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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.

bmc molecular biology

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