Outline Nov. 8 Types of Gene Regulation • Review the lactose (lac) operon – Predicting phenotypes of partial diploids • Gene regulation can occur at various steps Examples of other operons: – The amount of product depends on • arabinose (ara) operon What are the similarities and • rate of mRNA synthesis (transcription), • arginine (arg) operon differences in the • mRNA degradation, – A repressible operon control of these • protein synthesis (translation) etc. operons? • tryptophan (trp) operon • Prokaryotes commonly control – Two kinds of regulation • Repression transcription • Attenuation Terminology Types of Gene Regulation • Constitutive genes are always expressed – Tend to be vital for basic cell functions (often called • Repressors and Activators are proteins that housekeeping genes) bind to DNA and control transcription. – Those genes are said to be repressible or • Inducible genes are normally off, but can be turned inducible on when substrate is present – Common for catabolic genes (i.e. for the utilization of particular resources) • Inhibitors and Inducers: small “effector” • Repressible genes are normally on, but can be molecules that bind to repressors or turned off when the end product is abundant activators – Common for anabolic (biosynthesis) genes Operons Organization of the lac operon • In Prokaryotes, functionally related genes are regulated as a unit, called an One promoter operon. One operator Controls 3 • Operons consist of: enzymes – Several structural genes – ONE promoter and one terminator – A control site (operator) – A separate regulator gene (codes for protein that binds to operator) Repressor 1 Overview of the lac Operon Glucose must be absent • Gene is normally off – There is no transcription because a repressor binds to the • “Catabolite repression” control site – A separate regulatory mechanism controls the • When lactose is present, it inactivates the repressor, binding of RNA polymerase to the promoter. allowing transcription to begin. – Produces both lacZ (to break down lactose) and lacY (to let it into the cell) – If glucose is present, there is little cAMP, so the • When lactose is used up, the repressor is again free activator complex (CAP-cAMP) can not bind to the to bind to DNA, and halt transcription. promoter region. • Glucose must be absent. If glucose is present, transcription doesn’t start. Cells respond quickly to Some practice available sugars • What is the phenotype: Add lactose A B Add glucose lacI+ lacO+ lacZ- lacY+ / F’ lacI- lacO+ lacZ+ lacY- 1250 1000 – Will there be B-galactosidase activity? • With lactose? Without lactose? 750 – Will there be permease activity? !-galactosidase activity 500 • With lactose? Without lactose? – Are the genes inducible (is there a difference with 250 and without lactose)? 0 0 1 2 3 4 5 Time (h) More practice Even More practice lacI+ lacO+ lacZ- lacY+ / F’ lacI- lacO+ lacZ+ lacY- • Make a table: – Will there be B-galactosidase activity? -lactose + lactose Interpretation • With lactose? Without lactose? – B-gal – Will there be permease activity? – Permease • With lactose? Without lactose? 2 Which is more effective in regulating lac: What effects do you predict for repressor or CAP? mutations in the operator? • What is the phenotype for: “Northern Blot” detects mRNA on a gel lacI+ lacOc lacZ- lacY+ / F’ lacI+ lacO+ lacZ+ lacY- 1 2 3 4 – What does the operator do? – What will be the consequence if it is non-functional? – Will the mutation be cis-dominant, trans-dominant, or 1: no sugars recessive? 2: lactose 3: glucose 4: glucose+lactose -lactose + lactose Interpretation –B-gal –Permease Arabinose operon Arabinose operon Arabinose binds to the Transcription is repressor and “unlocks” normally off transcription (“Keep the operon turned off, until there is something to metabolize”) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. araO2 araC Fig. 14.1A2raC (proTteinE Art) Where is the likely mutation? Arabinose binding domain Linker region Pc DNA binding domain 1. AraB is expressed when arabinose is P araB araA araD a CAP site BAD raO 1 araI added, but not araA. (a) Operon inhibited in the absence of arabinose 2. Enzymes are never expressed, even n Loop broken D o a i r t a p i when arabinose added to the medium. r A c a s r n a a r a Arabinose T r B a a C r a araO2 cAMP AD P B CAP l P a c CAP site r a RNA ara polymerase O1 (b) Operon activated in the presence of arabinose 3 Arginine: a repressible operon Repressible and Inducible operons • Arginine is an essential amino acid. Arginine biosynthesis Lactose degradation Transcription is normally on. • When excess arginine is present, it binds to the repressor and changes its shape. Then the repressor binds to the operator and blocks arginine synthesis. • (“Don’t synthesize arginine if plenty is already available”) Repression Induction Brock Biology of Microorganisms, vol. 9, Chapter 7 The trp Operon The trp operon Also a promoter and a special “leader” peptide, • trp is another example of a repressible trpL operon 5 genes: E, D, C, B, A • Contains genes for the synthesis of tryptophan • Normally on; If the end product (tryptophan) is abundant, the operon is turned off. Same order as enzymes for trp synthesis Trp operon • Two regulation mechanisms, repression and attenuation • Repressor (trpR) is activated by tryptophan – Changes shape so it can bind to the operator. – 70x reduction in synthesis • As with lac and arabinose, the repressor protein is produced by another gene (trpR) far away 4 Four Regions in the Leader Attenuation Sequence can pair • Attenuation depends on an interaction between transcription and translation in a “leader sequence” at the beginning of the operon. – 10x reduction The leader has several trp codons P O trpR trpE trpD trpC trpB trpA Leader (trpL) Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Trp common; forms termination loop Trp low; transcription occurs Termination loop Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Attenuation is common in other operons that synthesize amino acids. Here are some other leader sequences: Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. 5.