Announcements

1. Reading Ch. 15: skim btm 425-4262. Look over problems Ch. 15: 5, 6, 7.

Review of Last Lecture

I. RNA processing in eukaryotes

II. Translation of mRNA into protein - tRNA and ribosomes

III. Three steps of translation

IV. First evidence that proteins are important to heredity

Outline of Lecture 26Regulating gene expression in prokaryotes

I. Lactose metabolism - the lac operon, an inducible system

II. Trp operon, a repressible system

In E. coli, some proteins are found at 5-10 copies/cell; others are found at 100,00 copies.

How does a cell regulate the levels or amounts of different proteins at different times - in response to the environment?

•Operon: a series of gene coding regions (usually products with related functions) under the control of a single gene regulatory unit.

•Allows rapid response to changing environmental conditions.

Background on gene organization

lac operon

3 structural genes

Bacterial Strategy

• If glucose is present,

– then use glucose as a carbon source.

• If glucose is not present, and if lactose is present,

– then use lactose (indirectly) as carbon source.

Levels of enzymes needed to use lactose as carbon source increase dramatically when lactose is present; enzymes are inducible and lactose is the inducer.

The -galactosidase Enzyme

lacZ gene->

lacY gene ->Permease(entry of lactoseinto cell)

lacA ->transacetylase

lac operon, a polycistronic mRNA

Could the cell make varying amounts of lacZ and lacA RNA?

Genes involved in Lactose Metabolism

Repressor Enzyme lactose ?- removes Regulator transporter toxicProtein byproducts

Normally binds to Operator when lactose is absent,but when lactose is present, comes off.

The Operon Model - components

R

L

pol

R

R

pol

pol

Lac operon when no lactose is present

R

R

pol

pol

R R

pol

L

L

R

R

pol pol

pol pol

Learning Check

pol

R

L

Will transcription and translation of Z, Y,and A enzymes occur?

What would happen if a wild-type copy of I was added?

glucose lactose b-galactosidase

+ - -

+ + -

- - -

- + +

What happens when cell has both lactose and glucose?

How does cell prevent breaking lactose down into glucose and galactose? Catabolite Activating Protein (CAP)

Catabolite Repression of lac Operon -/+ Glucose

Therefore, for maximal transcription, repressor must be bound by repressor and CAP must bind CAP-binding site

Goal: efficiency, don’t waste energy converting lactose, when glucose available

Glucose Inhibits Formation of cAMP from ATP

glucose

lac operon regulatory binding sites

CAP site promoter operator lac coding

Glu cAMP lactose lac rep

+ - -

+ - +

CAP-cAMP lac rep

- + -

CAP-cAMP mRNA

- + +

RNA polymerase

3-D structure of Repressor bound to

Operator and CAP bound to

Promoter

Lewis et al. (1996) Science 271:1247

glucose absentlactose absent

CAP

Repressor

Polymerasebindshere

DNA

II. Tryptophan Operon

It makes sense for E. coli to synthesize the enzymes to make the amino acid tryptophan ONLY when:

1. The tryptophan concentration is low

AND

2. The tryptophan-charged tRNA is low

Tryptophan Operon - Repressor Bindswhen tryptophan is present

mRNA leader sequence involved inAttenuation - the cast of characters

HighTryptophan-tRNA-->>TerminatesTranscription

Low Tryptophan-tRNA-->>TranscriptionContinues

Translation Event controlsTranscription Event !?!?