Part:BBa_K1996000
EtnP-Putative promoter region for ethylene oxidation genes in Mycobacterium NBB4
Putative promoter for suspected ethylene-oxidising genes sourced from Mycobacterium NBB4
Usage and Biology
This part was obtained from the ethylene metabolism operon in Mycobacterium NBB4. It is thought to be a promoter that regulates bidirectional transcription of gene involved in ethylene metabolism (Figure 1). The enzymes required for ethylene metabolism are contained within an operon organised in two bidirectional gene clusters. One gene cluster encodes the catabolic enzymes required to convert ethylene into acetyl-CoA, the final product of the degradation pathway which is subsequently directed to the tricarboxylic acid cycle. The other gene cluster encodes the enzymes responsible for synthesising coenzyme M, a cofactor required for the function of the second enzyme in the metabolic pathway.
Figure 1. Schematic of the ethylene metabolism operon in Mycobacterium NBB4. The two bidirectional gene clusters are indicated by the curved brackets. Between the two clusters is the putative promoter for their transcription, EtnP.
EtnR1 binding to EtnP DNA has been demonstrated in an electrophoretic mobility shift assay (EMSA), where it was found that EtnR1 binding to EtnP notably inhibits the migration of the DNA through the gel, indicating a strong binding affinity between EtnR1 protein and EtnP DNA (Figure 2).
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Figure 2. Electrophoretic mobility shift assay for EtnP DNA. The 250 bp region of EtnP was PCR amplified and 100 ng of DNA incubated with 6 mg of protein. The protein-DNA mixtures were run on a 3% agarose gel at 150 V for 100 minutes and stained with Gel Green overnight for visualisation. Lane 1: NEB 100 bp ladder. Lane 2: EtnP DNA only. Lane 3: EtnR1 protein only. Lane 4: EtnR1 protein and EtnP DNA. Lane 5: BSA protein and EtnP DNA.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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