LECTURE CONNECTIONS

16 | Control of Gene Expression

© 2009 W. H. Freeman and Companyin Prokaryotes

Genes and Regulatory Elements

• Structural genes: encode proteins that are used in metabolism or biosynthesis or that play an structural role in the cell.

• Regulatory genes: encoding products that interact with other sequences and affect the transcription and translation of these sequences

• Regulatory elements: DNA sequences that are not transcribed but play a role in regulating other nucleotide sequences

Genes and Regulatory Elements

• Constitutive expression: continuously expressed under normal cellular conditions (encode essential cellular functions)

• Positive control: stimulate gene expression

• Negative control: inhibit gene expression

Level of Gene Regulation

Genes can be regulated at a number of points along the pathway:

- Alteration of gene structure (DNA methylation/ changes in chromatin)

- Transcription

- mRNA processing

- RNA stability (rate mRNA is degraded)

- Translation

- Posttranslational modification

DNA-Binding Proteins

• Much of gene regulation is accomplished by proteins that bind to DNA sequences and affect their expression.

• These regulatory proteins generally have discrete functional parts, called domains.

• Domains: 60 ~ 90 amino acids, responsible for binding to DNA, forming hydrogen bonds with DNA

DNA-Binding Proteins

• Distinctive types of DNA-binding proteins based on the motif

• Motif: within the binding domain, a simple structure that fits into the major groove of the DNA

Concept Check 1

How do amino acids in DNA-binding proteins interact with DNA?

a. By forming covalent bonds with DNA base

b. By forming hydrogen bonds with DNA base

c. By forming covalent bonds with sugars

Concept Check 2

What is the difference between a structural gene and a regulator gene?

a. Structural genes are transcribed into mRNA, but regulator genes are not.

b. Structural genes have complex structures; regulator genes have simple structure.

c. Structural genes encode proteins that function in the structure of the cell; regulator genes carry out metabolic reactions.

d. Structural genes encode proteins; regulator genes control the transcription of structural genes.

16.3 Operons Control Transcription in Bacterial Cells

• A group of bacterial structural genes that are transcribed together (along with the promoter and additional sequences that control transcription) is called an operon.

• Operon: promoter + additional sequences that control transcription (operator) + structure genes

• Regulator gene: DNA sequence encoding products that affect the operon function, but are not part of the operon

Negative and Positive Control; Inducible and Repressible Operons

• Inducible operons: Transcription is usually off and needs to be turned on.

• Repressible operons: Transcription is normally on and needs to be turned off.

Negative and Positive Control; Inducible and Repressible Operons

• Negative inducible operons: The control at the operator site is negative.

The binding of the regulator protein to the operator inhibit transcription.

Such operons are usually off and need to be turned on -by an inducer- so the transcription is called inducible.

• Inducer: small molecule that binds to the repressor and turns on the transcription

Negative and Positive Control; Inducible and Repressible Operons

• Negative repressible operons: Some operons with negative control are repressible, meaning that transcription normally takes place and must be turned off.

• Corepressor: a small molecule that binds to the repressor and makes it capable of binding to the operator to turn off transcription.

Negative and Positive Control; Inducible and Repressible Operons

• With positive control, a regulatory protein is an activator: it binds to DNA and stimulates transcription.

• Positive inducible operon: transcription is normally turned off because the regulator protein (an activator) is produced in an active form.

• Positive repressible operon: transcription normally takes place and has to be repressed. Transcription is inhibited when a substance besomes attached to the activator and renders it unable to bind to DNA.

Concept Check 3

In a negative repressible operon, the regulator protein is synthesized as .

a. an active activator

b. an inactive activator

c. an active repressor

d. an inactive repressor

The lac Operon of E. coli

• A negative inducible operon

• Inducer: allolactose

• lacI: repressor encoding gene

• lacP: operon promoter

• lacO: operon operator

The lac Operon of E. coli

• Structural genes

• lacZ: encoding β-galactosidases• lacY: encoding permease• lacA: encoding transacetylase

• The repression of the lac operon never completely shuts down transcription.

The repression never completely shuts down.

Concept Check 4

In the presence of allolactose, the lac repressor _________ .

a. binds to the operator

b. binds to the promoter

c. cannot bind to the operator

d. binds to the regulator gene

lac Mutations

• Partial diploid: full bacterial chromosome + an extra piece of DNA on F plasmid

• Structural gene mutations: affect the structure of the enzymes, but not the regulations of their synthesis

Partial diploids with lacZ+ and lacY− on the bacterial chromosome and lacZ− and lacY+ in the plasmid functioned normally producing β-galactosidase and permease.

lac Mutations

• Regulator gene mutations: lacI− leads to constitutive transcription of three structure genes.

• lacI+ is dominant over lacI− and is trans acting. A single copy of lacI+ brings about normal regulation of lac operon.

• lacI+lacZ−/lacI−lacZ+ produce fully functional β-galactosidase.

lac Mutations

• Operator mutations: lacOc (C = constitutive)

• lacOc is dominant over lacO+, which is cis acting.

Cis-acting: able to control the expression of genes only when on the same piece of DNA only.

Trans-acting: able to control the espression of genes on other DNA molecules.

• lacI+ lacO+ lacZ- and lacI+ lacOc lacZ+ produce fully functional β-galactosidase constitutively.

(If the operator is mutated, the repressors can not bind to it!)

lac Mutations

• Promoter mutations

• lacP−: cis acting

• These mutations interfere with the binding of RNA polymerase to the promoter.

• lacP- mutations don’t produce lac enzymes either in the presence or in the absence of lactose.

Positive control and catabolite repression

• Catabolite repression: using glucose when available, and repressing the metabolite of other sugars.

• This is a positive control mechanism: The positive effect is activated by catabolite activator protein (CAP).

cAMP is binded to CAP, together CAP–cAMP complex binds to a site slightly upstream from the lac gene promoter.

Positive control and catabolite repression

• cAMP – adenosine-3′,5′-cyclic monophosphate

• The concentration of cAMP is inversely proportional to the level of available glucose.

Concept Check 4

What is the effect of high levels of glucose on the lac operon?

a. Transcription is stimulated.

b. Little transcription takes place.

c. Transcription is not affected.

d. Transcription may be stimulated or inhibited, depending on the levels of lactose.

The trp Operon of E. coli

• A negative repressible operon

• Five structural genes

• trpE, trpD, trpC, trpB, and trpA – five enzymes together convert chorismate to tryptophane.