chapter 13 regulation of gene activity. humans and nemotodes have about the same number of genes...

Chapter 13

Regulation of

Gene Activity

• Humans and nemotodes have about the same number of genes roughly 20,500

• So how can a complex organism produce the proteins they require?

• By regulation of pre-mRNA splicing to produce many proteins from a single gene

• In 1961, Jocob and Monod showed that the bacteria Escherichia coli could regulate the expression of genes

• They received the Nobel prize for the “operon model” to express gene regulation in prokaryotes

• Operon includes:

• Promoter which is a short sequence of DNA where RNA polymerase first attaches to begin transcription

• Operator which is a short portion of DNA where an active repressor binds

• When active repressor binds to operator, RNA polymerase cannot attach to promoter and no transcription

• Structural genes are one to several genes coding for primary structure of enzymes in metabolic pathway transcribed as a unit

• Regulator gene, usually located outside operon and controlled by own promoter, codes for a repressor

that controls whether the operator is active or not

-Some operons in E. coli are usually in “on” rather than “off” condition

-Trp operon, the regulator codes a repressor that ordinarily is unable to attach to operator

-RNA polymerase is able to bind to promoter and structural genes are expressed

-Then five enzymes promote anabolic pathway for synthesis of amino acid tryptophan

-If tryptophan is present, it binds to the repressor, changes its shape and binds to operon

Structural genes are not expressed

• Entire unit is called a repressor operon

• Tryptophan is the corepressor

• Repressible operons are usually involved in anobolic pathways

Fig. 13.1

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regulator gene promoter operator structural genes

DNARNA polymerase

RNA polymerase cannot bind to promoter.

mRNA

enzymesinactive repressor

a. Tryptophan absent. Enzymes needed to synthesize tryptophan are produced.

DNA

inactive repressor

b. Tryptophan present. Presence of tryptophan prevents production of enzymes used to synthesize tryptophan.

tryptophan

active repressor

5 3

• Bacteria metabolism is efficient

• If a protein or enzyme is not needed, genes to make them are inactive

• If lactose is not present, enzymes for lactose catabolism are not active

• If E coli are denied glucose and given lactose, it immediately makes the three enzymes needed to metabolize lactose

• The three structural genes needed are adjacent to one another and under control of a single promoter and operon

• Lac operator repression usually binds to operator and prevents transcription

• Lactose binds to repressor, changes its shape that prevents its binding to promoter

• RNA polymerase binds to promoter and carries out transcription of enzymes for lactose metabolism

• Presence of lactose brings about expression of genes and is called inducer

• Entire unit is called inducible operon

• Inducible operons are usually necessary for catabolic pathways

Fig. 13.2Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

regulator gene promoter operator structural genes

DNA

RNA polymerase cannot bind to promoter.

RNA polymerase can bind to promoter.

active repressor

active repressor

mRNA

enzymes

active repressor

inactive repressor

b. Lactose present. Enzymes needed to take up and use lactose are produced only when lactose is present.

a. Lactose absent. Enzymes needed to take up and use lactose are not produced.

lactose

DNA

5 3

• E coli prefers using glucose

• A molecule called cyclic AMP (cAMP) accumulates when glucose is absent

• cAMP binds to a molecule called catabolite activator protein (CAP) and the complex attaches to site next to lac promoter

• This bends DNA exposing the promoter to RNA polymerase

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adenine

OH

CH2

cyclic AMP(cAMP)

O

P

5

3

Fig. 13.3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

DNA

inactive CAP

inactive CAP

active CAP

a. Lactose present, glucose absent (cAMP level high)

b. Lactose present, glucose present (cAMP level low)

DNA

cAMP

promoterCAP binding site

RNA polymerase bindsfully with promoter.

