Pasternak C, Assemat K, Breton A M, Clement-Metral J D, Klug G
Laboratoire de Technologie Enzymatique, Université de Technologie de Compiègne, France.
Mol Gen Genet. 1996 Feb 5;250(2):189-96. doi: 10.1007/BF02174178.
The structural gene (trxA) coding for thioredoxin in the photosynthetic bacterium Rhodobacter sphaeroides has been cloned and sequenced previously. In the present study, the role of oxygen in trxA expression in R. sphaeroides Y was investigated using mRNA analyses and plasmid-borne trxA'-lacZ+ translational and transcriptional fusions. Northern analysis revealed a trxA-specific transcript of approximately 420-460 nucleotides, indicating that trxA is transcribed as a single gene. By studying the beta-galactosidase activity in strains harboring various phi(trxA'-lacZ+) fusion constructs, the promoter region of the trxA gene was localized within a 64-bp region located 97 nucleotides upstream of the trxA initiator codon. A single trxA transcription initiation site was mapped by primer extension, 27 bp upstream of the trxA gene. Based on these results and the DNA sequence analysis, we propose that a sigma70 consensus sequence serves as a trxA promoter. Results from oxygen shift experiments, as deduced from both mRNA analysis and fusions of the trxA promoter region to lacZ indicate that transcription of the R. sphaeroides trxA gene is regulated by high oxygen tension. DNA sequences involved in this oxygen regulation were also localized in the 64-bp region containing the trxA promoter. Based on our findings the hypothetical biological function of thioredoxin from R. sphaeroides is discussed.
光合细菌球形红杆菌中编码硫氧还蛋白的结构基因(trxA)此前已被克隆和测序。在本研究中,利用mRNA分析以及质粒携带的trxA'-lacZ+翻译和转录融合,研究了氧气在球形红杆菌Y株trxA表达中的作用。Northern分析显示了一个约420 - 460个核苷酸的trxA特异性转录本,表明trxA作为一个单基因被转录。通过研究携带各种phi(trxA'-lacZ+)融合构建体的菌株中的β-半乳糖苷酶活性,trxA基因的启动子区域被定位在trxA起始密码子上游97个核苷酸处的一个64 bp区域内。通过引物延伸在trxA基因上游27 bp处定位了一个单一的trxA转录起始位点。基于这些结果和DNA序列分析,我们提出一个sigma70共有序列作为trxA启动子。从mRNA分析以及trxA启动子区域与lacZ的融合推导得出的氧气转换实验结果表明,球形红杆菌trxA基因的转录受高氧张力调节。参与这种氧气调节的DNA序列也定位在包含trxA启动子的64 bp区域内。基于我们的发现,讨论了球形红杆菌硫氧还蛋白的假设生物学功能。