Fahr M J, Sriprakash K S, Hatch T P
Department of Microbiology and Immunology, University of Tennessee, Memphis 38163.
Plasmid. 1992 Nov;28(3):247-57. doi: 10.1016/0147-619x(92)90056-g.
Transcription of the 7.5-kb cryptic plasmid of Chlamydia trachomatis serovar L2 was investigated. Faint, diffuse transcripts of about 1.6 and 2.2 kb and intense short transcripts of about 250 and 430 bases were identified by Northern blot analysis. The short transcripts were found to have a common 5' end corresponding to bp 501 relative to the unique BamHI site of the plasmid and to terminate at different downstream sites. Putative promoter sequences of TTGCCA and TATATT, which closely resemble the consensus recognition site of Escherichia coli sigma 70, were identified at the -35 and -10 positions upstream from the 5' end of the short transcripts made in chlamydia. Transcripts of similar sizes were also expressed from this promoter in E. coli harboring a recombinant plasmid encoding the short transcripts. The short transcripts encode a common open reading frame (ORF) of 34 codons; however, a strong ribosome binding site was not found in the vicinity of the initiator codon, and it is not known whether the transcripts are translated in vivo. A large ORF of 330 codons, which has been shown to encode a hypothetical protein containing conserved domains of recombinase-like proteins, is antisense to the short transcripts. Transcripts encoding the large ORF could not be detected directly by Northern blot or primer extension analysis. However, transcripts were detected by polymerase chain reaction amplification of the large ORF cDNA and when Southern blots of single-stranded antisense DNA for the large ORF were probed with radiolabeled RNA synthesized by host-free chlamydial reticulate bodies. Thus, both strands of the chlamydial plasmid are transcribed in the region encoding the short transcripts. We propose that the short transcripts play a regulatory role as antisense RNAs.
对沙眼衣原体L2血清型7.5kb隐蔽质粒的转录进行了研究。通过Northern印迹分析鉴定出约1.6和2.2kb的微弱、弥散转录本以及约250和430个碱基的强烈短转录本。发现这些短转录本具有一个共同的5'末端，对应于相对于质粒独特BamHI位点的第501个碱基对，并在不同的下游位点终止。在衣原体中产生的短转录本5'末端上游的-35和-10位置鉴定出了与大肠杆菌σ70共有识别位点非常相似的TTGCCA和TATATT推定启动子序列。在携带编码短转录本的重组质粒的大肠杆菌中，类似大小的转录本也从该启动子表达。短转录本编码一个34个密码子的共同开放阅读框（ORF）；然而，在起始密码子附近未发现强核糖体结合位点，并且尚不清楚这些转录本在体内是否被翻译。一个330个密码子的大ORF与短转录本呈反义，已证明它编码一种含有重组酶样蛋白保守结构域的假定蛋白。通过Northern印迹或引物延伸分析无法直接检测到编码大ORF的转录本。然而，通过对大ORF cDNA进行聚合酶链反应扩增以及当用无宿主衣原体网状体合成的放射性标记RNA探测大ORF的单链反义DNA的Southern印迹时，检测到了转录本。因此，衣原体质粒的两条链在编码短转录本的区域都被转录。我们提出短转录本作为反义RNA发挥调节作用。
