Advanced Molecular Genetics-Biology 566 Lactose Operon and Cooperativity
	Index CourseInfo LogIn Syllabus References Other Resources

Development

"A human is made of cells with widely differing characteristics--compare, for example, muscle, blood, and neural cells."

"What distinguishes hands and feet, for example, is not solely (or even largely) the expression of different genes, but rather expression of common genes, but at different times, in different places, and in different combinations."

"Very often the choice of which genes are expressed in a given cell depends on signals received from its environment."

Cells secrete sonic hedgehog -> Induces adjacent cells to develop into motor neurons.

Cells secrete sonic hedgehog -> Induces adjacent cells to express genes for limb development.

"Many genes respond to combinations of signals"
 
 

Evolution

Several examples of diversity have been shown to be related to changes in expression patterns of a single gene.

"According to one popular view, changes in patterns of gene expression (rather than evolution of new genes) have had an important, perhaps even determinative, role in generating much of that diversity."

"… a relatively small number of genes" (32,000 in humans) "and signals have generated an astounding panoply of organisms. Thus, the regulatory machinery must be such that it readily throws up variations -- new patterns of gene expression -- for selection to work on."
 
 

Regulation

"The most pervasive form of gene regulation--from bacteria to higher eukaryotes--involves the initiation of transcription by RNA polymerase."

Gene regulation probed by mutations which fall into two classes.

Regulatory genes -> protein products responsible for transcription.

Operators -> DNA sites near target genes where transcription machinery binds.
 
 

Allostery

Allostery means "other shape". Regulatory proteins often are influenced by the binding of other proteins or ligands to sites other than their DNA binding sites. Ligand binding at these sites can result in alternate shapes and activity which allows transcriptional responsiveness to changing environmental conditions.

Cooperativity

Life depends on the specificity of enzymes.

Each enzyme typically catalyzes just one reaction. Recognition occurs at the "Active Site". Example - b-galactosidase.

Some ligands are found on many catalyzed and non-catalyzed molecules. Example - tyrosines phosphorylated by a tyrosine kinase.
Additional specificity needed.

How is specificity achieved? - Often there are many substrates. Substrate choice - determined by recruitment.

Recruitment involves additional binding at alternative sites separate from the substrate recognition site.

Recruitment often tethers the substrate in close proximity to the substrate binding site such that it has more opportunities to interact with the binding site. This is effectively like increasing the local concentration of the substrate near the binding site which results in greater activity.

Take, for instance, a DNA binding protein. It has a specific site binding site on the DNA and depending on the concentration of the protein and affinity of the protein to the DNA site, it will spend a certain percentage of its lifetime associated with that site. Fig. 1a.

Figure 1. Recruitment example.

A. Single monomer. B. Dimer with both bound. C. Dimer with one bound. Tethering increases concentration to second site.

If two binding sites are adjacent on the DNA and the DNA binding protein can form multimers then the resident time of the DNA binding protein increases. The resident time is usually higher at each site than if the protein only existed as monomers. This is because the tethering of the second protein near the DNA binding site effectively raises the local concentration.

What effects do multiple binding sites have on the resident time of a protein with its target?

Cooperativity between protein subunits can result in allosteric changes that affect the affinity of adjacent subunits for their targets. For example, hemoglobins molecules are composed of four subunits. Each subunit has the ability to bind oxygen. As the first oxygen molecule binds to one of the hemoglobin subunits, it changes its confirmation such that it alters the shape of the adjacent subunits such that these subunits have a higher affinity for oxygen binding. As the second oxygen binds, the allosteric change again increases the affinity of adjacent empty subunits. The dramatic difference between oxygen transport by a myoglobin (single protein) and hemoglobin (4 cooperative allosteric subunits) can be seen below.

Spreadsheet example.

RNA Polymerase

2a, b, b' subunits make up the core enzyme.

Alternative s factors provide specificity for different classes of promoters.

Figure 1.1 the E. coli RNA polymerase.

Upon binding of the RNA polymerase to the promoter the activity of the polymerase can be further regulated by the interaction.

Regulated recruitment: "RNA polymerase bearing the s70 subunit is constitutively active; activators work by recruiting that enzyme to specific genes."

Polymerase Activation: "RNA polymerase bearing the s54 subunit binds to specific genes in a stable but inactive state. Activation of a particular gene requires ... an allosteric change in the polymerase and that change is induced by the activator."

Promoter Activation: "RNA polymerase bearing s70 also binds a small set of genes that form a stable, inactive complex. Activation requires that the promoter undergo a conformational change that is induced by the activator."

Promoter Strength

Basal transcription - dependant on intrinsic promoter strength (-10, -35 in bacteria), conformation to the consensus recognition sequence.

Figure 1.5 Sequence elements in promoters recognized by the holoenzyme containing s70.

Repressors exclude polymerase from promoter site

Activators stabilize polymerase at promoter site

Is strength of a promoter due to recruitment and coopertivity or to induced allosteric changes?

Activator bypass experiments:

Figure 1.8 Activation through heterologous protein:protein interactions.

Figure 1.9 Direct tethering of polymerase.

Increase in concentration of polymerase in the abscence of activator.

Properties of DNA Binding Proteins

1. Protein binds to one side of the DNA

2. Protein is usually a multimer, dimer or greater, of either the same protein or a mixture.

3. Protein binding domain is usually a Helix-Turn-Helix.

4. Protein usually binds in major groove of DNA.

5. Protein hydrogen bonds to specific bases in DNA.

6. Basic proteins interact with negative charges of phosphates at interface.

7. DNA binding site is usually symmetrical.

The Four Classes of Regulation

Positive regulation - binding of regulatory protein to DNA results in transcription.

Negative regulation - binding of regulatory protein to DNA results in no transcription.

Induction - binding of a ligand to regulatory protein results in transcription.

Repression - binding of a ligand to regulatory protein results in no transcription.
 

Figure 1.2 The lac operon.

Figure 1.3 Three states of the lac operon.

  More on Coopertivity

"Cooperativity is a powerful tool for creating sensitive switches" that allow integration of multiple signals.

Created 2004 by CA Rinehart for CLASSROOM USE ONLY. References for source material used here may be found in References .

Index • CourseInfo LogIn • Syllabus • References • Other Resources