Question

You plate colonies of E. coli with the lac operon genotype of I+P+OCZ+CAP+ on 1) a medium containing only glucose, and 2) a medium containing only lactose. You isolate mRNA from bacteria from each medium and do a Northern blot with a probe for β-galactosidase. Draw the results.

Answer 1

Structure of wild type lac operon-

lacI+

CAP site

Promoter

lac Operator

lac Z+

Lac Y+

lacA+

lacI gene encodes the repressor which blocks the transcription of lac structural genes (Z,Y,A) when bound at the lac operator site.

CAP site is the site where 2 molecules of CAP protein and 4 molecules of c-AMP in a complex binds. This binding of CAP-cAMP at the CAP site facilitates the binding of RNA polymerase to the promotor. RNA polymerase can't bind the promoter efficiently without the aid of CAP-cAMP complex

(CAP- catabolite activator protein, cAMP- cyclic AMP)

Promotor is the region where RNA polymerase binds for transcription of lac structural genes to take take place.

lac operator is the site between promoter and lac structural genes that interacts with  the regulatory protein, repressor and controls the transcription of the operon.

lacZ- encodes β-galactosidase.

lacY - encodes Permease

lacA - encodes transacetylase

The lac operon genotype of the E.Coli in the question is- I⁺P⁺O^CZ⁺CAP⁺

The structure of lac operon of genotype I⁺P⁺O^CZ⁺CAP⁺ is-

lacI+

CAP site

Promoter

lac Oc

lac Z+

Lac Y+

lacA+

This shows there is a mutation in the lac operator, lac O^c mutation. The lac O^c mutation leads to constitutive expression of the lac operon genes. This is due to base- pair alterations of the operator DNA sequence which doesn't let it bind to the repressor protein. Hence, the structural genes are constitutively expressed in case of lac O^c mutation both in the presence or absence of the repressor protein.

It is mentioned in the question, that E.Coli are grown on two different mediums - 1) a medium containing only glucose, and 2) a medium containing only lactose.

When E. Coli are grown on a medium containing only glucose, catabolite repression or glucose effect occurs. In this, the lac operon is expressed at only very low levels. This is because glucose causes the amount of cAMP in the cell to be reduced greatly. Hence, insufficient numbers of CAP- cAMP complexes are  available to bind at the CAP site. As a result, RNA polymerase is not recruited properly to the lac promoter, can't bind efficiently at the promoter and the lac genes are not transcribed. This low level of transcription is unaffected by mutations in operator (lac O^c) or by the absence of repressor proteins.

When E.Coli are grown on a medium containing only lactose, then positive regulation of lac operon takes place. This is possible because in this case cAMP is present in sufficient numbers to bind CAP and form CAP-cAMP complexes which are able to bind at the CAP site efficiently. As a result CAP-cAMP complexes are able to recruit RNA polymerase at the lac promoter efficiently and hence transcription of structural genes is initiated.

So, it can be concluded that-

• When E.Coli having lac operon of genotype I⁺P⁺O^CZ⁺CAP⁺ are grown in medium containing glucose, low or no transcription of structural genes like  β-galactosidase take place, even in the presence of lac O^c mutation, as binding of RNA polymerase is not facilitated due to insufficient availability of CAP-cAMP complexes.
• When E.Coli having lac operon of genotype I⁺P⁺O^CZ⁺CAP⁺ are grown in medium containing lactose, transcription of structural genes like  β-galactosidase take place due to the availability of CAP-cAMP complexes. Binding of these complexes at the CAP site facilitates binding of RNA polymerase at the lac promoter efficiently and and hence initiate transcription of structural genes. This transcription in this case is constitutive and the structural genes like β-galactosidase are constitutively expressed due to lac O^c mutation, which doesn't let repressor protein to bind at the operator.

Results of Northern blot with a probe for β-galactosidase-

• Lane -1: Glucose in medium- No bands are visible in the blot. This is because of no or very low level of transcription of β-galactosidase in E.Coli grown on medium containing glucose only.
• Lane-2: Lactose in medium- Band visible on the blot. Expression of β-galactosidase visible on the blot around 120KDa size. This is because of transcription of β-galactosidase gene in the presence of lactose only.

Image of Northern blot with a probe for β-galactosidase-

Lane I Lane 2 RNA Ladder (in kDa) 180 130 sexforecedent of ß galactoridade too 75 63 35 28 iz