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

<p>Abstract</p> <p>Background</p> <p>In <it>Mycobacterium tuberculosis </it>and in <it>Mycobacterium smegmatis </it>the <it>furA</it>-<it>katG </it>loci, encoding the FurA regulatory protein and the KatG catalase-peroxidase, are highly conserved. In <it>M. tuberculosis furA-katG </it>constitute a single operon, whereas in <it>M. smegmatis </it>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 <it>furA </it>gene, corresponds to transcription initiation from the <it>furA </it>promoter; the second one is the <it>katG </it>mRNA 5' end, located in the terminal part of <it>furA</it>.</p> <p>Results</p> <p>In this work we demonstrate by in vitro transcription and by RNA polymerase Chromatin immunoprecipitation that no promoter is present in the <it>M. smegmatis </it>region covering the latter 5' end, suggesting that it is produced by specific processing of longer transcripts. Several DNA fragments of <it>M. tuberculosis </it>and <it>M. smegmatis </it>were inserted in a plasmid between the <it>sigA </it>promoter and the <it>lacZ </it>reporter gene, and expression of the reporter gene was measured. A polypurine sequence, located four bp upstream of the <it>katG </it>translation start codon, increased beta-galactosidase activity and stabilized the <it>lacZ </it>transcript. Mutagenesis of this sequence led to destabilization of the mRNA. Analysis of constructs, in which the polypurine sequence of <it>M. smegmatis </it>was followed by an increasing number of <it>katG </it>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 <it>katG </it>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.</p> <p>Conclusion</p> <p>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 <it>furA-katG </it>mRNA is transcribed from the <it>furA </it>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.</p

Characterization of the oriI and oriII Origins of Replication in Phage-Plasmid P4

In the Escherichia coli phage-plasmid P4, two partially overlapping replicons with bipartite ori sites coexist. The essential components of the oriI replicon are the α and cnr genes and the ori1 and crr sites; the oriII replicon is composed of the α gene, with the internal ori2 site, and the crr region. The P4 α protein has primase and helicase activities and specifically binds type I iterons, present in ori1 and crr. Using a complementation test for plasmid replication, we demonstrated that the two replicons depend on both the primase and helicase activities of the α protein. Moreover, neither replicon requires the host DnaA, DnaG, and Rep functions. The bipartite origins of the two replicons share the crr site and differ for ori1 and ori2, respectively. By deletion mapping, we defined the minimal ori1 and ori2 regions sufficient for replication. The ori1 site was limited to a 123-bp region, which contains six type I iterons spaced regularly close to the helical periodicity, and a 35-bp AT-rich region. Deletion of one or more type I iterons inactivated oriI. Moreover, insertion of 6 or 10 bp within the ori1 region also abolished replication ability, suggesting that the relative arrangement of the iterons is relevant. The ori2 site was limited to a 36-bp P4 region that does not contain type I iterons. In vitro, the α protein did not bind ori2. Thus, the α protein appears to act differently at the two origins of replication

The mRNA of and is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation-0

Asmid pMV261, which carries the BCG promoter, and plasmid pMYS648, in which the 437–552 region has been substituted to the promoter region. The samples were run on a 5% denaturing polyacrylamide gel. Molecular weight markers (MWM) run in the same gel are indicated on the right. The about 300 bp transcript synthesized by pMV261 is indicated by an arrow.<p><b>Copyright information:</b></p><p>Taken from "The mRNA of and is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation"</p><p>http://www.biomedcentral.com/1471-2199/9/33</p><p>BMC Molecular Biology 2008;9():33-33.</p><p>Published online 4 Apr 2008</p><p>PMCID:PMC2358910.</p><p></p

The mRNA of and is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation-1

to the promoter. The lines below the map correspond to different regions amplified by a pair of oligonucleotides. Fragment A: -689/-492; B: -81/91 (promoter); C: 374/557; D: 1155/1344. Below the lines, the values of the ratio of each fragment relative to the DNA.<p><b>Copyright information:</b></p><p>Taken from "The mRNA of and is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation"</p><p>http://www.biomedcentral.com/1471-2199/9/33</p><p>BMC Molecular Biology 2008;9():33-33.</p><p>Published online 4 Apr 2008</p><p>PMCID:PMC2358910.</p><p></p

