3/18/2014

1

Mechanisms of Eukaryotic Gene Regulation

2 Terms before we get started: Types of Chromatin

• Heterochromatin:

– Highly condensed

– Not expressed = Off!

• Euchromatin:

– Less condensed

– Available for transcription = On!

3/18/2014

2

Fig. 16-21a

DNA double helix (2 nm in diameter)

Nucleosome (10 nm in diameter)

Histones Histone tail

H1

DNA, the double helix Histones Nucleosomes, or “beads on a string” (10-nm fiber)

Fig. 16-21b

30-nm fiber

Chromatid (700 nm)

Loops Scaffold

300-nm fiber

Replicated chromosome (1,400 nm)

30-nm fiber Looped domains (300-nm fiber)

Metaphase chromosome

Animation embedded

3/18/2014

3

Fig. 18-6

DNA

Signal

Gene

NUCLEUS

Chromatin modification

Chromatin

Gene available

for transcription

Exon

Intron

Tail

RNA

Cap

RNA processing

Primary transcript

mRNA in nucleus

Transport to cytoplasm

mRNA in cytoplasm

Translation

CYTOPLASM

Degradation

of mRNA

Protein processing

Polypeptide

Active protein

Cellular function

Transport to cellular

destination

Degradation

of protein

Transcription

Prokaryotic gene regulation

is at the level of transcription,

for the most part =

The Operons!

Eukaryotic gene regulation

occurs at multiple levels –

see this figure. ALL the

boxes represent possible

control points.

Fig. 18-7

Histone tails

DNA

double helix

(a) Histone tails protrude outward from a nucleosome

Acetylated histones

Amino acids available for chemical modification

(b) Acetylation of histone tails promotes loose chromatin structure that permits transcription

Unacetylated histones

Histone Acetylation

(Chromatin modification Mechanism)

Acetyl groups = turn on!

De-acetylate = turn off!

3/18/2014

4

DNA Methylation

• DNA methylation, the addition of methyl groups to certain bases in DNA (usually cytosine), is associated with reduced transcription in some species

• DNA methylation can cause long-term inactivation of genes in cellular differentiation

(Chromatin modification mechanism)

Methyl groups = turn off!

De-methylate = turn on!

Epigenetic Inheritance

• Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells

• The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance

• (…watching a video on this later this week…)

3/18/2014

5

Fig. 18-6

DNA

Signal

Gene

NUCLEUS

Chromatin modification

Chromatin

Gene available

for transcription

Exon

Intron

Tail

RNA

Cap

RNA processing

Primary transcript

mRNA in nucleus

Transport to cytoplasm

mRNA in cytoplasm

Translation

CYTOPLASM

Degradation

of mRNA

Protein processing

Polypeptide

Active protein

Cellular function

Transport to cellular

destination

Degradation

of protein

Transcription

The Roles of Transcription Factors

• To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors

• General transcription factors are essential for the transcription of all protein-coding genes

• In eukaryotes, high levels of transcription of particular genes depend on control elements interacting with specific transcription factors

3/18/2014

6

• Proximal control elements are located close to the promoter

• Distal control elements, groups of which are called enhancers, may be far away from a gene or even located in an intron

Enhancers and Specific Transcription Factors

• An activator is a protein that binds to an enhancer and stimulates transcription of a gene

• Bound activators cause mediator proteins to interact with proteins at the promoter

Fig. 18-9-3

Enhancer TATA box

Promoter Activators

DNA Gene

Distal control element

Group of mediator proteins

DNA-bending protein

General transcription factors

RNA polymerase II

RNA polymerase II

Transcription initiation complex RNA synthesis

3/18/2014

7

Fig. 18-10

Control elements

Enhancer

Available activators

Albumin gene

(b) Lens cell

Crystallin gene expressed

Available activators

LENS CELL NUCLEUS

LIVER CELL NUCLEUS

Crystallin gene

Promoter

(a) Liver cell

Crystallin gene not expressed

Albumin gene expressed

Albumin gene not expressed

Fig. 18-6

DNA

Signal

Gene

NUCLEUS

Chromatin modification

Chromatin

Gene available

for transcription

Exon

Intron

Tail

RNA

Cap

RNA processing

Primary transcript

mRNA in nucleus

Transport to cytoplasm

mRNA in cytoplasm

Translation

CYTOPLASM

Degradation

of mRNA

Protein processing

Polypeptide

Active protein

Cellular function

Transport to cellular

destination

Degradation

of protein

Transcription

3/18/2014

8

RNA Processing

• In alternative RNA splicing, different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

