Chapter 13 ppt

(1)

Biology

Sylvia S. Mader

Michael Windelspecht

Chapter 13 Regulation of

Gene Expression

Lecture Outline

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(2)

Outline

(3)

13.1 Prokaryotic Regulation

• Bacteria do not require the same enzymes

all the time

• Enzymes are produced as needed

• François Jacob and Jacques Monod

(1961) proposed the

operon

model to

explain regulation of gene expression in

prokaryotes



An operon is a group of structural and

regulatory genes that function as a single unit

(4)

Prokaryotic Regulation

• An operon consists of three components



Promoter

• DNA sequence where RNA polymerase first attaches

• Short segment of DNA



Operator

• DNA sequence where active repressor binds

• Short segment of DNA



Structural Genes

• One to several genes coding for enzymes of a metabolic

pathway

• Transcribed simultaneously as a block

• Long segment of DNA



A

regulatory gene

that codes for a repressor protein

• The regulatory gene is normally located outside the operon

• The repressor protein controls whether the operon is active or

(5)

Prokaryotic Regulation

• The

trp

Operon



The regulator codes for a repressor



If tryptophan (an amino acid) is absent:

• Repressor is unable to attach to the operator (expression is

normally “on”)

• RNA polymerase binds to the promoter

• Enzymes for synthesis of tryptophan are produced



If tryptophan is present:

• It combines with the repressor protein as its corepressor

• Repressor becomes functional when bound to tryptophan

• Repressor blocks synthesis of enzymes in the pathway for

(6)

The

trp

Operon

When the repressor

binds to the operator,

transcription is prevented.

active

repressor

structural genes

regulator gene

(7)

The

trp

Operon

7

3′ 5′

regulator gene promoter operator structural genes

DNA

mRNA inactive

repressor enzymes mRNA RNA polymerase

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

RNA polymerase cannot bind to promoter.

active repressor tryptophan

DNA

mRNA inactive repressor

(8)

Prokaryotic Regulation

• The

lac

Operon



The regulator codes for a repressor



If lactose (a sugar that can be used for food)

is absent:

• The repressor attaches to the operator

• Expression is normally “off”



If lactose is present:

• It combines with the repressor and renders it unable to bind

to operator

(9)

The

lac

Operon

9 regulatory gene

promoter operator

lactose metabolizing genes

DNA

mRNA

active repressor

a.

RNA polymerase bound to promoter.

enzymes

mRNA

lactose

active

repressor

inactive

repressor

mRNA

DNA

(10)

Prokaryotic Regulation

• Further control of the

lac

operon



E. coli

preferentially breaks down glucose



The

lac

operon is maximally activated only in the

absence of glucose



When glucose is absent

• Cyclic AMP (cAMP) accumulates

• cAMP binds to catabolite activator protein (CAP)

• CAP, when bound to cAMP, binds to a site near the

lac

promoter

• When CAP is bound, RNA polymerase binds better to the

promoter

(11)

Prokaryotic Regulation

• Further control of the

lac

operon



When glucose is present

• There is little cAMP in the cell

• CAP is inactive

(12)

Action of CAP

DNA

inactive CAP

(13)

Action of CAP

DNA

inactive CAP

promoter operator

cAMP

(14)

Action of CAP

DNA

inactive CAP

promoter operator

cAMP

(15)

Action of CAP

15 DNA

inactive CAP

active CAP

promoter operator

cAMP

(16)

Action of CAP

DNA

inactive CAP

active CAP

promoter operator

cAMP

(17)

DNA

inactive CAP

active CAP

RNA polymerase binds

fully with promoter.

promoter operator

CAP binding site

cAMP

Action of CAP

17

(18)

Action of CAP

DNA

inactive CAP

active CAP

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

DNA cAMP

promoter CAP binding site

RNA polymerase binds fully with promoter.

