Regulation of Gene Action

The basis of cell differentiation is generegulation: different sets of genes are turned onand off in different cells. (There are othermechanisms as well but this is our focus.)

E.g. globin genes are expressed only inerythroblasts and are turned off in muscle cells.Myosin genes are on in muscle cells but off inerythrocytes.

Progression through the cell cycle also requiresturning different sets of genes on and off atdifferent stages.

Bacteria and single-celled eukaryotes undergocell differentiation. This includes respondingto the availability of different nutrients.

I will discuss some of the most basic aspects;Dr. Restifo will give more detail.

Levels of Expression and Control

The expression of any genebegins with transcriptionwhich can be regulated. Theexpression of protein-codinggenes requires severaladditional steps and can beturned off at any step.However, I will focus only onthe control of transcription.

DNA

mRNA

Polypeptide

Protein

transcriptionmRNA stability

translation(ribosomes,tRNAs, aminoacids, etc.)

foldingstabilityaggregationlocalization

Transcription Control in ProkaryotesNegative Regulation

Negative regulationinvolves a proteinrepressor that binds to arepressor binding siteand prevents binding ofthe transcriptioncomplex.

Inducible system: offunless inducermolecule binds to andinactivates repressor.

Repressible system: onunless co-repressorbinds to inactiveaporepressor to formactive repressor.

Negative Regulation Examples

Inducible system: lactose operon in E. coli

E. coli can cleave lactose into glucose + galactose to use forcarbon and energy sources. This requires the enzyme β-galactosidase, and also galactoside permease to import the lactoseinto the cell.

If there is no lactose in the medium, E. coli does not make eitherβ-galactosidase or galactoside permease. Synthesis is blocked atthe transcription level. If lactose is added to the medium, thesynthesis of both molecules is induced.

lac OperonOperon encodes• lacZ: β-galactosidase which cleaves lactose

into glucose and galactose.• lacY: lactose permease which brings lactose

into the cell• lacA: thiogalactoside transacetylase, not

required for growth on lactose, unknownfunction but sequence is conserved so it isimportant.

Other important players:• lacP: Promoter, binds RNA polymerase to

start transcription• lacO: Operator, binds repressor• lacI: Repressor gene, encodes repressor

protein

β-galactosidase and lactose permease are madeonly if lactose is present; only if they areneeded.They are induced by their substrate.

Simple Model of lac System

lacI P O Z Y A

Repressorprotein

Inducer

RNA polymerase

Basic features of lac operon were deduced from mutant phenotypes byFrancois Jacob, Jacques Monod, and collaborators in 1960s by studying thephenotypes of mutants.

lac Operon Mutants

Francois Jacob, Jacques Monod, Andre LwoffNobel Prize 1965

“Commandant Malivert”of the Resistance, Legion ofHonor

Jacob in medicalbattalion in NorthAfrica, wounded inaction

lac Operon Mutants

Repressor can’t bind inducer;super-repressorlacI s

Promoter can’t bind RNApolymerase; operon not

transcribedlacP -

lacO can’t bind repressor;constitutive (always on)lacO c

Null mutation in lacYlacY -

Null mutation in lacZlacZ -

NatureGenotype

Studied the interactions of different mutant alleles in partial diploidswhich have the bacterial chromosome plus a plasmid with some genes.Plasmids = small DNA molecules that use their own replication origins toreplicate independently of the cell chromosome; have the own origin ofreplication. Usually not required for cell function; some may be present inmany copies.

Studying Interactions of lac Operon Mutants

ChromosomeF’ lac plasmid

lac operonlacZ + lacY -

lac operonlacZ - lacY +

Cell genotype F’ lacZ - lacY + / lacZ + lacY - Cell phenotype Lac + (NOTE: other genes assumed to be wild type if not specified.)

(DNA molecules notto scale)

Phenotypes of lac Operon Mutants in Diploids

Jacob, Monod, and collaborators deduced how the lac operon is controlled from these dataand from the map position of the mutants. Note lacO and lacP mutants only affect expressionof lac genes on the same chromosome, while lacI mutants can operate at a distance, fromanother chromosome.

lac Operon: Second Level of Regulation(Things are always more complicated than we’d like.)

When glucose is present, β-galactosidase etc. are notmade.

cAMP = cyclic AMPCRP = cAMP receptorprotein

Glucose inhibits synthesisof cAMP.

cAMP+CRP must bebound to promoter in orderfor lac transcription

lac Operon StructuralDetails

Negative Regulation Examples

Inducible lac operon: operon turned ononly when lactose is available as acarbon and nitrogen source.

Repressible system: tryptophanoperon in E. coli

Tryptophan is needed all the time bygrowing cells, so trp genes should be onall the time, until cells have madesufficient tryptophan (or it is providedin the medium).

Chorismic acid

Anthranilic acid

PRA

CDRP

InGP

Indole

L-Tryptophan

ASase trpE

PRTase trpD

InGPSase trpC

TSaseB trpB

TSaseA trpA

Negative Regulation ExamplesRepressible system: tryptophan operon in E. coli

Tryptophan is needed all the time by growing cells, so trp genes should be on all thetime, until cells have made sufficient tryptophan.

trpA-E genes are in an operon. OK if only needed to make tryptophan.Has operator trp o and promoter trp-p and attenuator trp a.

Negative Regulation of trp Operon

Tryptophan levels low:aporepresssor proteincomplex (encoded by distantgenes) can’t bind topromoter and transcriptionis on.

Tryptophan levels high:tryptophan binds toaporepressor to form activerepressor which binds topromoter and shuts offtranscription.

Attenuation mechanismstops transcription in leaderregion unless there issufficient Trp-tRNA in thecell, provides fine-tuning oftryptophan synthesis.