Hart C M, Roberts J W
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
J Biol Chem. 1991 Dec 15;266(35):24140-8.
By substituting template segments encoding AU-rich, GU-rich, and CA-rich transcripts for natural sequences upstream of the phage lambda rho-dependent tR1 termination site, we demonstrate that cytidines are required in the upstream RNA for rho-dependent termination to occur. These results are extended through in vitro mutagenesis of a template encoding an inactive AU-rich upstream sequence: certain mutant templates encoding new cytidines are able to activate rho-dependent termination. Cytidines must be dispersed over a region of the transcript in order for rho to be activated, although no specific pattern of cytidines appears to be required. The results show that no local clustering or regular spacing of cytidines is necessary and that cytidines are not used as a "ruler" to determine the location of termination sites. Rho is somewhat sensitive to the relative positions of cytidines since slightly different nascent transcripts activate rho termination activity to various degrees. A model is presented in which hexameric rho binds 78 nucleotides of contiguous RNA in a primary site, such that each monomer interacts with at least one cytidine somewhere in the 13 nucleotides allotted to the monomeric primary site.
通过将编码富含AU、富含GU和富含CA转录本的模板片段替换为噬菌体λrho依赖性tR1终止位点上游的天然序列，我们证明上游RNA中的胞嘧啶对于rho依赖性终止的发生是必需的。这些结果通过对编码无活性富含AU上游序列的模板进行体外诱变得到扩展：某些编码新胞嘧啶的突变模板能够激活rho依赖性终止。胞嘧啶必须分散在转录本的一个区域上才能激活rho，尽管似乎不需要特定的胞嘧啶模式。结果表明，胞嘧啶不需要局部聚集或规则间隔，并且胞嘧啶不用作确定终止位点位置的“尺子”。由于稍有不同的新生转录本会不同程度地激活rho终止活性，因此rho对胞嘧啶的相对位置有些敏感。我们提出了一个模型，其中六聚体rho在一个主要位点结合78个连续RNA核苷酸，使得每个单体在分配给单体主要位点的13个核苷酸中的某处与至少一个胞嘧啶相互作用。
