Outline Nov. 8 Types of Regulation • Review the (lac) – Predicting of partial diploids • Gene regulation can occur at various steps Examples of other : – The amount of product depends on • (ara) operon What are the similarities and • rate of mRNA synthesis (), • arginine (arg) operon differences in the • mRNA degradation, – A repressible operon control of these • synthesis () etc. operons? • (trp) operon • commonly control – Two kinds of regulation • Repression transcription • Attenuation

Terminology Types of Gene Regulation

• Constitutive are always expressed – Tend to be vital for basic functions (often called • and Activators are 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 : 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 • In Prokaryotes, functionally related genes are regulated as a unit, called an One operon. One operator Controls 3 • Operons consist of: – Several structural genes – ONE promoter and one – A control site (operator) – A separate (codes for protein that binds to operator)

1 Overview of the lac Operon must be absent

• Gene is normally off – There is no transcription because a repressor binds to the • “” 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 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 • What is the : 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? 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 be cis-dominant, trans-dominant, or 1: no sugars recessive? 2: lactose 3: glucose 4: glucose+lactose -lactose + lactose Interpretation –B-gal


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”)

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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 of , vol. 9, Chapter 7


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.