Fig. 18-11

or

RNA splicing

mRNA

Primary RNA transcript

Gene – introns and exons

Exons

DNA

3/18/2014

9

Fig. 18-6

DNA

Signal

Gene

NUCLEUS

Chromatin modification

Chromatin

Gene available

for transcription

Exon

Intron

Tail

RNA

Cap

RNA processing

Primary transcript

mRNA in nucleus

Transport to cytoplasm

mRNA in cytoplasm

Translation

CYTOPLASM

Degradation

of mRNA

Protein processing

Polypeptide

Active protein

Cellular function

Transport to cellular

destination

Degradation

of protein

Transcription

mRNA Degradation

• The life span of mRNA molecules in the cytoplasm is a key to determining protein synthesis

• Eukaryotic mRNA is more long lived than prokaryotic mRNA

• The mRNA life span is determined in part by sequences in the leader and trailer regions – UTR’s

3/18/2014

10

Fig. 18-6

DNA

Signal

Gene

NUCLEUS

Chromatin modification

Chromatin

Gene available

for transcription

Exon

Intron

Tail

RNA

Cap

RNA processing

Primary transcript

mRNA in nucleus

Transport to cytoplasm

mRNA in cytoplasm

Translation

CYTOPLASM

Degradation

of mRNA

Protein processing

Polypeptide

Active protein

Cellular function

Transport to cellular

destination

Degradation

of protein

Transcription

Protein Processing and Degradation

• After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control

• Proteasomes are giant protein complexes that bind protein molecules and degrade them

3/18/2014

11

Fig. 18-12

Proteasome and ubiquitin to be recycled Proteasome

Protein fragments (peptides) Protein entering a

proteasome

Ubiquitinated protein

Protein to be degraded

Ubiquitin

Fig. 18-6

DNA

Signal

Gene

NUCLEUS

Chromatin modification

Chromatin

Gene available

for transcription

Exon

Intron

Tail

RNA

Cap

RNA processing

Primary transcript

mRNA in nucleus

Transport to cytoplasm

mRNA in cytoplasm

Translation

CYTOPLASM

Degradation

of mRNA

Protein processing

Polypeptide

Active protein

Cellular function

Transport to cellular

destination

Degradation

of protein

Transcription

3/18/2014

12

Concept 18.3: Noncoding RNAs play multiple roles in controlling gene expression

• Only a small fraction of DNA codes for proteins, rRNA, and tRNA

• A significant amount of the genome is transcribed into noncoding RNAs

• Noncoding RNAs regulate gene expression at two points: degrade mRNA or block its translation

• MicroRNAs (miRNAs) are small single-stranded RNA molecules that can bind to mRNA

• The phenomenon of inhibition of gene expression by RNA molecules is called RNA interference (RNAi)

• RNAi is caused by small interfering RNAs (siRNAs)

• siRNAs and miRNAs are similar but form from different RNA precursors

• Prokaryotic and eukaryotic cells use small RNA’s to regulate gene expression

3/18/2014

13

Fig. 18-13

miRNA- protein complex (a) Primary miRNA transcript

Translation blocked

Hydrogen bond

(b) Generation and function of miRNAs

Hairpin miRNA

miRNA

Dicer

3

mRNA degraded

5

2 ways that miRNA’s

interfere with gene

expression:

Summary of Several Eukaryotic Gene Regulation Mechanisms

• Genes in highly compacted

chromatin are generally not

transcribed.

Chromatin modification

• DNA methylation generally reduces transcription.

• Histone acetylation seems to

loosen chromatin structure,

enhancing transcription.

Chromatin modification

Transcription

RNA processing

Translation mRNA degradation

Protein processing and degradation

mRNA degradation

• Each mRNA has a characteristic life span, determined in part by sequences in the 5’ and 3’ UTRs.

• Protein processing and degradation by proteasomes are subject to regulation.

Protein processing and degradation

• Initiation of translation can be controlled via regulation of initiation factors.

Translation

or mRNA

Primary RNA transcript

• Alternative RNA splicing:

RNA processing

• Coordinate regulation:

Enhancer for

liver-specific genes

Enhancer for

lens-specific genes

Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription.

Transcription

• Regulation of transcription initiation: DNA control elements bind specific transcription factors.

3/18/2014

14

Summary Roles of Small RNA’s in Gene Regulation of Eukaryotic Cells

Chromatin modification

RNA processing

Translation mRNA degradation

Protein processing

and degradation

mRNA degradation

• miRNA or siRNA can target specific mRNAs

for destruction.

• miRNA or siRNA can block the translation

of specific mRNAs.

Transcription

• Small RNAs can promote the formation of

heterochromatin in certain regions, blocking

transcription.

Chromatin modification

Translation

Concept 18.5: Cancer results from genetic changes that affect cell cycle

control

• The gene regulation systems that go wrong during cancer are the very same systems involved in embryonic development

3/18/2014

15

Oncogenes and Proto-Oncogenes

• Oncogenes are cancer-causing genes

– Analogy: Foot on the gas pedal!

