Wu C J, Janssen G R
Department of Microbiology, Miami University, Oxford, Ohio 45056, USA.
Mol Microbiol. 1996 Oct;22(2):339-55. doi: 10.1046/j.1365-2958.1996.00119.x.
The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine-Dalgarno homology. The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Streptomyces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon. Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E. coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine-Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation. Addition of four nucleotides (5'-AUGC-3') onto the 5' end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence. Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E. coli and S. lividans.
酒红色链霉菌紫霉素磷酸转移酶（vph）mRNA包含一个具有传统Shine-Dalgarno同源性的非翻译前导序列。通过将vph编码序列连接到链霉菌或大肠杆菌启动子的转录起始位点，去除了vph前导序列，使得转录将在vph起始密码子的第一个位置开始。对mRNA的分析表明，在天蓝色链霉菌和大肠杆菌中，转录主要在vph AUG翻译起始密码子的A处起始；表达无前导序列vph mRNA的细胞对紫霉素具有抗性，这表明Shine-Dalgarno序列或前导序列中包含的其他特征对于vph翻译不是必需的。在无前导序列vph mRNA的5'末端添加四个核苷酸（5'-AUGC-3'）导致从vph起始密码子和添加序列中包含的AUG三联体开始翻译起始。vph序列与Tn5 neo报告基因的翻译融合表明，vph编码序列的前16个密码子足以指定在大肠杆菌和天蓝色链霉菌中表达新霉素抗性的翻译起始位点和阅读框。