RNA polymerase does promoter operator

(19)

13.2 Eukaryotic Regulation

• A variety of mechanisms

• Five primary levels of control:



Nuclear levels

• Chromatin Structure

• Transcriptional Control

• Posttranscriptional Control



Cytoplasmic levels

• Translational Control

• Posttranslational Control

(20)

Levels at Which Control of Gene

Expression Occurs in Eukaryotic Cells

polypeptide chain Posttranslational control Posttranscriptional control Transcriptional control Translational control nuclear pore mRNA pre- mRNA

intron exon histones nuclear envelope Chromatin structure 3 3 5 5

(21)

Eukaryotic Regulation

• Chromatin Structure



Eukaryotic DNA is associated with histone proteins

• Together make up

chromatin



Nucleosomes

• DNA is wound around groups of eight molecules of histone

proteins

• Looks like beads on a string

• Each bead is called a nucleosome



The levels of chromatin packing are determined by

degree of nucleosome coiling

(22)

Chromatin Structure Regulates

Gene Expression

euchromatin heterochromatin

nucleolus

DNA

histone protein a. Darkly stained heterochromatin and lightly stained euchromatin

H2A H2B

H3

(23)

Eukaryotic Regulation

• Euchromatin



Loosely coiled DNA



Transcriptionally active

• Heterochromatin



Tightly packed DNA



Transcriptionally inactive

• Barr Body



Females have two X chromosomes, but only one is

active



The other X chromosome is tightly packed along its

entire length and is inactive



The inactive X chromosome is called a Barr body

(24)

X-Inactivation in Mammalian

Females

Coats of tortoiseshell

cats have patches

of orange and black.

One X chromosome is inactivated in

each cell. Which one is by chance.

Females have two

X chromosomes.

active X chromosome

inactive X

active X chromosome

allele for

orange color

allele for

black color

cell division

Barr bodies

(25)

Eukaryotic Regulation

• Transcriptional Control



Transcription is controlled by proteins called

transcription factors

• Bind to

enhancer

DNA

– Regions of DNA where factors that regulate transcription

can also bind

• Transcription factors are always present in the cell,

but most likely have to be activated before they will

bind to DNA

(26)

Eukaryotic Transcription Factors

promoter DNA

enhancer

transcription activator

mediator proteins

RNA polymerase

transcription factor complex

gene

(27)

Eukaryotic Regulation

• Posttranscriptional control

operates on the

primary mRNA transcript

• Given a specific primary transcript:



Excision of introns can vary



Splicing of exons can vary



Determines the type of mature transcript that leaves

the nucleus

• May also control the speed of mRNA transport

from nucleus to cytoplasm



Will affect the number of transcripts arriving at rough

ER and, therefore, the amount of gene product

realized per unit time

(28)

Alternative Processing of pre-mRNA

intron

cap

mRNA

RNA

splicing

poly-A

tail

exon

intron

RNA

splicing

exon

cap

A

B

C

D E

A B C D E

A

B

C

D E

A

B

D E

3

5

5 pre-mRNA

mRNA

pre-mRNA poly-A

tail

3

(29)

Eukaryotic Regulation

• Translational Control

- Determines the degree

to which mRNA is translated into a protein

product

• Features of the mRNA affect whether translation

occurs and how long the mRNA remains active



Presence of 5′ cap



Length of poly-A tail on 3′ end

• MicroRNAs

(miRNAs) regulate translation by

causing the destruction of mRNAs before they

can be translated

(30)

Eukaryotic Regulation

• Posttranslational Control

- Affects the activity of

a protein product

• Posttranslational control is accomplished by

regulating



Activation

(31)

13.3 Gene Mutations

• A

gene mutation

is a permanent change

in the sequence of bases in DNA.

• The effects of a gene mutation can range

from



No effect on protein activity to



Complete inactivation of the protein

• Germ-line mutations occur in sex cells

• Somatic mutations occur in body cells

(32)

Gene Mutations

• Spontaneous mutations



Chemical changes in DNA that lead to mispairing during

replication



Movement of transposons from one chromosomal location to

another



Replication Errors

• DNA polymerase

– Proofreads new strands

– Generally corrects errors

• Overall mutation rate is 1 in 1,000,000,000 nucleotide pairs

replicated

• Induced mutations



Caused by

mutagens

such as radiation and organic chemicals



Many mutagens are also

carcinogens

(cancer-causing)

(33)

The Ames Test For Mutagenicity

bacterial

strain

(requires

histidine)

Control

Suspected

chemical

mutagen

bacterial

strain

(requires

histidine)

Plate onto petri plates

that lack histidine.

