Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension
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- 作者：Margaret E. Glasner ; Catherine C. Yen ; Eric H. Ekland ; and David P. Bartel
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：December 19, 2000
- 年：2000
- 卷：39
- 期：50
- 页码：15556 - 15562
- 全文大小：112K
- 年卷期：v.39,no.50(December 19, 2000)
- ISSN：1520-4995
文摘
In support of the idea that certain RNA molecules might be able to catalyze RNA replication,a ribozyme was previously generated that synthesizes short segments of RNA in a reaction modeled afterthat of proteinaceous RNA polymerases. Here, we describe substrate recognition by this polymeraseribozyme. Altering base or sugar moieties of the nucleoside triphosphate only moderately affects itsutilization, provided that the alterations do not disrupt Watson-Crick pairing to the template. Correctlypaired nucleotides have both a lower Km and a higher kcat, suggesting that differential binding and orientationeach play roles in discriminating matched from mismatched nucleotides. Binding of the pyrophosphateleaving group appears weak, as evidenced by a very inefficient pyrophosphate-exchange reaction, thereverse of the primer-extension reaction. Indeed, substitutions at the 
hars/gamma.gif" BORDER=0 >-phosphate can be tolerated, althoughpoorly. Thio substitutions of oxygen atoms at the reactive phosphate exert effects similar to those seenwith cellular polymerases, leaving open the possibility of an active site analogous to those of proteinenzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve thevery fast kcat of the parent ribozyme when the substrate of the polymerase (GTP) is replaced by an extendedsubstrate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotidesof the ligase. This suggests that the GA dinucleotide, which had been deleted when converting the ligaseinto a polymerase, plays an important role in orienting the 5'-terminal nucleoside. Polymerase constructsthat restore this missing orientation function should achieve much more efficient and perhaps more accurateRNA polymerization.
hars/gamma.gif" BORDER=0 >-phosphate can be tolerated, althoughpoorly. Thio substitutions of oxygen atoms at the reactive phosphate exert effects similar to those seenwith cellular polymerases, leaving open the possibility of an active site analogous to those of proteinenzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve thevery fast kcat of the parent ribozyme when the substrate of the polymerase (GTP) is replaced by an extendedsubstrate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotidesof the ligase. This suggests that the GA dinucleotide, which had been deleted when converting the ligaseinto a polymerase, plays an important role in orienting the 5'-terminal nucleoside. Polymerase constructsthat restore this missing orientation function should achieve much more efficient and perhaps more accurateRNA polymerization.