Womble D D, Sampathkumar P, Easton A M, Luckow V A, Rownd R H
J Mol Biol. 1985 Feb 5;181(3):395-410. doi: 10.1016/0022-2836(85)90228-1.
The minimal replicon of the 90,000 base-pair IncFII R plasmid NR1 consists of a 2700 base-pair region of the DNA. Minireplicator plasmids consisting of the 2700 base-pair minimal replicon plus a 2200 base-pair region coding for chloramphenicol acetyltransferase (cat) were used as templates for in vitro transcription. Six RNA transcripts were synthesized from these templates in vitro. We have determined the directions of transcription and the approximate sites of initiation and termination of each of the in vitro RNA transcripts. One RNA transcript was synthesized from the cat gene, while the other five were transcribed from the minimal replicon. Four of the RNA transcripts also were identified by quantitative hybridization of RNA synthesized in vivo from these minireplicator plasmids. The strengths of the promoters for the RNA transcripts were estimated by the relative rates of transcription both in vitro and in vivo. Transcription from convergent promoters reduced the rate of RNA synthesis in vivo in both directions. In vivo, a significant fraction of the cat mRNA was extended past its in vitro termination point. Transcription of mutants that have altered plasmid copy number and/or incompatibility properties also were examined. The possible roles of each of the transcripts as mRNA and their involvement in regulation of DNA replication are discussed.
90,000碱基对的IncFII R质粒NR1的最小复制子由一段2700碱基对的DNA区域组成。由2700碱基对的最小复制子加上一段编码氯霉素乙酰转移酶（cat）的2200碱基对区域组成的微型复制体质粒被用作体外转录的模板。从这些模板在体外合成了六种RNA转录本。我们已经确定了体外RNA转录本各自的转录方向以及起始和终止的大致位点。一种RNA转录本由cat基因合成，而其他五种则从最小复制子转录而来。其中四种RNA转录本也通过对这些微型复制体质粒在体内合成的RNA进行定量杂交得以鉴定。通过体外和体内的相对转录速率估计了RNA转录本启动子的强度。来自汇聚启动子的转录降低了体内两个方向的RNA合成速率。在体内，相当一部分cat mRNA延伸超过了其体外终止点。还研究了具有改变的质粒拷贝数和/或不相容性特性的突变体的转录情况。讨论了每种转录本作为mRNA的可能作用及其在DNA复制调控中的参与情况。
