Identification and characterization of promoters regulating tuf expression in Chlamydia trachomatis serovar F
Identification and characterization of promoters regulating tuf expression in Chlamydia trachomatis serovar F
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DOI：
10.1006/abbi.2000.1854
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发表时间：
2000-07-01
影响因子：
3.9
通讯作者：
Zhang, YX
中科院分区：
文献类型：
--
作者：
Shen, L;Shi, Y;Zhang, YX
沙眼衣原体专性胞内细菌的基因表达范围从感染性EB形式的零到分裂的RE形式的高。衣原体的基因调控机制知之甚少，仅有少数几个启动子序列被鉴定。本研究的目的是检测沙眼衣原体F血清型中翻译所需的一组基因的表达：InfA(编码起始因子1)、tRNA(Thr)、TUF(编码延伸因子Tu)和tRNA(Trp)。引物延伸分析表明，TUF在3种不同的mRNAs中均有表达，这些转录产物的启动子序列分别为P1(tRNA(Thr)上游)、P2(InfA内)和P3(InfA上游)，定量RT-PCB法分析表明，感染后16h(Pi)P1转录物最丰富，而P2转录物在24hpi占主导地位。P1和P3在启动子融合载体和衣原体体外转录系统中的表达情况表明，P3在启动子融合载体和衣原体体外转录系统中均有活性。定点突变证实了-35和-10六聚体在P1和P3启动子中的重要性。P2在大肠杆菌中的活性很弱，在体外转录系统中不活跃，这表明P2转录本是从较长的转录本中加工出来的，或者P2的表达需要一个在大肠杆菌或体外转录系统中不存在的sigma或转录因子。我们的数据表明，在发育周期中，多个过程在调节KRF基因表达方面发挥了作用，(C)2000学术出版社。
Gene expression in the obligate intracellular bacterium Chlamydia trachomatis ranges from nil in the infectious EB form to high in the dividing RE form. Little is known about the mechanisms of gene regulation in chlamydiae and only a few promoter sequences have been characterized. The purpose of our study was to examine the expression of a cluster of genes that are required for translation in C. trachomatis serovar F: infA (encoding Initiation Factor 1), tRNA(Thr), tuf (encoding Elongation Factor Tu), and tRNA(Trp). Primer extension analysis indicated that tuf is expressed in three different mRNAs, Putative promoter sequences for these transcripts were defined as P1 (upstream of tRNA(Thr)), P2 (within infA) and P3 (upstream of infA), Quantitative RT-PCB analysis revealed that P1 transcripts were most abundant at 16 h postinfection (pi), whereas P2 transcripts predominated at 24 h pi. P3 was active at all times pi; however, transcription terminated upstream of tuf at early times pi and continued through tuf at later times, P1 and P3 were active in Escherichia coli, as assessed by CAT expression in promoter-fusion vectors and a chlamydial in vitro transcription system. Site-specific mutagenesis confirmed the importance of the -35 and -10 hexamers in the P1 and P3 promoters. P2 was weakly active in E. coli and inactive in the in vitro transcription system, indicating either that the P2 transcript is processed from a longer transcript or that P2 expression requires a sigma or transcription factor which is not present in E. coli or the in vitro transcription system. Our data suggest that multiple processes play a role in the regulation of krf gene expression during the developmental cycle, (C) 2000 Academic Press.