Gamper M, Ganter B, Polito M R, Haas D
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
J Mol Biol. 1992 Aug 20;226(4):943-57. doi: 10.1016/0022-2836(92)91044-p.
Anaerobic growth of Pseudomonas aeruginosa on arginine depends on the arcDABC operon encoding the enzymes of the arginine deiminase pathway. The co-ordinate, anaerobic induction of these enzymes requires the FNR-like regulatory protein ANR, which activates the arc promoter lying upstream from arcD. By Northern hybridization experiments, three abundant arcA, arcAB and arcABC transcripts and three minor arcDA, arcDAB and arcDABC transcripts could be detected. The 5' ends of the arcA, arcAB and arcABC mRNAs were determined by S1 and primer extension mapping. These 5' ends appear to be generated by endonucleolytic cleavage (processing) in arcD mRNA rather than by a second promoter; this was concluded from the effects of insertion and deletion mutations in arcD. Intergenic inverted repeats between arcA and arcB as well as between arcB and arcC were shown to be involved in the formation of 3' ends of arc transcripts. Deletion of either intergenic region in the P. aeruginosa chromosome led to the loss of the arcA or arcAB transcript, respectively. Dot blot experiments revealed that arc mRNAs extracted from the wild-type strain had similar chemical half-lives in the arcA, arcB and arcC regions, ranging from 16 to 13 minutes. The half-life of arcD mRNA, by contrast, was significantly shorter, suggesting that this mRNA segment may be destabilized by the processing cuts within arcD. Deletion of the putative intergenic stem-loop structures did not result in a dramatic loss of arc mRNA stability. Thus, the intergenic hairpin structures do not contribute importantly to the overall mRNA stability; they might act primarily as partial transcription terminators and locally protect the 3' ends from exonuclease action. The expression levels of the four Arc proteins correlated approximately with the relative abundance of the corresponding mRNA segments. In conclusion, mRNA processing and, presumably, partial termination of transcription contribute to differential gene expression within the arc operon.
铜绿假单胞菌在精氨酸上的厌氧生长依赖于编码精氨酸脱亚氨酶途径中各种酶的arcDABC操纵子。这些酶的协同厌氧诱导需要类FNR调节蛋白ANR，它可激活位于arcD上游的arc启动子。通过Northern杂交实验，可检测到三种丰富的arcA、arcAB和arcABC转录本以及三种少量的arcDA、arcDAB和arcDABC转录本。通过S1和引物延伸图谱确定了arcA、arcAB和arcABC mRNA的5'端。这些5'端似乎是由arcD mRNA中的内切核酸酶切割（加工）产生的，而不是由第二个启动子产生的；这是根据arcD中插入和缺失突变的影响得出的结论。arcA与arcB之间以及arcB与arcC之间的基因间反向重复序列被证明与arc转录本3'端的形成有关。在铜绿假单胞菌染色体中删除任何一个基因间区域分别导致arcA或arcAB转录本的缺失。斑点印迹实验表明，从野生型菌株中提取的arc mRNA在arcA、arcB和arcC区域具有相似的化学半衰期，范围从16到13分钟。相比之下，arcD mRNA的半衰期明显更短，这表明该mRNA片段可能因arcD内的加工切割而不稳定。删除假定的基因间茎环结构并未导致arc mRNA稳定性的显著丧失。因此，基因间发夹结构对整体mRNA稳定性的贡献不大；它们可能主要作为部分转录终止子，并局部保护3'端免受核酸外切酶作用。四种Arc蛋白的表达水平与相应mRNA片段的相对丰度大致相关。总之，mRNA加工以及推测的转录部分终止有助于arc操纵子内的差异基因表达。
