The katG mRNA of Mycobacterium tuberculosis and Mycobacterium smegmatis is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation.
The katG mRNA of Mycobacterium tuberculosis and Mycobacterium smegmatis is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation.
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DOI：
10.1186/1471-2199-9-33
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发表时间：
2008-04-04
影响因子：
--
通讯作者：
Ghisotti D
中科院分区：
文献类型：
--
作者：
Sala C;Forti F;Magnoni F;Ghisotti D
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。In M.结核分枝杆菌furA-katG是一个操纵子，而结核分枝杆菌furA-katG是一个操纵子。在两个物种中，已经鉴定了特定的5'端：第一个位于furA基因的上游，对应于从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。在这项工作中，我们证明了在体外转录和RNA聚合酶染色质免疫沉淀，没有启动子存在于M。覆盖后一个5'端的smeglobin区域，表明它是由较长转录物的特异性加工产生的。M.结核和M.将smeglobulin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的聚嘌呤序列增加了β-半乳糖苷酶活性并稳定了lacZ转录本。该序列的突变导致mRNA的不稳定。构建体的分析，其中M. smeglycine之后是增加数量的katG密码子，证明mRNA稳定性需要至少20个氨基酸的翻译。为了定义katG转录物的5'加工的要求，我们在该区域中产生了几个突变并分析了转录物的5'末端：与多嘌呤序列的距离似乎不影响加工，切割点周围的序列也不影响加工。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。这是分枝杆菌中首次报道的病例，其中多嘌呤序列和翻译起始均显示有助于mRNA稳定性。furA-katG mRNA从furA启动子转录并立即加工;该加工被切割位点处的双链RNA阻止，表明负责切割的核糖核酸内切酶切割单链RNA。
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. 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. 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.
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