Incubate overnight

bacterial

(34)

Gene Mutations

• Point Mutations



Involve a change in a single DNA nucleotide



Change one codon to a different codon



Effects on the protein vary:

• Nonfunctional

• Reduced functionality

• Unaffected

• Frameshift Mutations



One or two nucleotides are either inserted or deleted

from DNA



The protein is always rendered nonfunctional

• Normal :

THE CAT ATE THE RAT

• After deletion:

THE ATA TET HER AT

(35)

Point Mutations in Hemoglobin

35 No mutation

His Leu Thr Pro Glu Glu

C A C G T G G G T G A G G T C T C C T C

Val

A

3

5 b. Normal red blood cell

c. Sickled red blood cell

(36)

Point Mutations in Hemoglobin

b. Normal red blood cell

c. Sickled red blood cell

No mutation

His Leu Thr Pro Glu Glu

(normal protein)

His

C A C G T G G G T G A G G T C T C C T C

Val

His Leu Thr Pro Glu Glu Val

C A C G T

A

G A G T G A G G T C T C C T C

A

3

5

(37)

Point Mutations in Hemoglobin

37 b. Normal red blood cell

c. Sickled red blood cell

Leu Thr Pro Val Glu

(abnormal protein)

Glu

Val

No mutation

His Leu Thr Pro Glu Glu

(normal protein)

His

C A C G T G G G T G A G G T C T C C T C

Val

His Leu Thr Pro Glu Glu Val

C A C G T

A

G A G T G A G G T C T C C T C

A

3

5

Val His

C A C G T G G A G T G A G G T C

A

C C T C

(38)

Point Mutations in Hemoglobin

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

C T C C T C T G G A G T C A C G T G G A G

C T C C T C T G G A G T C A C G T G A G

Val His Leu Thr Pro Glu Glu

C T C C A C T G G A G T C A C G T G G A G

Val His Leu Thr Pro Glu

C T C C A T G G A G T

C A C G T G G A G T Val His Leu Thr Pro Stop

A

Val

(39)

Gene Mutations

• Development of cancer involves a series of

accumulating mutations

• Proto-oncogenes – Stimulate cell division



Mutated proto-oncogenes become oncogenes that

are always active

• Tumor suppressor genes – inhibit cell division

• Mutations in oncogene and tumor suppressor

genes:

• Stimulate the cell cycle uncontrollably

• Lead to tumor formation

(40)

Cell Signaling Pathway That Stimulates a

Mutated Tumor Suppressor Gene

receptor

_plasma

membrane

(41)

Cell Signaling Pathway That Stimulates a

Mutated Tumor Suppressor Gene

41 receptor

inhibiting growth factor

plasma

membrane

nucleus

mutated tumor suppressor gene

(42)

Cell Signaling Pathway That Stimulates a

Mutated Tumor Suppressor Gene

receptor

inhibiting growth factor

plasma

membrane

signal

transducers

nucleus

(43)

Cell Signaling Pathway That Stimulates a

Mutated Tumor Suppressor Gene

43 receptor

inhibiting growth factor

plasma

membrane

signal

transducers

transcription factor

nucleus

(44)

Cell Signaling Pathway That Stimulates a

Mutated Tumor Suppressor Gene

protein that is

unable to inhibit

the cell cycle

or promote

apoptosis

receptor

inhibiting growth factor

plasma

membrane

signal

transducers

transcription factor

(45)

Cell Signaling Pathway That Stimulates

an Oncogene

receptor

cytoplasm

plasma

membrane

nucleus

(46)

Cell Signaling Pathway That Stimulates

an Oncogene

receptor

stimulating growth factor

cytoplasm

plasma

membrane

nucleus

(47)

Cell Signaling Pathway That Stimulates

an Oncogene

receptor

stimulating growth factor

cytoplasm

plasma

membrane

signal

transducers

nucleus

oncogene

(48)

Cell Signaling Pathway That Stimulates

an Oncogene

receptor

stimulating growth factor

cytoplasm

plasma

membrane

signal

transducers

transcription factor

nucleus

(49)

Cell Signaling Pathway That Stimulates

an Oncogene

receptor

stimulating growth factor

cytoplasm

plasma

membrane

signal

transducers

transcription factor

nucleus

protein that

overstimulates

the cell cycle

oncogene