Question

How does arabinose affect GFP on an ara operon?

a. What does arabinose play as on an ara operon?

b. Positive and negative regulation?

c. Assuming the ara operon ascts like a CAP oper, draw the oper in precense and absence of arabinose

Answer 1

Answer: GFP stands for Green Fluorescent Protein. It basically involves a concept of biotechnology whereby, araA, araB and araD genes of E.coli are replaced by a bioluminescence gene from a jellyfish. This gene is introduced in the bacteria using a specialized plasmid vector called pGLO. Then those bacteria that have successfully taken up the vector are grown in the LB media containing ampicillin and arabinose sugar. In the presence of arabinose, there will be a conformational change in araC gene that will now allow the binding of RNA polymerase and trancribe the gene of bioluminescence (taken from jellyfish) which will produce GFP that is capable of glowing in UV rays.

So, according to the question, arabinose does affect the structure of araC gene, only then it would be possible to recruit the RNA polymerase to trancribe the gene and allows its binding at promoter region. No such protein will be produced in the absence of arabinose.

Answer-a: In an ara operon, arabinose act as an inducer molecule. It act as a carbon source and a source of energy for E.coli. In the presence of arabinose only, the structural genes ara-B, A & D are transcribed whose products are helpful in utilizing arabinose for the bacteria. However, in te absence of arabinose, these structural genes remain switched off.

Answer-b:

(i) Positive Regulation- Positive regulation is achieved by the presence of arabinose and absence of glucose. As a result, it increases the level of cAMP. So, now, Ara-C and CAP site acts as activators and they work together. Ara-C acts as activator in presence of arabinose which leads to a conformational change in its structure because arabinose binds to the dimer structure and as a result, Arac-arabinose complex falls off from the operator region which causes breaking of the DNA loop. To avoid this, AraC-arabinose complex binds to DNA half sites AraL1 and AraL2 sites . Upon binding, it aesily makes room for the RNA polymerase to sit on promotor site and stast transcribing the structural genes. Second mode of regulation is by CAP site. CAP site only act as an activator in the absence of glucose. In glucse absent conditions, CAP protein and cAMP binds between AraL1 and Operator-2 and it opens up the DNA loop that enhances the affinity of AraC protein for AraL2 site and further promote binding of RNA polymerase on promotor region and transcription of the structural genes.

(ii) Negative Regulation- In the absence of arabinose, Dimer of AraC does not break at all which therefore acts like a repressor molecule. As a result it forms a loop of DNA that fails to recruit RNA polymerase and structural genes are not transcribes and the operon is said to be in switched off state.

Answer-c: Source of the diagrammatic picture is internet. Link is attached herewith: " https://www.google.co.in/search?rlz=1C2RLNS_enIN767IN768&biw=1366&bih=662&tbm=isch&sa=1&ei=RuAWW6W9O5uR9QPnholA&q=CAP+in+ara+operon&oq=CAP+in+ara+operon&gs_l=img.3...10891.30846.0.31093.48.29.6.0.0.0.249.3730.0j23j2.25.0....0...1c.1.64.img..18.18.2433...0j0i24k1j0i67k1j0i30k1j0i5i30k1j0i8i30k1.0.ikaHHDhxhkw#imgrc=PX8hOJ3ppLYkGM: "