Question

Draw what the results for a Northern blot, probing for ara a mRNA, would look like for E. coli cells grown under the following conditions: 1) arabinose absent and glucose present, 2) arabinose present and glucose absent, 3) arabinose present and glucose present. Explain your reasoning.

Answer 1

General concept:

Ara operon is consist of ara B, araA and ara D genes. ara B codes for ribulokinase, ara A codes for an isomerase and ara D codes for an epimerase. All these enzymes are needed to catalyze the conversion of arabinose to a metabolic intermediate D-xylulose-5-phosphate.

So, if there are arabinose present in the growth medium these bacteria will take up arabinose and express the operon to catalyse the subsequent reactions, if not these genes are in off- state where the gene expression from this operon shuts down. When both glucose and arabinose is present in the medium the cell will use up all the glucose first and then it will start braking down the arabinose. Unless all the glucose are used up the ara operon will be in repressed state.

Northern blotting is used to identify the gene expression by using probes specific for experimental mRNA. If the probes bind with more mRNA then the gene is upregulated and if less mRNA is present then less probe will bind to the mRNA which implies the gene is downregulated.

Gene regulation:

The repression of ara operon is controlled by another gene product Ara C which dimerize and bind to the operator region of the ara operon and the operon expression stops.

But when arabinose is present this arabinose will bind to the Ara C and arabinose bound Ara C will activate gene expression from ara operon by binding to the another operator region. (So, Ara C act as both activator and repressor.) When glucose level decreases cAMP will increase and this cAMP will bind to CAP and cAMP bound CAP will activate ara operon.

Answer:

1) As arabinose absent, the ara operon will be in repressed state. None of the genes will be expressed from this operon. So, if you do northern blotting for ara A mRNA, the result will be negative.

2) As arabinose present, the operon will be in active state and all the genes from this operon will express. So, you will get positive result for northern blotting.

3) As both glucose and arabinose is present, initially the ara operon will be in repressed state. So, if you take up sample at the early stage of the experiment you will get negative result for northern blotting. But in the late stage when all the glucose will be used up and the cells will express ara operon to catalyze arabinose. So, in the late culture cells the northernn blotting result will be positive.