Transcript 041610_gene Regulation

The Central Dogma
(Francis Crick, 1958)
(Transcription)
DNA
(Gene)

(Translation)
RNA

Protein
(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 induced
by 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
1. 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)
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:
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 lac operon – An inducible operon
Contains the genes involved in the uptake and breakdown
of the disaccharide lactose
The linkage is a
betagalactoside
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 betagalactoside 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
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
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
Breeding
DNA is a strand of genes, much
like a strand of pearls. Traditional
breeding combines many genes
at once.
Commercial variety
Traditional donor
Desired Gene
New variety
(many genes are transferred)
=
X
(crosses)
Desired gene
Biotechnology
Using biotechnology, a single
gene may be added to the strand.
Commercial variety
Desired gene
(transfers)
New variety
(only desired gene is transferred)
=
Desired gene
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:
1. Read and understand lecture .ppt files and animations
2. Review these materials and use audio file of lectures to self-clarify doubts
3. 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
1. Regulatory (repressor) Gene
I+ or I- or IS
2. Operator Gene
O+ or OC
3. Structural Genes
Z+ or ZY+ or YA+ or A-
The lac operon – An inducible operon
beta-galtosidase
activity
Genotype
I+O+Z+
I+O+ZI–O+Z+
I+OcZ+
IsO+Z+
IsOcZ+
Lactose present
betagalactosidase
activity
Lactose absent
allostearic
protein
The lac operon – An inducible operon
beta-galtosidase
activity
Genotype
I+O+Z+
I+O+ZI–O+Z+
I+OcZ+
IsO+Z+
IsOcZ+
Lactose present
+
betagalactosidase
activity
Lactose absent
The lac operon – An inducible operon
beta-galtosidase
activity
Genotype
I+O+Z+
I+O+ZI–O+Z+
I+OcZ+
IsO+Z+
IsOcZ+
Lactose present
+
betagalactosidase
activity
Lactose absent
-
The lac operon – An inducible operon
beta-galtosidase
activity
Genotype
I+O+Z+
I+O+ZI–O+Z+
I+OcZ+
IsO+Z+
IsOcZ+
Lactose present
+
betagalactosidase
activity
Lactose absent
-
The lac operon – An inducible operon
beta-galtosidase
activity
Genotype
Lactose present
betagalactosidase
activity
Lactose absent
I+O+Z+
+
-
I+O+Z-
-
-
I–O+Z+
I+OcZ+
IsO+Z+
IsOcZ+
The lac operon – An inducible operon
beta-galtosidase
activity
Genotype
Lactose present
betagalactosidase
activity
Lactose absent
I+O+Z+
+
-
I+O+Z-
-
-
I–O+Z+
+
+
I+OcZ+
IsO+Z+
IsOcZ+
The lac operon – An inducible operon
beta-galtosidase
activity
Genotype
Lactose present
betagalactosidase
activity
Lactose absent
I+O+Z+
+
-
I+O+Z-
-
-
I–O+Z+
+
+
I+OcZ+
+
+
IsO+Z+
IsOcZ+
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 lac operon – An inducible operon
beta-galtosidase
activity
Genotype
Lactose present
betagalactosidase
activity
Lactose absent
I+O+Z+
+
-
I+O+Z-
-
-
I–O+Z+
+
+
I+OcZ+
+
+
IsO+Z+
-
-
IsOcZ+
The lac operon – An inducible operon
beta-galtosidase
activity
Genotype
Lactose present
betagalactosidase
activity
Lactose absent
I+O+Z+
+
-
I+O+Z-
-
-
I–O+Z+
+
+
I+OcZ+
+
+
IsO+Z+
-
-
IsOcZ+
+
+