3: Prokaryotic Regulation of Genetic Expression

Prokaryotic Regulation of Genetic Expression

Regulation of transcription: Operons and operators

Vocabulary

structural genes: genes which encode information for a protein

“gene expression”= transcription (+ translation when describing structural genes)

for our class discussion, we will presume that when a structural gene is transcribed, the mRNA will be translated into protein thus “gene expression” results in protein production

-ase= common ending for enzymes

lactose: also called “milk sugar”, lactose is a disaccharide of glucose and galactose joined by a glycosidic bond.

lactose= galactose-glucose

promoter: special DNA sequence to which RNA polymerase binds to start transcription

operon: DNA sequence encoding promoter and operator sites and structural genes they control

operator: special DNA sequence to which repressor proteins may bind to block transcription by RNA polymerase; the “on-off” switch for transcription

allosteric proteins: proteins which can have 2 different forms

lac repressor protein: an allosteric protein which has 2 forms:

an active form in which the repressor bind to the lac operator and blocks transcription of the lac operon structural genes when lactose is absent and …an inactive form which cannot bind the lac operator. The inactive form is dominant when lactose is present and the inducer allolactose binds to the lac repressor protein.

Intro survival notes:

Gene expression and protein synthesis are costly to cells (requires lots of “building blocks” and energy.

(From Bauman) ...most bacterial genes are expressed constantly= constitutive expression. These genes include genes for tRNAs, rRNAs and structural genes for proteins constantly needed for example, ribosomal proteins and enzymes used in glycolysis.

However some gene products may be required only under certain conditions. For example, the gene encoding the enzyme beta-galactosidase need only be expressed if lactose is present in the environment. Beta-galactosidase is the bacterial equivalent of human “lactase”. It catalyzes hydrolysis of the glycosidic bond between the residues of glucose and galactose in milk sugar, lactose:

Lactose-> beta-galactosidase -> glucose + galactose

Bacteria which can turn on and turn off transcription of genes like beta-galactosidase will have a survival advantage over other bacteria which cannot regulate gene expression (Why?)

Regulation of Bacterial Gene Expression: transcription control

1. Three types of gene expression

• constitutive: continual transcription (and continual synthesis of encoded proteins)
• inducible: made only when substrate or signal molecule is present e.g., enzymes for lactose transport/metabolism in E. coli
• repressible: produced only when signal molecule is scarce

2. Operon: group of genes whose transcription is coordinately turned on or off; under control of operator and promoter

3. Operator: specific DNA sequence which lies between promoter and 1st codon of gene. Repressor proteins bind operator and

block ability of RNA polymerase to bind promoter/ transcribe “downstream” structural genes. Repressor proteins are allosteric proteins. The have one binding site for a DNA operator sequence and a second binding site for an “inducer” molecule for e.g. allolactose in the lac operon ( or a “corepressor” molecule e.g. trp operon )

Inducible operons. Gene transcription turned only when substrate/signal is present. Usually genes for catabolic enzymes example: E. coli lac operon. Jacob and Monod 1961

1. Lac operon consists of 3 structural genes (lacZ, Y and A) , promoter and operator.

Lac operon is inducible.

Structural gene gene product (enzyme/transport protein)

lacZ beta-galactosidase

lacY lactose transport protein/ galactoside permease

lacA galactoside transacetylase

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2. Regulatory/repressor gene, lacI

LacI is the lac repressor protein.

The lac repressor protein is a DNA binding protein, which when active can bind to the lac operator, blocking transcription/expression of lacZ, Y and A in absence of lactose (survival note: it would be wasteful for the bacterium to make these proteins if lactose is not present). Lac repressor gene has its own promoter and is constitutively expressed. The lac repressor gene is NOT part of the lac operon

3. lac operon when NO LACTOSE AVAILABLE (fill-in cartoon below, double dotted lines represent a single strand of DNA)

DNA ___________________________________________________________

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lac I Promoter_lac Operator_lac lacZ lacY lacA

How should your cartoon look?

lac repressor protein binds lac operator in absence of lactose, blocks transcription of lac genes by RNA polymerase. Note: all repression is “leaky”, that is repressor binds and releases operator in a concentration dependent manner. When lactose is absent, most repressor proteins are in the active form, blocking most (but not all) transcription of the lac structural genes)

4. ADD LACTOSE: (How does your cartoon differ from above?)lactose-> inducer allolactose binds to allosteric site on lac repressor, causes it to change shape so it can no longer bind operator.

DNA ___________________________________________________________

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lac I Promoter_lac Operator_lac lacZ lacY lacA

DNA ___________________________________________________________

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lac I Promoter_lac Operator_lac lacZ lacY lacA

5. Now RNA polymerase can start transcribing the lac genes and cell can makes transport protein and beta-galactosidase, cell starts transporting and breaking down lactose at high rate

6. When lactose used up, allolactose levels drop/release lac repressor, repressor regains shape, binds operator, turns off lac gene transcription

DNA ___________________________________________________________

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lac I Promoter_lac Operatorlac lacZ lacY lacA

What would happen if…..

What if….

E.coli had a mutation so that the lac repressor could not bind DNA?

“ “ had a mutation so that the mutant lac repressor protein could NOT bind allolactose, the inducer?

“” had a mutation so that the operator could not bind normal lac repressor protein?

Deleted section from previous semesters:

I. Regulation of metabolism: 2 ways

A. Change activity of enzymes: allosteric sites/allosteric enzymes

1.binding sites other than active site=allosteric sites, bind “effectors”

2. binding of effector changes 3-D shape of enzyme

3. Two kinds of effectors:

a. allosteric activators: bind allosteric site, change enzyme so that it is MORE ACTIVE/turned on/activate enzyme

b. allosteric inhibitors: bind allosteric site, change enzyme shape so it is less active/turned off /inhibit enzyme e.g.,: end product inhibition

4. Provides rapid response to changes in substrates, need for endproducts

B. Change expression of genes that is regulate transcription (or even translation)