RNA polymerase doesnot bind fully with promoter.

promoter operator

operator

CAP binding site

• Each cell in multicellular eukaryote, has a copy of all genes

• Different genes are actively expressed in different cells

• Types of control in eukaryotic cells:

• 1) Chromatin structure- Chromatin packing is used to keep genes turned off by preventing access to RNA polymerase

• In nucleus, loosely condensed chromatin is available for transcription

• Part of epigenetic inheritance, the transcription of genetic information outside coding sequence of a gene

• 2) Transcriptional control is the degree to which a gene is transcribed into mRNA determines amount of gene product

• Transcription factors may promote or repress transcription

• 3) Posttranscriptional control involves mRNA processing and how fast mRNA leaves the nucleus

• Can determine type of protein product made and amount of gene product made in a given time

• 4) Translational control occurs in cytoplasm and affects when translation begins and how long it continues

• Any influence on the persistence of 5’ cap and 3’ poly-A tail affect length of translation

• Excised introns are involved in regulatory system and affect life span of mRNA

• 5) Posttranslational control occurs in cytoplasm after protein synthesis

• Only functional protein is an active gene product

Fig. 13.4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

functional protein

plasmamembrane

polypeptide chain

Posttranslationalcontrol

Posttranscriptional control

Transcriptional control

Translationalcontrol

nuclear pore

mRNA

pre-mRNA

intron exon

histones

nuclear envelope

Chromatinstructure

3

3

5

5

• Highly condensed heterochromatin is inaccessible to RNA polymerase

• It appears as darkly stained portions within nucleus in electron micrographs

• Example of heterochromatin is the Barr body in mammalian females

• It is an inactive X chromosome that does not produce gene products

• In females one X chromosome transcribes genes and the other becomes a Barr body

• Which X is inactive depends on which X chromosome that cell received

• One X comes from father and the other from the mother

• Conditions in human females include: ocular albinism, Duchanne muscular distrophy, X-linked hereditory absence of sweat glands

Fig. 13.6

Coats of tortoiseshellcats have patchesof orange and black.

One X chromosome is inactivated ineach cell. Which one is by chance.

Females have twoX chromosomes.

active X chromosome

inactive X

inactive X

active X chromosome

allele fororange color

allele forblack color

cell division Barr bodies

© Chanan Photo 2004

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

• Term euchromatin is used for the more loosely packed active chromatin

• In herterochromatin, the histone tails tend to bear methyl groups (-CH3)

• In euchromatins, the histone tails tend to be acetylated and have attached acetyl groups (-COCH3)

• When euchromatin is transcribed, chromatin remodeling complex pushes aside the histone portion of nucleosome so access to DNA is not barred

• Also affects gene expression by adding acetyl or methyl groups to histone tails

• Epigenetic inheritance concerns the pattern of inheritance that does not depend on only the genes

• If a histone is methylated, the DNA may also be methylated

• Genomic imprinting occurs when either the mother’s or father’s allele is methylated during gamete formation

• If inherited, the gene is not expressed

• Transcriptional control is the most critical of all controls

• No operons like those in prokaryotic cells have been found in eukaryotes

• Every cell contains transcriptional factors, proteins that help regulate transcription

• In eukaryotes, transcription activators are DNA binding proteins that speed transcription

• They bind to a region of DNA called enhancer that can be far away from promoter

• A hairpin loop in the DNA brings the transcription activators attached to enhancers into contact with transcriptional factor complex

• Transcription factors, activators,and repressors are always present in nucleus, but have to be activated before they bind to DNA

Fig. 13.7 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

promoter

DNA

enhancer

transcriptionactivator

mediator proteins

mRNA transcription

RNA polymerase

transcriptionfactor complex

gene

• During pre-mRNA splicing, introns (noncoding regions) are excised, and exons (expressed regions) are joined together to form mRNA

• Sometimes an exon is skipped or an intron is included

• Results in mature mRNA that has an altered sequence, and protein encoded differs

Fig. 13.8

intronintron

intron

cap

protein product 1

mRNA

RNA splicing

poly-Atail

exon intron

protein product 2

RNA splicing

exon

a. b.

