Schiebel W, Haas B, Marinković S, Klanner A, Sänger H L
Max-Planck-Institut für Biochemie, Martinsried, Germany.
J Biol Chem. 1993 Jun 5;268(16):11858-67.
The catalytic properties of electrophoretically homogeneous RNA-directed RNA polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue were studied with the aid of oligonucleotides of defined sequence. It was found that RdRP catalyzes in vitro the transcription of short single-stranded RNA and DNA molecules into precisely complementary RNA copies up to the full length of these templates. The transcription of RNA- and DNA-oligonucleotide templates was equally effective. Differences in transcription efficiency were found to depend on nucleotide sequence rather than on the RNA or DNA nature of the single-stranded nucleic acid. Double-stranded nucleic acids such as poly(A).poly(U) and a double-stranded DNA 14-mer were not transcribed. The RdRP-directed transcription could be primed because RNA and DNA dinucleotides and trinucleotides complementary to the 3'-terminal nucleotides of the template were extended by the enzyme. The unprimed transcription was shown to start preferentially at the 3'-terminal nucleotides of the template. RdRP is capable of adding a single noncomplementary nucleotide to the 3' terminus of about 50% of the runoff transcripts. AMP was preferred over GMP, whereas CMP and UMP were terminally added at very low frequency.
借助特定序列的寡核苷酸，研究了来自番茄叶片组织的电泳纯RNA指导的RNA聚合酶（RdRP，EC 2.7.7.48）的催化特性。发现RdRP在体外催化短单链RNA和DNA分子转录为精确互补的RNA拷贝，直至这些模板的全长。RNA和DNA寡核苷酸模板的转录同样有效。发现转录效率的差异取决于核苷酸序列，而不是单链核酸的RNA或DNA性质。双链核酸如聚（A）·聚（U）和双链DNA 14聚体不被转录。RdRP指导的转录可以被引发，因为与模板3'末端核苷酸互补的RNA和DNA二核苷酸及三核苷酸可被该酶延伸。未引发的转录显示优先从模板的3'末端核苷酸开始。RdRP能够在约50%的连续转录本的3'末端添加单个非互补核苷酸。AMP比GMP更受青睐，而CMP和UMP在末端添加的频率非常低。
