Eom Chi Yong, Park Sang Tae, Kim Eungbin, Ro Young Tae, Kim Si Wook, Kim Young Min
Department of Biology, Yonsei University, Seoul, Korea.
Mol Cells. 2002 Oct 31;14(2):245-54.
Three structural genes that consist of a dnaK operon in a restricted facultative methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726 were cloned and characterized. The genes were clustered in the transcription order grpE-dnaK-dnaJ. The cloned grpE, dnaK, and dnaJ genes had open-reading frames of 474, 1,926, and 1,116 nucleotides, coding for proteins with calculated molecular masses of 17,390, 69,761, and 41,050, respectively. The overall identities in the deduced amino acid sequences of GrpE, DnaK, and DnaJ with those of the Escherichia coli homologs were 45.2, 74.5, and 61.2%, respectively. Northern blot analyses with grpE-, dnaK-, and dnaJ-specific probes revealed that the three genes are co-transcribed as a 4.0-kb mRNA. A primer extension analysis revealed that the transcription of the dnaK operon started at the nucleotide A that is located 28 bp upstream of the grpE start codon. The transcription start site was preceded by a putative promoter region 15'-CCCCGCTTGAA(13-bp)CCCCAATTT-3'], which is highly homologous to the consensus sequences of the E. coli sigma32-type heat shock promoter. The putative promoter worked under both normal and heat shock conditions in E. coli. The nature of the nucleotide sequence in the second half of the -35 region played a critical role during transcription. The heat shock mRNA was maximally produced at about 10 min after transfer of the Methylovorus sp. strain SS1 from 30 to 42 degrees C. The dnaK operon was also induced by ethanol, hydrogen peroxide, and NaCl shocks. The cloned dnaK operon complemented the E. coli dnaK mutant.
在严格兼性甲基营养菌嗜甲基菌属（Methylovorus sp.）菌株SS1 DSM 11726中，克隆并鉴定了由dnaK操纵子组成的三个结构基因。这些基因按grpE - dnaK - dnaJ的转录顺序成簇排列。克隆的grpE、dnaK和dnaJ基因具有474、1926和1116个核苷酸的开放阅读框，分别编码计算分子量为17390、69761和41050的蛋白质。GrpE、DnaK和DnaJ推导氨基酸序列与大肠杆菌同源物的总体同一性分别为45.2%、74.5%和61.2%。用grpE、dnaK和dnaJ特异性探针进行的Northern印迹分析表明，这三个基因作为一个4.0 kb的mRNA共转录。引物延伸分析表明，dnaK操纵子的转录起始于位于grpE起始密码子上游28 bp的核苷酸A。转录起始位点之前是一个假定的启动子区域[15'-CCCCGCTTGAA(13 bp)CCCCAATTT-3']，它与大肠杆菌sigma32型热休克启动子的共有序列高度同源。该假定启动子在大肠杆菌的正常和热休克条件下均起作用。-35区域后半部分的核苷酸序列性质在转录过程中起关键作用。嗜甲基菌属菌株SS1从30℃转移到42℃后约10分钟，热休克mRNA产生量最大。dnaK操纵子也受到乙醇、过氧化氢和NaCl冲击的诱导。克隆的dnaK操纵子可互补大肠杆菌的dnaK突变体。
