Verma A, Kinger A K, Tyagi J S
Department of Biotechnology, All India Institute of Medical Sciences, New Delhi.
Gene. 1994 Oct 11;148(1):113-8. doi: 10.1016/0378-1119(94)90243-7.
A functional analysis of Mycobacterium tuberculosis 16S ribosomal RNA (rRNA) transcription and processing was undertaken in this study. RNA:DNA hybridizations indicated that the maximum transcriptional activity of rRNA-encoding genes (rDNA) corresponded to the earliest period of exponential growth. Transcription start points (tsp) were mapped by primer extension analysis of RNA from M. tuberculosis H37Rv and M. tuberculosis H37Ra. An identical pattern of rRNA transcription and processing was exhibited in laboratory-grown cultures of M. tuberculosis H37Rv and H37Ra. One promoter represents the structural equivalent of the Escherichia coli rrn P2 promoter. The precursor transcripts are processed into mature 16S rRNA through a pathway that includes recognition of RNA secondary structure by ribonuclease III (RNase III) in the stem structure surrounding the 16S rRNA indicating that at least this RNA processing step is conserved in mycobacteria and E. coli. The 16S rDNA promoter region from H37Rv was cloned upstream from the promoterless chloramphenicol (Cm) acetyltransferase (CAT)-encoding gene (cat) in a shuttle plasmid vector, pSD7. The promoter-fusion construct, pSD7.16S, was characterized by CAT assays, measurement of percent survival in Cm-containing medium and in vivo transcription analysis in M. smegmatis. The M. smegmatis transformant exhibited a CAT activity of 16,669 nmol/min per mg protein, suggesting that the 16S promoter was of exceptionally high strength. Two tsp utilized in M. tuberculosis were also employed in M. smegmatis. The cat mRNA synthesized under the direction of the ribosomal promoter was less stable, as compared to genome-derived rRNA.
本研究对结核分枝杆菌16S核糖体RNA（rRNA）的转录和加工进行了功能分析。RNA:DNA杂交表明，rRNA编码基因（rDNA）的最大转录活性与指数生长期的最早阶段相对应。通过对结核分枝杆菌H37Rv和结核分枝杆菌H37Ra的RNA进行引物延伸分析，确定了转录起始点（tsp）。结核分枝杆菌H37Rv和H37Ra的实验室培养物中呈现出相同的rRNA转录和加工模式。一个启动子代表了大肠杆菌rrn P2启动子的结构等效物。前体转录本通过一条途径加工成成熟的16S rRNA，该途径包括核糖核酸酶III（RNase III）在围绕16S rRNA的茎结构中识别RNA二级结构，这表明至少这个RNA加工步骤在分枝杆菌和大肠杆菌中是保守的。将来自H37Rv的16S rDNA启动子区域克隆到穿梭质粒载体pSD7中无启动子的氯霉素（Cm）乙酰转移酶（CAT）编码基因（cat）的上游。通过CAT测定、含Cm培养基中的存活率测量以及耻垢分枝杆菌中的体内转录分析，对启动子融合构建体pSD7.16S进行了表征。耻垢分枝杆菌转化体表现出的CAT活性为每毫克蛋白质16,669 nmol/分钟，表明16S启动子具有极高的强度。结核分枝杆菌中使用的两个tsp也在耻垢分枝杆菌中使用。与基因组衍生的rRNA相比，在核糖体启动子指导下合成的cat mRNA稳定性较差。
