Shuman S, Moss B
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
J Biol Chem. 1988 May 5;263(13):6220-5.
Transcription termination in vitro by vaccinia RNA polymerase is dependent on a trans-acting factor, VTF, that is associated with, if not identical to, the vaccinia mRNA capping enzyme. VTF-induced termination occurs approximately 50 nucleotides downstream of a signal sequence TTTTTNT in the non-transcribed templated strand; thus the cognate sequence UUUUUNU is expressed in the nascent RNA. To address the role of the nascent RNA in chain termination, the effects of nucleotide base analog substitutions were studied. Incorporation of bromo- (Br) UMP or iodo- (I) UMP into RNA abrogated factor-dependent termination without preventing the synthesis of read-through transcripts. Substitution of either ITP or 7'-methylguanosine for GTP did not inhibit factor-dependent termination, nor did the substitution of BrCTP or ICTP for CTP. The early transcripts synthesized in vitro were sensitive to RNase T2 but resistant to RNase H, indicating an absence of extensive hybridization of RNA product to the DNA template. Substitution of BrUTP for UTP did not alter the nuclease sensitivity of the transcripts, suggesting that increased stability of RNA:DNA hybrid structures did not account for the analog effects. These results are consistent with a model in which recognition of the primary sequence UUUUUNU in nascent RNA by the polymerase and/or VTF is required for transcription termination.
牛痘病毒RNA聚合酶在体外的转录终止依赖于一种反式作用因子VTF，该因子即便与牛痘病毒mRNA加帽酶不完全相同，也与之相关。VTF诱导的终止发生在非转录模板链上信号序列TTTTTNT下游约50个核苷酸处；因此，新生RNA中会表达同源序列UUUUUNU。为了探究新生RNA在链终止中的作用，研究了核苷酸碱基类似物取代的影响。将溴尿苷单磷酸（BrUMP）或碘尿苷单磷酸（IUMP）掺入RNA会消除因子依赖性终止，但不会阻止通读转录本的合成。用肌苷三磷酸（ITP）或7'-甲基鸟苷取代鸟苷三磷酸（GTP）不会抑制因子依赖性终止，用溴胞苷三磷酸（BrCTP）或碘胞苷三磷酸（ICTP）取代胞苷三磷酸（CTP）也不会。体外合成的早期转录本对核糖核酸酶T2敏感，但对核糖核酸酶H有抗性，这表明RNA产物与DNA模板不存在广泛杂交。用溴尿苷三磷酸（BrUTP）取代尿苷三磷酸（UTP）不会改变转录本对核酸酶的敏感性，这表明RNA:DNA杂交结构稳定性的增加并不能解释类似物的作用。这些结果与一种模型一致，即转录终止需要聚合酶和/或VTF识别新生RNA中的一级序列UUUUUNU。
