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Lecture 10

Regulation of Gene Expression

The Lactose Operon

Bacterial genes performing similar functions are often organized together in a linear sequential order. This adjacent group of genes is called an "operon". Transcription of an operon occurs under the control of a promoter, and other regulatory proteins may determine whether the set of genes is transcribed or not. Multiple protein coding regions on a single bacterial mRNA can be translated sequentially. Regulating the expression of a cluster of genes (such as an operon) is an easy way to control an entire metabolic pathway. An example of a coordinately regulated set of genes is the lactose (lac) operon.

The lactose operon of E. coli consists of three structural genes under coordinate control (FIGURE): the Z gene encoding beta-galactosidase, the Y gene encoding permease, and the A gene encoding transacetylase. Beta-galactosidase is an enzyme that breaks down the more complicated sugar lactose into two simpler sugars glucose and galactose. Permease is protein that transports lactose into the cell. Transacetylase has a known, but not essential, enzymatic activity. Transcription commences at a promoter to the left of the Z gene and transcribes a 5,200 nucleotide messenger RNA molecule (mRNA), ending at a terminator just beyond the A gene. Translation of the large polygenic (polycistronic) messenger RNA produces all three proteins.

The enzyme beta-galactosidase hydrolyzes the disaccharide lactose to the monosaccharides glucose and galactose. Since galactose can be subsequently converted to glucose by a series of other biochemical reactions, lactose ultimately provides two molecules of glucose for energy. The concentration of beta-galactosidase is very low in cells normally (basal level); but when lactose is the sole carbon source, the concentration of the enzyme is elevated markedly (a process called induction). After all the lactose is metabolized, beta-galactosidase eventually returns to a very low level in the cell. The study of the induction of beta-galactosidase has been made easier by the use of a non-metabolized inducer isopropyl beta-thiogalactoside, also called IPTG, and a non-inducing substrate, called Xgal, which turns BLUE when hydrolyzed by beta-galactosidase. The lactose operon is one of the most intensely studied genetic regulatory systems and is extraordinarily useful in genetic engineering.

The Promoter of the Lactose Operon

Since the promoter differs from the ideal -35 sequences and -10 sequences, RNA polymerase cannot bind efficiently to the promoter without the assistance of Catabolite Activator Protein (CAP):

 

 -35 Sequences

  -10 Sequences

 Ideal promoter

 TTGACA

 TATAAT

 Lactose operon promoter

 TTTACA

 TATGTT

Some promoter up-mutants (mutant promoters that "work" better) that make the -35 box and/or the -10 region more like the ideal promoter sequence no longer require CAP assistance.

CAP Protein Assists Binding of RNA Polymerase at the Promoter

The promoter of the lactose operon is a very weak promoter and RNA polymerase does not bind well. A positive activator called CAP (Catabolite Activator Protein) binds to a specific sequence immediately next to the promoter and helps RNA polymerase to bind to the promoter. CAP itself must bind a small molecule called cAMP (cyclic AMP: where the phosphate is attached to both the 3'-hydroxyl and the 5'-hydroxyl of ribose) in order to bind to its site in the lactose operon:

 
Catabolite Activator Protein (CAP)

 Type of Regulation

 cAMP bound to CAP

 DNA binding

 Transcription

 Positive

Yes

 Yes

 Yes

No

 No

 No

It once was thought that the levels of cAMP varied in E. coli under certain conditions, but we know now that is not the case (see Catabolite Repression below). Since there are always adequate concentrations of cAMP in E. coli, CAP is bound to its binding site at all times (even during repression by the lac repressor).

How does CAP work? CAP needs to "touch" RNA polymerase since the spacing between the CAP site and the promoter is important for recruiting RNA polymerase to the promoter: insertion of 5 base pairs between the two sites eliminates positive control by CAP, but insertion of 10 base pairs between the two sites restores positive control to about 20% (thus, the orientation between CAP and RNA polymerase is thought to be important; that is, they must bind to the same "side" of the DNA molecule).

The Repressor Prevents Transcription of the Lactose Operon (FIGURE)

The lactose operon is under the control of the adjacent I gene which encodes the lactose repressor. The I gene is transcribed constitutively from its own promoter (it is also a "poor" promoter; only a few molecules of repressor are ever present in E. coli). The repressor binds to a site, called the operator, which overlaps the promoter. If the repressor is bound to the operator, RNA polymerase cannot bind to the promoter. The repressor can be removed from the operator by binding a small molecule called an inducer (either the natural inducer, allolactose, or the synthetic inducer, IPTG). In the absence of glucose, lactose enters the cell and is converted by the very few beta-galactosidase molecules in the cell to allolactose, the natural inducer of the lac operon. Allolactose binds to the repressor and changes the shape of the repressor so that it no longer can bind to the operator. This removes the repressor from the operator. Inducer-bound repressor does not bind to the operator site. CAP then loads RNA polymerase onto the promoter, and transcription of the lac operon begins. This results in more than a 1000-fold increase in the three coordinately regulated enzymes: beta-galactosidase, permease, and transacetylase. The lac operon cannot be induced in the presence of glucose because glucose prevents the entry of lactose into the cell; that is, repressor remains bound to the operator when both glucose and lactose are present (see Catabolite Repression below).

Lac Repressor

 Type of Regulation

Inducer bound to Repressor

 DNA binding

 Transcription

 Negative

 Yes

 No

 Yes

No

 Yes

 No

Catabolite Repression

E. coli grown on glucose do not metabolize lactose (or several other sugars either) until all of the glucose in the medium is consumed. This is called catabolite repression. It once was thought that catabolite repression was due to low levels of cAMP in the cell which, in turn, affected the binding of CAP to its binding site in the lactose operon. However, cAMP levels are the same in cells grown on glucose alone or on lactose alone. Glucose represses the lactose operon of E. coli by preventing the entry of lactose by permease. In the presence of glucose there is never enough lactose inside the uninduced cells to be transformed by the very few molecules of beta-galactosidase into the true inducer allolactose.