• Proto-oncogenes are the corresponding normal cellular genes that are responsible for normal cell growth and division

• Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle

– Analogy: Putting the pedal to the metal!

Fig. 18-20

Normal growth- stimulating protein in excess

DNA

Proto-oncogene

Normal growth-stimulating protein in excess

Normal growth- stimulating protein in excess

Hyperactive or degradation- resistant protein

Point mutation:

Oncogene Oncogene

within a control element within the gene

3/18/2014

16

Tumor-Suppressor Genes • Tumor-suppressor genes help prevent uncontrolled

cell growth

– Analogy: Foot on the brake pedal!

• Mutations that decrease protein products of tumor-suppressor genes may contribute to cancer onset

– Analogy: Taking the foot of the brake pedal!

• Tumor-suppressor proteins

– Repair damaged DNA

– Control cell adhesion

– Inhibit the cell cycle in the cell-signaling pathway

• Suppression of the cell cycle can be important in the case of damage to a cell’s DNA

• The p53 gene prevents a cell from passing on mutations due to DNA damage

• Mutations in the p53 gene prevent suppression of the cell cycle

3/18/2014

17

Interference with Normal Cell-Signaling Pathways …add to lecture…

• Mutations in the ras proto-oncogene and p53 tumor-suppressor gene are common in human cancers

• Mutations in the ras gene can lead to production of a hyperactive Ras protein and increased cell division

Fig. 18-21a

Receptor

Growth factor

G protein GTP

Ras

GTP

Ras

Protein kinases (phosphorylation cascade)

Transcription factor (activator)

DNA

Hyperactive Ras protein (product of oncogene) issues signals on its own

MUTATION

NUCLEUS

Gene expression

Protein that stimulates the cell cycle

(a) Cell cycle–stimulating pathway

1 1

3

4

5

2

3/18/2014

18

Fig. 18-21b

MUTATION

Protein kinases

DNA

DNA damage in genome

Defective or

missing

transcription

factor, such

as p53, cannot

activate

transcription

Protein that

inhibits

the cell cycle

Active form of p53

UV light

(b) Cell cycle–inhibiting pathway

2

3

1

Fig. 18-21c

(c) Effects of mutations

EFFECTS OF MUTATIONS

Cell cycle not

inhibited

Protein absent

Increased cell

division

Protein

overexpressed

Cell cycle

overstimulated

3/18/2014

19

The Multistep Model of Cancer Development

• Multiple mutations are generally needed for full-fledged cancer; thus the incidence increases with age

• At the DNA level, a cancerous cell is usually characterized by at least one active oncogene and the mutation of several tumor-suppressor genes

Fig. 18-22

EFFECTS OF MUTATIONS

Malignant tumor

(carcinoma)

Colon

Colon wall

Loss of tumor-

suppressor gene Activation of

oncogene

Loss of

tumor-suppressor

gene

Loss of

tumor-suppressor

gene p53

Additional

mutations

Larger benign

growth (adenoma)

Small benign

growth (polyp)

Normal colon

epithelial cells

5

4 2

3

1

3/18/2014

20

Summary of Several Eukaryotic Gene Regulation Mechanisms

• Genes in highly compacted

chromatin are generally not

transcribed.

Chromatin modification

• DNA methylation generally reduces transcription.

• Histone acetylation seems to

loosen chromatin structure,

enhancing transcription.

Chromatin modification

Transcription

RNA processing

Translation mRNA degradation

Protein processing and degradation

mRNA degradation

• Each mRNA has a characteristic life span, determined in part by sequences in the 5’ and 3’ UTRs.

• Protein processing and degradation by proteasomes are subject to regulation.

Protein processing and degradation

• Initiation of translation can be controlled via regulation of initiation factors.

Translation

or mRNA

Primary RNA transcript

• Alternative RNA splicing:

RNA processing

• Coordinate regulation:

Enhancer for

liver-specific genes

Enhancer for

lens-specific genes

Bending of the DNA enables activators to contact proteins at the promoter, initiating transcription.

Transcription

• Regulation of transcription initiation: DNA control elements bind specific transcription factors.

Summary Roles of Small RNA’s in Gene Regulation of Eukaryotic Cells

Chromatin modification

RNA processing

Translation mRNA degradation

Protein processing

and degradation

mRNA degradation

• miRNA or siRNA can target specific mRNAs

for destruction.

• miRNA or siRNA can block the translation

of specific mRNAs.

Transcription

• Small RNAs can promote the formation of

heterochromatin in certain regions, blocking

transcription.

Chromatin modification

Translation