of defined length were used to evaluate the contribution of primer and template length and sequence to the efficiency of nucleotide incorporation without the complication of RNA structure. Interestingly, thermodynamic stability of the duplex region of these primer/templates was more important for efficient nucleotide incorporation than either primer or template length. Surprisingly, products greater than unit length formed in all reactions regardless of length or sequence. Neither a distributive nor a processive slippage mechanism could be used to explain completely the formation of long products. Rather, the data were consistent with a template-switching mechanism. All of the nucleotide could be polymerized during the course of the reaction. However, very few primers could be extended, suggesting an inverse correlation between the efficiency of primer utilization and that of nucleotide incorporation. Therefore, the greatest fraction of incorporated nucleotide derives from a small fraction of enzyme when radioactive nucleotide and homopolymeric primer/template substrates are employed. The impact of these results on mechanistic studies of 3D(pol)-catalyzed nucleotide incorporation and RNA recombination are discussed.
AB - We have performed a systematic, quantitative analysis of the kinetics of nucleotide incorporation catalyzed by poliovirus RNA-dependent RNA polymerase, 3D(pol). Homopolymeric primer/templates of defined length were used to evaluate the contribution of primer and template length and sequence to the efficiency of nucleotide incorporation without the complication of RNA structure. Interestingly, thermodynamic stability of the duplex region of these primer/templates was more important for efficient nucleotide incorporation than either primer or template length. Surprisingly, products greater than unit length formed in all reactions regardless of length or sequence. Neither a distributive nor a processive slippage mechanism could be used to explain completely the formation of long products. Rather, the data were consistent with a template-switching mechanism. All of the nucleotide could be polymerized during the course of the reaction. However, very few primers could be extended, suggesting an inverse correlation between the efficiency of primer utilization and that of nucleotide incorporation. Therefore, the greatest fraction of incorporated nucleotide derives from a small fraction of enzyme when radioactive nucleotide and homopolymeric primer/template substrates are employed. The impact of these results on mechanistic studies of 3D(pol)-catalyzed nucleotide incorporation and RNA recombination are discussed.
UR - http://www.scopus.com/inward/record.url?scp=0033613849&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0033613849&partnerID=8YFLogxK
U2 - 10.1074/jbc.274.5.2706
DO - 10.1074/jbc.274.5.2706
M3 - Article
C2 - 9915801
AN - SCOPUS:0033613849
SN - 0021-9258
VL - 274
SP - 2706
EP - 2716
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 5
ER -