Identification and characterization of rel promoter element of Mycobacterium tuberculosis.
The rel gene is responsible for the maintenance of the level of (p)ppGpp in bacteria under nutrient starvation. This phenomenon known as stringent response plays an important role during survival of the microorganisms in stationary phase. We have cloned 1.6 kb upstream sequence of rel gene of Mycobacterium tuberculosis in a shuttle vector pSD5B containing promoterless lacZ gene and promoter activity was observed in Mycobacterium smegmatis cells by blue/white selection and was measured by beta-galactosidase assay. In order to delineate the minimal promoter element of rel gene, a 200 bp fragment from this 1.6 kb upstream sequence was further cloned in promoterless lacZ shuttle vector pSD5B and promoter activity was observed in M. smegmatis cells in similar way. The 200 bp promoter fragment was found to be mycobacterium specific and did not respond when transformed in Escherichia coli. The +1 transcription start site was determined by primer extension method. The -10 promoter region was identified to be TATCCT. The three T bases when mutated, showed a remarkable decrease in the lacZ expression thus confirming the -10 region. The translation start site has also been identified by site directed frame shift mutagenesis. It appears that this rel promoter can be used for expression of proteins in mycobacteria.
Jain
Vikas
V
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
Sujatha
Subbanna
S
Ojha
Anil Kumar
AK
Chatterji
Dipankar
D
D024506:5' Flanking Region / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018387:Codon, Initiator / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008025:Ligases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics; D024363:Transcription Initiation Site; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D018387:Codon, Initiator; D001616:beta-Galactosidase; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases