Albrechtsen B, Squires C L, Li S, Squires C
Department of Biological Sciences, Columbia University, New York, NY 10027.
J Mol Biol. 1990 May 5;213(1):123-34. doi: 10.1016/S0022-2836(05)80125-1.
We have used a plasmid antitermination test system to examine the response of an Escherichia coli rRNA operon antiterminator to transcription through Rho-dependent and Rho-independent terminator-containing fragments. We also monitored transcription through multiple copies of a terminator to explore the mechanism of rrn antitermination. Four principal observations were made about antitermination and transcriptional terminators. (1) The rrn antiterminator mediated efficient transcription through Rho-dependent terminators. (2) Under the influence of the rrn antiterminator, RNA polymerase transcribed through two and three copies of the Rho-dependent 16 S----terminator with nearly the same efficiency as through one. (3) The antiterminator had less effect on fragments containing Rho-independent terminators; the rpoC t fragment and three fragments derived from the rrnB terminator region stopped antiterminated transcription. Four other Rho-independent terminator fragments were weakly antiterminated in our test system. (4) Surprisingly, the strength of these terminator fragments was not strongly related to properties such as the -delta G or number of trailing uridine residues of their canonical Rho-independent structures, but appears to be related to additional downstream terminators. We have drawn the following conclusions from these experiments. First, that ribosomal antitermination primarily reverses Rho-dependent termination by modifying the RNA polymerase elongation complex. Transcription through a 1700 nucleotide, multiple terminator sequence showed that the antiterminator caused persistent changes in the transcription process. Second, that fragments derived from the Rho-independent rrnB and rpoBC terminator regions can effectively stop antiterminated transcription. Third, that efficient in vivo termination may often involve regions with complex multiple terminators.
我们使用了一种质粒抗终止测试系统，来检测大肠杆菌rRNA操纵子抗终止子对通过含Rho依赖性和Rho非依赖性终止子片段的转录的反应。我们还监测了通过多个终止子拷贝的转录，以探究rrn抗终止的机制。关于抗终止和转录终止子，我们有四项主要观察结果。（1）rrn抗终止子介导了通过Rho依赖性终止子的高效转录。（2）在rrn抗终止子的影响下，RNA聚合酶转录通过两份和三份Rho依赖性16S----终止子，其效率与通过一份时几乎相同。（3）抗终止子对含Rho非依赖性终止子的片段影响较小；rpoC t片段和源自rrnB终止子区域的三个片段阻止了抗终止转录。在我们的测试系统中，其他四个Rho非依赖性终止子片段被弱抗终止。（4）令人惊讶的是，这些终止子片段的强度与其典型Rho非依赖性结构的-ΔG或尾随尿苷残基数量等特性没有很强的相关性，而是似乎与额外的下游终止子有关。我们从这些实验中得出了以下结论。第一，核糖体抗终止主要通过修饰RNA聚合酶延伸复合物来逆转Rho依赖性终止。通过一个1700个核苷酸的多终止子序列的转录表明，抗终止子在转录过程中引起了持续的变化。第二，源自Rho非依赖性rrnB和rpoBC终止子区域的片段可以有效地阻止抗终止转录。第三，高效的体内终止可能经常涉及具有复杂多个终止子的区域。
