Qi F, Turnbough C L
Department of Microbiology, University of Alabama at Birmingham 35294, USA.
J Mol Biol. 1995 Dec 8;254(4):552-65. doi: 10.1006/jmbi.1995.0638.
Reiterative transcription is the repetitive addition of nucleotides to the 3' end of a nascent transcript due to slippage between the transcript and DNA template. Recently, we showed that pyrimidine-mediated regulation of pyrBI operon expression in Escherichia coli occurs, in part, through a mechanism in which induction of UTP-dependent reiterative transcription within the initially transcribed region prevents downstream extension of the nascent transcript to include structural gene sequences. In this study we demonstrate that pyrimidine-mediated regulation of codBA operon expression in E. coli also involves UTP-dependent reiterative transcription during initiation; however, the mechanism is different from that of the pyrBI operon. The initially transcribed region of the codBA promoter contains the sequence GATTTTTTG (non-template strand). Our results show that transcription is initiated primarily at the first two bases designated G7 and A8 (counting from the -10 region). When transcripts are initiated at position A8, UTP-dependent reiterative transcription always occurs within the run of T residues in the initially transcribed region. The AUUUUn (where n = 1 to > 15) transcripts produced by this reaction are not extended productively to include downstream codBA sequences. In contrast, most transcripts initiated at position G7 do not engage in reiterative transcription and can be elongated normally. Characterization of a codBA promoter mutation that prevents reiterative transcription showed that this reaction is required for virtually all pyrimidine-mediated regulation of operon expression and that UTP levels control the selection of the G7 and A8 transcriptional start sites. These results suggest a model for regulation in which high intracellular levels of UTP favor transcriptional initiation at position A8 and thus the accompanying reiterative transcription, which together preclude initiation at position G7. Low levels of UTP inhibit initiation at position A8 and the associated reiterative transcription, thereby allowing high levels of initiation at position G7 and operon expression. Our results also indicate critical sequence requirements for reiterative transcription, which are important for understanding the mechanism of this reaction as well as for identifying other promoters at which this reaction may occur. Of particular interest is the indication that an RNA:DNA hybrid forms during transcriptional initiation and the strength of this hybrid controls the extent of reiterative transcription.
重复转录是由于新生转录本与DNA模板之间的滑动，导致核苷酸重复添加到新生转录本的3'末端。最近，我们发现大肠杆菌中嘧啶介导的pyrBI操纵子表达调控部分是通过一种机制实现的，即在最初转录区域内由UTP依赖的重复转录诱导，阻止新生转录本向下游延伸以包含结构基因序列。在本研究中，我们证明大肠杆菌中嘧啶介导的codBA操纵子表达调控在起始阶段也涉及UTP依赖的重复转录；然而，其机制与pyrBI操纵子不同。codBA启动子的最初转录区域包含序列GATTTTTTG（非模板链）。我们的结果表明，转录主要起始于指定为G7和A8的前两个碱基（从-10区域开始计数）。当转录本在A8位置起始时，UTP依赖的重复转录总是发生在最初转录区域的T残基序列中。该反应产生的AUUUUn（其中n = 1至>15）转录本不会有效延伸以包含下游的codBA序列。相反，大多数在G7位置起始的转录本不会进行重复转录，并且可以正常延伸。对一个阻止重复转录的codBA启动子突变的表征表明，该反应实际上是所有嘧啶介导的操纵子表达调控所必需的，并且UTP水平控制着G7和A8转录起始位点的选择。这些结果提示了一种调控模型，即细胞内高浓度的UTP有利于在A8位置起始转录，从而伴随着重复转录，这两者共同阻止在G7位置起始转录。低水平的UTP抑制在A8位置起始转录以及相关的重复转录，从而允许在G7位置高水平起始转录和操纵子表达。我们的结果还表明了重复转录的关键序列要求，这对于理解该反应的机制以及识别可能发生该反应的其他启动子都很重要。特别值得关注的是，有迹象表明在转录起始过程中会形成RNA:DNA杂交体，并且这种杂交体的强度控制着重复转录的程度。
