BACKGROUND In Mycobacterium tuberculosis and in Mycobacterium smegmatis the furA-katG loci, encoding the FurA regulatory protein and the KatG catalase-peroxidase, are highly conserved. In M. tuberculosis furA-katG constitute a single operon, whereas in M. smegmatis a single mRNA covering both genes could not be found. In both species, specific 5' ends have been identified: the first one, located upstream of the furA gene, corresponds to transcription initiation from the furA promoter; the second one is the katG mRNA 5' end, located in the terminal part of furA. RESULTS In this work we demonstrate by in vitro transcription and by RNA polymerase Chromatin immunoprecipitation that no promoter is present in the M. smegmatis region covering the latter 5' end, suggesting that it is produced by specific processing of longer transcripts. Several DNA fragments of M. tuberculosis and M. smegmatis were inserted in a plasmid between the sigA promoter and the lacZ reporter gene, and expression of the reporter gene was measured. A polypurine sequence, located four bp upstream of the katG translation start codon, increased beta-galactosidase activity and stabilized the lacZ transcript. Mutagenesis of this sequence led to destabilization of the mRNA. Analysis of constructs, in which the polypurine sequence of M. smegmatis was followed by an increasing number of katG codons, demonstrated that mRNA stability requires translation of at least 20 amino acids. In order to define the requirements for the 5' processing of the katG transcript, we created several mutations in this region and analyzed the 5' ends of the transcripts: the distance from the polypurine sequence does not seem to influence the processing, neither the sequence around the cutting point. Only mutations which create a double stranded region around the processing site prevented RNA processing. CONCLUSION This is the first reported case in mycobacteria, in which both a polypurine sequence and translation initiation are shown to contribute to mRNA stability. The furA-katG mRNA is transcribed from the furA promoter and immediately processed; this processing is prevented by a double stranded RNA at the cutting site, suggesting that the endoribonuclease responsible for the cleavage cuts single stranded RNA.

中文翻译：

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结核分枝杆菌和耻垢分枝杆菌的 katG mRNA 在其 5' 端进行加工，并通过多嘌呤序列和翻译起始稳定。

背景在结核分枝杆菌和耻垢分枝杆菌中，编码FurA调节蛋白和KatG过氧化氢酶-过氧化物酶的furA-katG基因座是高度保守的。在结核分枝杆菌中，furA-katG 构成一个单一的操纵子，而在耻垢分枝杆菌中，没有发现覆盖两个基因的单一 mRNA。在这两个物种中，已经确定了特定的 5' 末端：第一个末端位于 furA 基因的上游，对应于来自 furA 启动子的转录起始；第二个是 katG mRNA 5' 端，位于 furA 的末端部分。结果 在这项工作中，我们通过体外转录和 RNA 聚合酶染色质免疫沉淀证明，在覆盖后 5' 端的耻垢分枝杆菌区域中不存在启动子，表明它是由较长转录物的特定加工产生的。将结核分枝杆菌和耻垢分枝杆菌的几个 DNA 片段插入质粒中 sigA 启动子和 lacZ 报告基因之间，并测量报告基因的表达。位于 katG 翻译起始密码子上游 4 bp 处的多嘌呤序列增加了 β-半乳糖苷酶活性并稳定了 lacZ 转录本。该序列的诱变导致 mRNA 的不稳定。对耻垢分枝杆菌的多嘌呤序列后跟越来越多的 katG 密码子的构建体的分析表明，mRNA 的稳定性需要翻译至少 20 个氨基酸。为了定义 katG 转录本 5' 处理的要求，我们在该区域创建了几个突变并分析了转录本的 5' 末端：与多嘌呤序列的距离似乎不影响加工过程，切割点周围的序列也不受影响。只有在加工位点周围产生双链区域的突变才能阻止 RNA 加工。结论 这是分枝杆菌中第一个报道的病例，其中显示多嘌呤序列和翻译起始都有助于 mRNA 的稳定性。furA-katG mRNA 从 furA 启动子转录并立即加工；这种加工被切割位点的双链 RNA 阻止，表明负责切割的内切核糖核酸酶切割单链 RNA。结论 这是分枝杆菌中第一个报道的病例，其中显示多嘌呤序列和翻译起始都有助于 mRNA 的稳定性。furA-katG mRNA 从 furA 启动子转录并立即加工；这种加工被切割位点的双链 RNA 阻止，表明负责切割的内切核糖核酸酶切割单链 RNA。结论 这是分枝杆菌中第一个报道的病例，其中显示多嘌呤序列和翻译起始都有助于 mRNA 的稳定性。furA-katG mRNA 从 furA 启动子转录并立即加工；这种加工被切割位点的双链 RNA 阻止，表明负责切割的内切核糖核酸酶切割单链 RNA。