The Central Dogma (Francis Crick, 1958)

The Central Dogma(Francis Crick, 1958) (Transcription) (Translation) DNA  RNA  Protein (Gene)(Phenotype) An informational process between the genetic material (genotype) and the protein (phenotype

Gene Regulation(Regulation of Gene Transcription) Chapter 16 Pages 433 - 476

Constitutive: needed all of the time Inducible: only needed some of the time

Gene Regulation in Bacteria • Constitutive genes/proteins • Genes always on and enzymes always made (needed) • Are not influenced by the external environment • Enzymes involved in the basic metabolism of the cell • Needed by the cell under all environments

Gene Regulation in Bacteria • However, bacteria must be able to adapt very rapidly to a changing environment • More so than eukaryotes (multicellular organisms) • For instance, they can make all 20 amino acids • If one amino acid is added to the external environment • It would clearly be more economical (energy wise) to take-up and use the amino acid rather than make it

Gene Regulation in Bacteria • Repressible genes/enzymes • Turn-off the genes (enzymes) needed to synthesize the amino acid from precursor molecules • Save energy

Gene Regulation in Bacteria • Inducible genes/enzymes • Must turn-on the genes (enzymes) needed to take-up and metabolize the amino acid • Genes that are not often needed, or may never be needed to be turned-on • The expression of these genes is inducedby the externally supplied compounds (the external environment)

Gene Regulation in Bacteria How are genes turned-on and turned–off? • Controlled by special proteins • Called regulatory proteins Regulatory proteins are of two types • Repressor proteins • Negative regulators (turn genes off) • Activator proteins • Positive regulators (turn genes on)

In the absence of the repressor, the gene is expressed (the gene is turned-on) The presence of the repressor protein prevents the expression of the controlled gene (the gene is turned-off)

In the absence of the activator, the gene is not expressed (the gene is turned-off) The presence of the activator protein causes the controlled gene to be expressed (the gene is turned-on)

Gene Regulation in Bacteria The operon model • Francois Jacob and Jacques Monod (1961)

The lac operon – An inducible operon • Glucose is used in energy metabolism • When glucose is present, there is no need to take-up and convert other sugars • It is much more efficient (energy-wise) to use glucose directly

The lac operon – An inducible operon • When glucose is not present • The bacteria must synthesize proteins to take-up other sugars • And the enzymes to metabolize these sugars into glucose

Operon is a group of structural genes whose transcription into a polycistronic mRNA is under the control of a separate regulatory gene (control gene) Transcription of the genes in the operon is coordinately regulated (All genes are turned-on or turned-off together)

The operon consists of: • Operator (o)– the binding site for the regulatory protein (control site) • Promotor(p)– the binding site for the RNA polymerase (control site) • Structural genes (S)– each encode an amino acid sequence of a polypeptide

The lac operon – An inducible operon Contains the genes involved in the uptake and breakdown of the disaccharide lactose

The linkage is a beta-galactoside bond • Glucose is used in metabolism, so other sugars must be converted to glucose • In order for the bacteria to use the lactose, they must first break the beta-galactoside linkage in the disaccharide lactose to produce one molecule of galactose and one of glucose • Lac operon contains the genes involved in the uptake and breakdown of the disaccharide lactose

When lactose is absent In an inducible operon, the regulatory protein is an active repressorand is made from a separate gene with its own promoter) Repressor binds to the operator, interferes with the binding of the RNA polymerase and prevent transcription of the structural genes. This is the default state.

Inducible Operon When lactose is present • Inducers: • Turn the genes on • bind to an active repressor, making the repressor inactive • Molecules from the environment (e.g., sugars, amino acids) • Almost always what needs to be metabolized by the enzymes coded for by the structural genes

Breeding Commercial variety New variety Traditional donor DNA is a strand of genes, much like a strand of pearls. Traditional breeding combines many genes at once. (many genes are transferred) = X (crosses) Desired Gene Desired gene Biotechnology Commercial variety New variety Desired gene Using biotechnology, a single gene may be added to the strand. (only desired gene is transferred) = (transfers) Desired gene New Course Listing Introduction to Biotechnology (PLS/MIC/MCB 340) Fall Semester, 2010 MWF 1:00-1:50, Chavez 303 Instructor: KA Feldmann. Over the past 25 years, Dr. Feldmann has worked at 3 different biotech companies. He is currently the Director of the School of Plant Sciences. Prerequisites: PLS 240 or MCB 181 or MIC 205A. Contact KAF @ 621-1977

EXAM IV IS ON APRIL 30, FRIDAY, 11-NOON • Topics included: lectures covered until 04/23/10 (next Friday) • That is, all the topics I covered and will cover except Genomics • 04/26/10 Monday is a back up slot, in case I could not finish on Friday • 04/28/10 Wednesday is a review lecture of all the second half topics • Preparation: • Read and understand lecture .ppt files and animations • Review these materials and use audio file of lectures to self-clarify doubts • If you still do not understand, seek help from preceptors, TA and me 4. Then, only then, try to answer questions found at the end of the book Chapters, practice problems and those I included in review lectures. 5. Do not start the preparation for the exam with these questions. 6. If you could not answer them, do not assume that • you are not good and smart enough for this course • this course is hard, • you are bound to fail, • the exam is going to be difficult and • Ravi is an out of touch Alien from another galaxy

Alleles for the genes in the lac operon Mutants in the regulatory (repressor) gene 1. lac I – • Defective repressor • Mutation in the allosteric site that binds to DNA (operator) Outcome: Repressor protein unable to bind to the operator DNA sequence Lac operon phenotype: Always transcribed (constitutive), whether the inducer is present or not

The lac operon – An inducible operon Contains the genes involved in the uptake and breakdown of the disaccharide lactose

Alleles for the genes in the lac operon Mutants in the regulatory (repressor) gene 2. lac I s • Defective repressor • Mutant in the allosteric site that binds with the inducer Outcome: Repressor protein unable to bind to the inducer (lactose) Lac operon phenoytpe: Genes are always (constitutively) turned-off

lac i s • Defective repressor • Protein unable to bind to the inducer • Super repressor – the genes can never be turned-on (no proteins produced)

Alleles for the genes in the lac operon Mutation in the operator DNA sequence 3. lac o c • Mutant operator • Operator will not bind any repressor • The repressor cannot recognize the operator Outcome: Repressor protein unable to bind to the operator DNA sequence Lac operon phenotype: Always transcribed (constitutive), whether the inducer is present or not

The repressor protein cannot recognize mutant operator

Alleles for the genes in the lac operon Alleles in the structural genes • 4.lacZ – • Mutant Beta-galactosidase gene • Similarly, lacY– and lacA- are mutants in permease and transacetylase genes Outcome: Make mutant proteins that are unable to metabolize lactose Lac operon phenotype: Lactose not metabolized

Alleles for the genes in the lac operon • Regulatory (repressor) Gene I+ or I- or IS • Operator Gene O+ or OC • Structural Genes Z+ or Z- Y+ or Y- A+ or A-

The lac operon – An inducible operon

allostearic protein

lac i s • Defective repressor • Protein unable to bind to the inducer • Super repressor – the lac operon genes can never be turned-on (consequently, no proteins are produced)

The Central Dogma (Francis Crick, 1958)