Question

1). What does the term ‘attenuation’ mean when used to describe regulation of the trp operon?

2). How does beta galactosidase play a role in cloning DNA into plasmids?

Answer 1

1) The trp operon refers to tryptophan operon. It consists of genes coding for enzymes required for the synthesis of amino acid called tryptophan. It is found in the bacteria Escherichia coli. When tryptophan levels are low, the operon gets switched on. When tryptophan levels are high, the operon gets switched off. The operon function may be either regulated by repression or by attenuation mechanism.

Regulation by repression: When tryptophan levels are high, it binds to repressor protein and changes its conformation. This helps the protein to bind the operator. This in turn inhibits the RNA polymerase from transcribing the genes. The operon there by gets switched off. When tryptophan levels are low, the repressor protein remains inactive and cannot bind to the operator. This allows transcription of the genes. The operon gets switched on.

Regulation by attenuation: This mechanism stops transcription midway when tryptophan levels are high. The trp operon has a leader sequence because of which mRNA transcribed will have region 1,2,3 and 4 at the 5'end of mRNA. Region 1 has 14 codons of which two adjacent codons are tryptophan codons. Region 3 is complementary to region 2 and 4. Therefore, it can form hairpin structures with either of these regions. Hairpin like structure formed by region 2 and 3 will not stop transcription while, hairpin like structure formed by region 3 and 4 will stop transcription. When tryptophan levels are high, the ribosome begin translation quickly at region 1 and blocks region 2. This results in formation of hairpin structure between region 3 and 4 and causes termination of transcription. When tryptophan levels are low, the ribosome waits for arrival of tRNA with tryptophan at the adjacent tryptophan codons. This results in formation of hairpin structure between region 2 and 3. So now there is no chance of formation of hairpin structure between region 3 and 4. This allows continuation of transcription.

2) Beta galactosidase is useful in blue white screening of the recombinant bacterial colonies and the principle involved is Insertional inactivation. Insertional inactivation is the inactivation of a plasmid gene (here beta glactosidase coding Lack Z gene) by insertion of a foreign gene resulting in the formation of recombinant DNA. This principle is used to determine if a plasmid has taken up gene of interest or not. . In this case if a plasmid has taken up the gene of interest, the Lac Z gene will not get expressed and there will be no synthesis of beta galactosidase enzyme. If a plasmid has not taken up the gene of interest, the Lac Z gene gets expressed and synthesis of enzyme beta galactosidase occurs.

The nutrient medium for growth of bacteria containing plasmid is substituted with X-galactose.The enzyme beta galactosidase can cleave X-galactose to produce a blue pigment. If the bacteria's plasmid has taken up gene of interest, the enzyme will not get synthesized resulting in formation of white colonies. If the bacteria's plasmid has not taken up the gene of interest, the enzyme gets synthesized resulting in formation of blue colonies.