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.
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.


CONCLUSIONS
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.
Sala
Claudia
C
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133, Milano, Italy. claudia.sala@epfl.ch
Forti
Francesca
F
Magnoni
Francesca
F
Ghisotti
Daniela
D
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002374:Catalase / Q000235:genetics*; D047369:Chromatin Immunoprecipitation; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010442:Peptide Chain Initiation, Translational / Q000502:physiology*; D011401:Promoter Regions, Genetic; D020871:RNA Stability / Q000502:physiology*; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis