Li C, Ichikawa J K, Ravetto J J, Kuo H C, Fu J C, Clarke S
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024-1569.
J Bacteriol. 1994 Oct;176(19):6015-22. doi: 10.1128/jb.176.19.6015-6022.1994.
We determined the DNA sequence of a 2,232-bp region immediately upstream of the pcm gene at 59 min on the Escherichia coli chromosome that encodes an L-isoaspartyl protein methyltransferase with an important role in stationary-phase survival. Two open reading frames of 477 and 1,524 bp were found oriented in the same direction as that of the pcm gene. The latter open reading frame overlapped the 5' end of the pcm gene by 4 bp. Coupled in vitro transcription-translation analysis of DNA containing the 1,524-bp open reading frame directly demonstrated the production of a 37,000-Da polypeptide corresponding to a RNA species generated from a promoter within the open reading frame. The deduced amino acid sequence showed no similarity to known protein sequences. To test the function of this gene product, we constructed a mutant strain in which a kanamycin resistance element was inserted at a BstEII site in the middle of its coding region in an orientation that does not result in reduction of Pcm methyltransferase activity. These cells were found to survive poorly in stationary phase, at elevated temperatures, and in high-salt media compared with parent cells containing the intact gene, and we thus designate this gene surE (survival). surE appears to be the first gene of a bicistronic operon also containing the pcm gene. The phenotypes of mutations in either gene are very similar and indicate that both gene products are important for the viability of E. coli cells under stressful conditions.
我们测定了大肠杆菌染色体上位于pcm基因上游紧邻的一段2232bp区域的DNA序列,该基因位于59分钟处,编码一种L-异天冬氨酰蛋白甲基转移酶,其在稳定期存活中起重要作用。发现两个分别为477bp和1524bp的开放阅读框,其方向与pcm基因相同。后一个开放阅读框与pcm基因的5'端重叠4bp。对含有1524bp开放阅读框的DNA进行体外转录-翻译偶联分析,直接证明产生了一种37000Da的多肽,对应于从开放阅读框内的一个启动子产生的一种RNA分子。推导的氨基酸序列与已知蛋白质序列无相似性。为了测试该基因产物的功能,我们构建了一个突变菌株,其中卡那霉素抗性元件以不导致Pcm甲基转移酶活性降低的方向插入其编码区中间的一个BstEII位点。与含有完整基因的亲本细胞相比,发现这些细胞在稳定期、高温和高盐培养基中存活能力较差,因此我们将该基因命名为surE(存活)。surE似乎是一个双顺反子操纵子的第一个基因,该操纵子也包含pcm基因。任一基因中的突变表型非常相似,表明这两个基因产物对压力条件下大肠杆菌细胞的生存力都很重要。