The mRNA of and is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation-5

Asmid pMV261, which carries the BCG promoter, and plasmid pMYS648, in which the 437–552 region has been substituted to the promoter region. The samples were run on a 5% denaturing polyacrylamide gel. Molecular weight markers (MWM) run in the same gel are indicated on the right. The about 300 bp transcript synthesized by pMV261 is indicated by an arrow.<p><b>Copyright information:</b></p><p>Taken from "The mRNA of and is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation"</p><p>http://www.biomedcentral.com/1471-2199/9/33</p><p>BMC Molecular Biology 2008;9():33-33.</p><p>Published online 4 Apr 2008</p><p>PMCID:PMC2358910.</p><p></p

The mRNA of and is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation-4

The m-fold structure prediction program of Zuker [41] and primer extension analyses were performed on RNAs extracted from mc155 transformed with the different plasmids, with oligonucleotide 809. Sequence reactions obtained with the same oligonucleotide were run in the same gel. The position of the promoter, the 443 processing site and the PPS region are indicated on the right. The arrows indicate the position of the cut on the sequence structure. The mutations are circled.<p><b>Copyright information:</b></p><p>Taken from "The mRNA of and is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation"</p><p>http://www.biomedcentral.com/1471-2199/9/33</p><p>BMC Molecular Biology 2008;9():33-33.</p><p>Published online 4 Apr 2008</p><p>PMCID:PMC2358910.</p><p></p

The mRNA of and is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation-2

the promoter, upstream of in pSM128. The gene is preceded by its ribosome binding site (44 nt from the cloning site). pMYS690 carries the 437–531 region of downstream of , containing the wild type PPS (GAAAGGAAA); pMYS692 carries the same region with the indicated mutations in the PPS (CTCTGGAGG). pMYT733 and pMYT735 carry the 405–531 region of downstream of . In pMYT733 carries the wild type PPS (GGAAGGAA), whereas in pMYT735 the PPS was substituted by CCTCCCTC. Beta-galactosidase activity and the half life of the mRNA were determined as indicated in the Methods section. Average beta-galactosidase activity in Miller Units of three to eight different replicas ± standard deviation is reported. The average relative increment in beta-galactosidase activity and in mRNA half life are also indicated. N.T. = not tested. Panel B. RNA was extracted from strains mc155(pMYS690) and mc155(pMYS692), and primer extension performed with oligonucleotide 809, internal to , as described in Methods. The DNA sequence obtained with the same oligonucleotide on plasmid pMYS690 was run in the same gel.<p><b>Copyright information:</b></p><p>Taken from "The mRNA of and is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation"</p><p>http://www.biomedcentral.com/1471-2199/9/33</p><p>BMC Molecular Biology 2008;9():33-33.</p><p>Published online 4 Apr 2008</p><p>PMCID:PMC2358910.</p><p></p

Mycobacterium tuberculosis FurA Autoregulates Its Own Expression

The furA-katG region of Mycobacterium tuberculosis, encoding a Fur-like protein and the catalase-peroxidase, is highly conserved among mycobacteria. Both genes are induced upon oxidative stress. In this work we analyzed the M. tuberculosis furA promoter region. DNA fragments were cloned upstream of the luciferase reporter gene, and promoter activity in Mycobacterium smegmatis was measured in both the presence and absence of oxidative stress. The shortest fragment containing an inducible promoter extends 45 bp upstream of furA. In this region, −35 and −10 promoter consensus sequences can be identified, as well as a 23-bp AT-rich sequence that is conserved in the nonpathogenic but closely related M. smegmatis. M. tuberculosis FurA was purified and found to bind upstream of furA by gel shift analysis. A ca. 30-bp DNA sequence, centered on the AT-rich region, was essential for FurA binding and protected by FurA in footprinting analysis. Peroxide treatment of FurA abolished DNA binding. Three different AT-rich sequences mutagenized by site-directed mutagenesis were constructed. In each mutant, both M. tuberculosis FurA binding in vitro and pfurA regulation upon oxidative-stress in M. smegmatis were abolished. Thus, pfurA is an oxidative stress-responsive promoter controlled by the FurA protein