cap

A B C D E

A B C D E

A B C

C

D E

A D EB

355

pre-mRNA

mRNA

pre-mRNA poly-Atail

3

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

• Translation control begins when processed mRNA reaches cytoplasm and before there is a protein product

• Includes presence or absence of 5’ cap and length of poly-A tail at 3’ end

• Micro RNAs (miRNAs) can regulate translation by causing the destruction of mRNAs before they can be translated

• Much like a dimmer switch on a light, miRNAs can fine-tune the expression of genes

Fig. 13.9Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

pre-mRNA

MicroRNA is cut froma pre-mRNA and binds withproteins to form RISC.

Complementary base pairingbetween RNAs allows RISCto bind to mRNA.

Translationis inhibited.

The mRNAis degraded.

mRNA

RISC(RNA-inducedsilencing complex)

microRNA(miRNA)

proteins

or

RISC

5

3

3

5

5

3

• A gene mutation is a permanent change in the sequence of bases in DNA

• Can range from no effect to complete inactivation

• Germ-line mutations occur in sex cells and can be passed to subsequent generations

• Somatic mutations occur in body cells and affect only a small number of cells in a tissue

• Somatic mutations are not passed on to future generations, but can lead to cancer

• Spontaneous mutations are associated with any number of normal processes

• The movement of transposons from one chromosomal

• location to another can disrupt a gene and lead to an abnormal product

• A base in DNA may undergo a chemical change that leads to a miss pairing during replication

• These mutations are rare because DNA polymerase proofreads the new strand against the old strand, detects most mismatched nucleotides, and usually replaces them with correct nucleotides

• Induced mutations are caused by mutagens, environmental factors that can alter base composition of DNA

• Includes radiation and organic chemicals

• Many mutations are also carcinogens (cancer-causing)

• Chemical mutagens are present in some food we eat and many industrial chemicals

• Tobacco smoke contains a number of carcinogenic organic chemicals

• One-third of all cancer deaths can be attributed to smoking

• Lung cancer is most frequent lethal cancer in the United States

• Ames test is used for mutagenicity of a chemical to be carcinogenic

• A histidine-requiring strain of bacteria is exposed to

• the chemical

• If the chemical is mutagenic, bacteria can grow without histidine

Fig. 13.10Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

bacterialstrain(requireshistidine)

Control

Mutation did not occurMutation occurred

Suspectedchemicalmutagen

bacterialstrain(requireshistidine) Plate onto petri plates

that lack histidine.

Incubate overnightbacterialgrowth

• Point mutations involve a change in a single DNA nucleotide with a possible change in a specific amino acid

• Frameshift mutations occur most often because one or more nucleotides are either inserted or deleted from DNA

• May form a completely new sequence of codons and nonfunctioning protein

• A single nonfunctioning protein can have a dramatic effect on the phenotype, because enzymes are often part of metabolic pathways

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(phenylalanine) (tyrosine) (melanin)A EA CB EB

Fig. 13.12

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b. Normal red blood cell

a.

c. Sickled red blood cell

No mutation

Val His Leu Thr Pro Glu Glu

(normal protein)

His His

(abnormal protein)

Glu Val

(incomplete protein)

Glu Stop

CTCCTCTGGAGTC A C G T G G A G

CTCCTCTGGAGTC A C G T G A G

Val His Leu Thr Pro Glu Glu

CTCCACTGGAGTC A C G T G G A G

Val His Leu Thr Pro Glu

CTCCATGGAGTC A C G T G G A G T

Val His Leu Thr Pro Stop

A

b, c: © Stan Flegler/Visuals Unlimited.

Val

3 5

• The development of cancer involves a series of accumulating mutations that can be different for each type of cancer

• The cell cycle occurs inappropriately when proto-oncogenes become oncogenes and tumor suppressor genes are no longer effective