Lactose Operon 1ui11

LACTOSE OPERON

PREPARED BY: • NORERLYDA BINTI HAMDAN (2009364241) • NUR SABARIAH BINTI ADNAN (2009946651) • NUR NABILAH BINTI ABU BAKAR (2009977639) • ERNIE SUHAIZA BINTI RADZI (2009301039)

The control of gene expression • Each cell in the human contains all the genetic material for the growth and development of a human • Some of these genes will be need to be expressed all the time • These are the genes that are involved in of vital biochemical processes such as respiration • Other genes are not expressed all the time • They are switched on an off at need

Operon • An operon is a group of genes that are transcribed at the same time. • They usually control an important biochemical process. • They are only found in prokaryotes.

Jacob, Monod & Lwoff © NobelPrize.org

The Lactose Operon  Consists of three genes (Z, Y, A) each involved in processing the sugar lactose  One of them is the gene for the enzyme βgalactosidase (Z)  This enzyme hydrolyses lactose into glucose and galactose  Formation of lac inducer (allolactose)

Lac Genes function • The lac Z gene which is responsible for making βgalactosidase – Β-galactosidase is the enzyme that hydrolyzes (breaks) the bond between glucose and galactose to make the disaccharide lactose

• Lac Y that makes the enzyme permease – Permease is a transport protein that carries lactose into the cell

• Lac A gene which is responsible for making transacetylase – Scientists are not sure of transacetylase’s function

RegulatoryGene Operon

i

p

o

z

y

a

DNA

m-RNA

Protein

repressor

Transacetylase

β-Galactosidase Permease

– Promoter: where RNA polymerase attaches to DNA sequence for transcription – Operator: where repressor attaches to DNA sequence to block transcription – Inducer: inactivates the repressor and removes it from the DNA (lactose is the inducer) – Repressor: attaches to the operator and blocks RNA polymerase from transcribing mRNA

How does Lactose Operon work? • Glucose not available, so cells need other source of energy such as lactose.

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Two forms of regulation 1) cis regulation of transcription (cis = adjacent) • refers to DNA sequences that regulate the transcription of downstream genes, through interaction with DNA binding proteins – Promoter: • RNA polymerase II binds to promoter • Initiates transcription of the lac genes – Operator • Binds a repressor which inhibits DNA synthesis • Overlaps with the promoter by about 9 bp

– CAP binding sites • Within the promoter sequence • Activated CAP binds to this sequence and results in more efficient binding of RNA polymerase II • Turns the operon “ON” • Without CAP bound there is a very small amount of the lac genes produced – still referred to as “OFF” • The promoter CAP binding sites are considered to be positive cis elements. Function in activating the operon. • The operator is a negative cis element. Function in deactivating the operon

2) Trans regulation of transcription (trans = across) • Proteins that regulate transcription by binding to cis elements a) Repressor – Has two binding sites – One is a DNA binding site, associates with operator as a tetramer – Inhibits RNA synthesis by blocking the action of RNA polymerase II • Forms a large complex next to the promoter and doesn’t allow enough space for the polymerase to bind – called “STERIC HINDERANCE”

– When the inducer (allolactose) binds the repressor (lacI) it undergoes a conformational change and can no longer bind the operator as a tetramer. Allows RNA polymerase II to bind. • Modification of conformation due to the binding of a small molecule – “ALLOSTERIC REGULATION” b) RNA polymerase - Transcribes the RNA c) CAP - Increases RNA polymerase binding efficiency & requires cyclic AMP to be active

Negative control of the lac operon The i gene encodes a repressor which, in the absence of lactose (top), binds to the operator (o) and blocks transcription of the three structural genes (z, βgalactosidase; y, permease; and a, transacetylase). Lactose induces expression of the operon by binding to the repressor (bottom), which prevents the repressor from binding to the operator. P = promoter; Pol = polymerase

Positive control of the lac operon Low levels of glucose activate adenylyl cyclase, which converts ATP to cyclic AMP (cAMP). Cyclic AMP then binds to the catabolite activator protein (CAP) and stimulates its binding to regulatory sequences of various operons concerned with the metabolism of alternative sugars, such as lactose. CAP interacts with the α subunit of RNA polymerase to activate transcription

Carbohydrates

Activator protein

Repressor protein

RNA polymerase

lac Operon

+ GLUCOSE + LACTOSE

Not bound to DNA

Lifted off operator site

Keeps falling off promoter site

No transcription

+ GLUCOSE - LACTOSE

Not bound to DNA

Bound to operator site

Blocked by the repressor

No transcription

- GLUCOSE - LACTOSE

Bound to DNA

Bound to operator site

Blocked by the repressor

No transcription

- GLUCOSE + LACTOSE

Bound to DNA

Lifted off Sits on the operator site promoter site

Transcription

Mutation Structural genes: • One functional copy of a gene; lactose is metabolized • Two non-functional copies of a gene present in the cell leads to a nonfunctional metabolic pathway – Remove lacY – lactose cannot enter the cell – Remove lacZ – lactose cannot be broken down into glucose and galactose Operator: • Normal – repressor can bind (O+) • Mutated – constitutive expression – negative regulation (expressed as Oor Oc) – Repressor can’t bind so the operator is desensitized to the lacI protein – Transcription occurs whether lactose is present or not

Promoter • Normal – RNA polymerase can bind (P+) • Mutated – RNA polymerase never binds (P-) Inhibitor • Normal – can bind the operator when no lactose is present. (I+) • Two mutants: 1) Mutated DNA binding domain – never binds operator, operon is not sensitive to inhibition by lacI protein. (I-) 2) Mutated allolactose binding site – (Is – super suppressor) loses the ability to associate with allolactose; the tetramer can never be removed from the operator in response to lactose entering the cell

References • Principle of Genetics fifth edition by D. Peter Snustad and Michael J. Simmons, 2010, Wiley and Sons. • Essentials of Genetics fifth edition by William S. Klug and Michael R. Cummings, 2005, Pearson Education Inc. • Website: www.wikipedia.com/lactose_operon • Website: pdf search engine

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