Lecture 10: Control of gene expression

1. One gene one enzyme hypothesis 2. Lac operon 3. Negative and positive regulation

Lac repressor bound to DNA

The Nobel Prize in Physiology or Medicine 1958

"for his discoveries concerning genetic recombination and the organization of the genetic material of bacteria"

"for their discovery that genes act by regulating definite chemical events"

George Wells Beadle 1/4 of the prize California Institute of Technology (Caltech)

Edward Lawrie Tatum 1/4 of the prize Rockefeller Institutute for Medical Research, NY

Joshua Lederberg 1/2 of the prize University of Wisconsin Madison, WI

Experiments of Beadle and Tatum, in 1940-ies, were the first to shed light on general mechanisms of the gene function. They studied mutations in the arginine biosynthetic pathway in Neurospora.

Arginine has two related substances: citrulline and ornithine that are converted into arginine by the pathway that must involve several enzymes:

Enz. X Enz. Y Enz. Z Precursor -----> Ornithine ------> Citrulline -----> Arginine

Search for mutants in amino acid biosynthesis

Growth of some arg- mutants can be supplemented not only by arginine, but also by related compounds: Mutant arg 1 arg 2 arg 3 Ornithine + Citrulline + + Arginine + + +

(plus means growth, and minus means no growth)

The compound which is made the latest (Arg) supports growth of the largest number of mutants The compound which is made the earliest (Orn) supports growth of smallest number of mutants

Enz. X Enz. Y Enz. Z Precursor -----> Ornithine ------> Citrulline -----> Arginine

how to explain the observed growth pattern?

Mutant arg 1 arg 2 arg 3

Ornithine + -

Citrulline + + -

Arginine + + +

- they hypothesized that each mutant is deficient in production of certain enzyme of the pathway, e.g. arg1 could not produce Enzyme X. Then this mutant cannot convert the precursor into ornithine. But given ornithine, it still can make citrulline and arginine.

Enz. X Enz. Y Enz. Z Precursor -----> Ornithine ------> Citrulline -----> Arginine

arg1

arg2+

arg3+

Mutant arg 1 arg 2 arg 3

Ornithine + -

Citrulline + + -

Arginine + + +

- mutant arg2 could not produce Enzyme Y. It can make ornithine (arg1+ allele) but cannot convert it into citrulline. Thats why its growth is not supported by ornithine. Citrulline, however, supports

Enz. X Enz. Y Enz. Z Precursor -----> Ornithine ------> Citrulline -----> Arginine

arg1+

arg2

arg3+

Mutant arg 1 arg 2 arg 3

Ornithine + -

Citrulline + + -

Arginine + + +

- mutant arg3 could not produce Enzyme Z. It can make ornithine and citrulline but cannot convert citrulline into arginine. Thats why its growth is not supported by ornithine or citrulline and is supported only by arginine.

Enz. X Enz. Y Enz. Z Precursor -----> Ornithine ------> Citrulline -----> Arginine

arg1+

arg2+

arg3

Mutant arg 1 arg 2 arg 3

Ornithine + -

Citrulline + + -

Arginine + + +

Enz. X Enz. Y Enz. Z Precursor -----> Ornithine ------> Citrulline -----> Arginine

arg1+

arg2+

arg3+

One gene one enzyme hypothesis: genes control biosynthetic pathways by encoding enzymes that catalyze specific steps of the pathways

Growth of E. coli with two energy sources: glucose and lactose

Phase II: utilization of lactose

Phase I: utilization of glucose

... nor is the metabolic chart

Ability of E.coli cells to utilize lactose depends on production of the enzyme -galactosidase
Phenotype Minimal Medium with glucose Minimal Medium with lactose

Lac+(inducible) Lac- (negative) Lac++ (constitutive)

- (no enzyme) +

+ (enzyme is produced) +

lac operon

A group of genes that are regulated together and are closely linked to each other is called an operon Genes of the same operon are transcribed together as a polycistronic (= polygenic) mRNA Lac operon is located at 8 min of the E. coli chromosome map. It contains three genes lacZ, lacY and lacA. The respective three enzymes of the lac operon are induced simultaneously.

lacP (promoter) and lacO (operator) are the operons regulatory regions

lacZ- or lacY- mutations cause Lac- phenotype

lac operon

An additional class of mutations was found close to lac operon. These mutations caused constitutive phenotype (Lac++), and it was concluded that they affect inducibility locus. Hence the name lacI. The lacI gene encodes lac repressor that inhibits the lac lacZ- or lacY- mutations cause Lac- phenotype operon. The operon is derepressed by lacI- mutations case Lac++ phenotype lactose in the medium.

The Nobel Prize in Physiology or Medicine 1965

"for their discoveries concerning genetic control of enzyme and virus synthesis"

Franois Jacob b. 1920

Jacques Monod 1910-1976

Andr Lwoff 1902-1994

lac repressor can reversibly attach to lac operon

Lac+ phenotype

lactose absent

lactose present

DNA binding site

Normal lac-repressor can interact with (bind to): 1) DNA (lac-operon) and 2) inducer (lactose)

lactose binding site

Mutant forms of lac repressor:

cannot attach to lac operon (lacI-) or cannot detach from it (lacIS)
Lac++ phenotype

lactose may be absent or present

Lac- phenotype

lactose may be absent or present

Mutant lacIS repressor is dominant to normal lacI+

Meroploid F+ cell
Lac- phenotype

F lacI+ / lacIS lacZ+ (lacIS is in cis to lacZ+) F lacIS / lacI+ lacZ+ (lacIS is in trans to lacZ+)

The same phenotype Lac-

Normal lacI+ repressor is dominant to mutant lacIMeroploid F+ cell

Lac+ phenotype

Normal lacI+ repressor is dominant to mutant lacIMeroploid F+ cell

Lac+ phenotype

F lacI+ / lacI- lacZ+ (lacI+ is in trans to lacZ+) F lacI- / lacI+ lacZ+ (lacI+ is in cis to lacZ+)

The same phenotype Lac+

Mutant lacOC operator in cis to lacZ is epistatic to all types of lac repressor
Lac++ phenotype

F lacI+ / lacOc lacZ+ F lacIS / lacOc lacZ+ F lacI- / lacOc lacZ+

Any type or configuration of lacI, provided that lacOC is in cis to lacZ+ the same phenotype Lac++

Mutant lacOC operator in trans to lacZ has no effect

F lacOc / lacO+ lacZ+

Now the phenotype depends on the type of the repressor present: no epistatic effect of lacOC on alleles of lacI

Carrot and whip on the molecular level

cyclic AMP, a regulator of many biological processes

the formulas are not for memorization

glucose cAMP

glucose

cAMP

Glucose is low

transcription is activated