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


• 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


• Repressible genes/enzymes– Turn-off the genes (enzymes) needed to

synthesize the amino acid from precursor molecules

– Save energy


• 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 induced

by the externally supplied compounds (the external environment)


How are genes turned-on and turned–off?• Controlled by special proteins

– Called regulatory proteins

Regulatory proteins are of two types1. Repressor proteins

– Negative regulators (turn genes off)2. 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)


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


• 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)

Promotor (p) – the binding site for the RNA polymerase (control site)

Operator (o) – the binding site for the regulatory protein (control site)

Structural genes (S) – each encode an amino acid sequence of a polypeptide

The operon consists of:

The lac operon – An inducible operonContains 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 repressor and 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

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

When lactose is present

Desired gene

BreedingDNA is a strand of genes, much like a strand of pearls. Traditional breeding combines many genes at once.

Traditional donor Commercial variety New variety

Desired GeneX =

(crosses)

(many genes are transferred)

BiotechnologyUsing biotechnology, a single gene may be added to the strand.

Desired gene Commercial variety New variety

(transfers)=

Desired gene

(only desired gene is transferred)

New Course ListingIntroduction to Biotechnology (PLS/MIC/MCB 340)

Fall Semester, 2010MWF 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 Genomics04/26/10 Monday is a back up slot, in case I could not finish on Friday04/28/10 Wednesday is a review lecture of all the second half topics

Preparation:

1. Read and understand lecture .ppt files and animations2. Review these materials and use audio file of lectures to self-clarify doubts3. 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 operonContains the genes involved in the uptake and breakdown of the disaccharide lactose

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 repressorProtein unable to bind to the inducerSuper repressor – the genes can never be turned-on (no proteins produced)

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 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


1. Regulatory (repressor) GeneI+ or I- or IS

2. Operator GeneO+ or OC

3. Structural GenesZ+ or Z-

Y+ or Y-

A+ or A-



beta-galtosidase activity

beta-galactosidase

activityGenotype Lactose present Lactose absent

I + O + Z +

I + O + Z -

I – O + Z +

I + O c Z +

I s O + Z +

I s O c Z +

allostearicprotein



beta-galactosidase


I + O + Z + +I + O + Z -

I – O + Z +

I + O c Z +

I s O + Z +

I s O c Z +



beta-galactosidase


I + O + Z + + -I + O + Z -

I – O + Z +

I + O c Z +

I s O + Z +

I s O c Z +



beta-galactosidase


I + O + Z + + -I + O + Z -

I – O + Z +

I + O c Z +

I s O + Z +

I s O c Z +



beta-galactosidase


I + O + Z + + -I + O + Z - - -I – O + Z +

I + O c Z +

I s O + Z +

I s O c Z +



beta-galactosidase


I + O + Z + + -I + O + Z - - -I – O + Z + + +I + O c Z +

I s O + Z +

I s O c Z +



beta-galactosidase


I + O + Z + + -I + O + Z - - -I – O + Z + + +I + O c Z + + +I s O + Z +

I s O c Z +

lac i s

Defective repressorProtein unable to bind to the inducerSuper repressor – the lac operon genes can never be turned-on (consequently, no proteins are produced)



beta-galactosidase


I + O + Z + + -I + O + Z - - -I – O + Z + + +I + O c Z + + +I s O + Z + - -I s O c Z +



beta-galactosidase


I + O + Z + + -I + O + Z - - -I – O + Z + + +I + O c Z + + +I s O + Z + - -I s O c Z + + +

The Central Dogma (Francis Crick, 1958)

Documents

genes enzymes

genes turnedon

controlled gene

genes onin

lac operon

group of structural

amino acid sequence

energygene regulation