The Lac operon, an example of a transcriptionally regulated system.

The most direct way to control the expression of a gene is to regulate its rate of transcription; that is, the rate at which RNA polymerase transcribes the gene into molecules of messenger RNA (mRNA).

E. coli break lactose down using two (there are 3, but our problem set only addressed two) enzymes: beta-galactosidase, which is encoded by the LacZ gene, and permease, which is encoded by the LacY gene. These genes and the regions that regulate them are called the Lac operon. Other important players in the lac operon are:

Operator (LacO) the binding site for the repressor

Promoter (LacP) the binding site for RNA polymerase

Repressor (LacI) the gene encoding for the lac repressor protein- in the absence of lactose the repressor protein binds to the operator and blocks binding of RNA polymerase at promoter

The genotypes are written in order of: repressor (i), promoter (p), operator (o), LacZ (z), LacY (y). If the organism in question is a partial diploid, the two chromosomes are written side by side, separated by a slash. Good copies of genes are indicated by a + sign.

Example: i+p-o+z+y+/i-p+o+z-y+

How to solve a lac operon question.

Look at the promoter first- do you have a good copy of the promoter (p+)? If not, RNA polymerase cannot get transcription started and that chromosome is a bust.

If you do have p+, next look at how the repressor and the operator interact. Here are a few scenarios:

i+p+o+ the repressor binds to the operator in the absence of lactose and inhibits transcription of the z and y genes. BUT, when lactose is around, it kicks the repressor off of the operator, and the z and y genes are expressed (look to see whether you have good copies of z and y, i.e. z+ and y+).

i^sp+o+ this is a super repressor- lactose cannot kick it off, so transcription of z and y FAILS whether lactose is around or not.

i^sp+o^c the super repressor meets the constitutive operator. The constitutive operator deflects any repressor (even a super repressor) and transcription OCCURS whether lactose is around to not.

One last thing to remember is that while p and o are regulatory regions and only affect the genes that they are sitting right next to (they act in cis), i codes for a diffusible regulatory protein, so it can affect both chromosomes (it can act in trans) of partial diploid. We didn’t have any examples of this in our problem set problem, so here is one:

i^sp-o^cz-y+/i-p+o+z+y- in this case, the first chromosome is a bust because of a bad promoter. The second chromosome has a bad repressor, but the protein produced by the super repressor from the first chromosome will bind to the operator of the second chromosome. In this case, beta-galactosidase is produced in the presence of lactose, but not in the absence of lactose. There is no functional LacY, so permease is never produced.

Lac operon

Element	Purpose
ß-gal (lacZ)	Gene that encodes for enzyme that cleaves lactose
lacZ^+	Normal gene
lacZ^-	Affects the structure of enzyme rendering it nonfunctional
Structural genes Permease (lacY)	Gene that encodes for permease that allows lactose to enter the cell
lacY^+	Normal gene
lacY^-	Affects the structure of enzyme rendering it nonfunctional
Trancetylase (lacA)	Unknown function
lacA^+	Normal gene
lacA^-	Affects the structure of enzyme rendering it nonfunctional
Operator (lacO)	Binding site for repressor
lacO^+	Normal operator
lacO^c	cis Constitutive operator, repressor cannot bind
Promoter (lacP)	Binding site for RNA polymerase
Regulatory genes lacP^+	Normal promoter
lacP^-	RNA polymerase cannot bind
Repressor (lacI)	Gene encoding lac repressor protein
lacI^+	trans Normal repressor
lacI^-	Repressor is not produced
lacI^s	Superrepressor, repressor cannot be inactivated by inducer

Cis-acting: Affects only genes on the same DNA molecule.

Trans-acting: Affects genes located on a different DNA molecule.