Lecture 3-1

Gene Regulation

Lac Operon
Experimental Concepts Model Utility

Lactose Metabolism
Lactose = galactose (1,4)-glucose (disaccharide; glycosidic bond) (1,4) linkage must be cleaved, then galactose and glucose are degraded Study of the genetics and physiology of lactose metabolism resulted in the operon concept Operon basic genetic regulatory unit Bacterial genes are regulated in response to physiological and external signals

Jacques Monod, early 1940s

Adaptation? Enzyme induction, gene induction, or genetic change?
Diauxie

preferential use of glucose

Enzymatic adaptation? Adaptive enzyme synthesis -single precursor enzyme could give rise to many enzymes by interaction with potential substrate -glucose could be inhibiting conversion of precursor to enzyme that can attack lactose

Early Genetic Studies with Lac

Lederberg
Used lac- mutants in the first genetic mapping experiments Had many synthetic -galactosides made (inducers and substrates) Included o-nitrophenyl--D-galactoside (ONPG)
When hydrolyzed, yellow-coloured o-nitrophenol is released (i.e. chromogenic) Assay for -galactosidase activity

Studied enzyme activity in adapted vs. unadapted cells

Lactose-grown cells had highest activity

Did not follow up these studies, since he was primarily interested in conjugation

Lactose Analogs
IPTG Isopropyl-1-thio--D-galactoside X-Gal 5-bromo-4-chloro-3-indolyl--Dgalactoside ONPG Orthonitrophenol--D-galactoside P-Gal Phenyl--D-galactoside TONPG Orthonitrophenyl--Dthiogalactoside Inducer, non-metabolizable Non-inducing chromogenic substrate, very sensitive, used in vivo Chromogenic substrate, used in vitro Non-inducible substrate, LacY not required for uptake, used to select for lacZ constitutive mutants Toxic to cells, but toxicity is dependent on LacY-mediated uptake, used to select for lacY- mutants

Monod
Synthesized a number of -galactosides
Some were inducers, but not substrates (i.e. gratuitous inducers)
Contradicted hypothesis that the potential substrated combined with the pre-enzyme, to form the active enzyme

INDUCTION IS INDEPENDENT OF UTILIZATION AS SUBSTRATE

Monod
Isolated Lac constitutive mutants by cycling experiments
Cycled culture between glucose and lactose Constitutive mutants that arose in the culture would not be subject to lag for utilization of lactose, and would therefore dominate the culture -GALACTOSIDASE FORMED IN ABSENCE OF SUBSTRATE

Adaptive enzyme hypothesis thrown out

Analysis of Constitutive Mutants from Cycling Experiment

Constitutive expression, even in absence of inducer (i-) Mapped close to lacZY These mutations were trans-recessive (i.e. could be repressed in trans by wt) Suggests that the wt synthesizes a diffusible repressor (product of lacI) Is the repressor a protein?
Yes, because suppressible nonsense mutations have been isolated

Where does the repressor act?

Isolated mutations that allowed constitutive expression in presence of active repressor
Did mutagenesis in presence of 2 copies of lac region (i.e. merodiploid) even if 1 copy of lacI mutated, 2nd un-mutated copy is present These mutations were not able to complement in trans, suggesting a site on the DNA Called lacO (operator) These lacOc mutants are cis-dominant

The Lac Operon

Lac- mutants were generated (no growth on lactose as sole carbon source
Most of these mapped to a specific locus

Used F'lac to study these (merodiploid analysis)

Merodiploid Analysis
1. F'lac / lac+ or F'lac+ / lac always Lac+
1. lac is recessive to lac+

2. F'lac / lac complementation analysis

2 complementation groups (lacZ & lacY)

m1 and m2 are in different genes

m1 and m2 are in the same gene

What are the functions of Z & Y?

1. ONPG assay
-galactosidase activity present in lacZ+, but not lacZ cells

2. Lactose uptake experiments (14C-lactose)

lactose not taken up by lacY cells, but taken up by lacZ cells