Identification of a novel mycobacterial transcriptional regulator and its involvement in growth rate dependence and stringent control.
A novel transcriptional regulator has been identified in the 400-bp upstream region of the guaA gene of Mycobacterium tuberculosis H37Rv that promotes the expression of lacZ gene in Mycobacterium smegmatis mc(2)155 and M. tuberculosis H37Rv but not in Escherichia coli DH5alpha. PCR-mediated deletion mutagenesis and cloning identified a 120-bp fragment upstream from the guaA gene to be the actual regulator. Primer extension analysis mapped the transcription start site to be the first 'G' residue of the translation start codon GTG of the guaA gene. Electrophoretic mobility shift assay showed strong binding of M. smegmatis RNA polymerase holoenzyme to the 400-bp fragment that expresses lacZ in mycobacterial species and a weak binding to the 280-bp fragment that expresses only in E. coli DH5alpha. Both promoter recombinants revealed varied response in the presence of purine nucleotides and exhibited down-regulation when subjected to amino acid starvation.
Kamalakannan
V
V
Department of Immunology, Tuberculosis Research Centre, Mayor V R Ramanathan Road, Chetput, Chennai 600 031, India.
Ramachandran
Geetha
G
Narayanan
Sujatha
S
Vasan
S K
SK
Narayanan
P R
PR
D001483:Base Sequence; D019731:Carbon-Nitrogen Ligases / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007763:Lac Operon; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000254:growth & development; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*
D011994:Recombinant Proteins; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing)