lecture 4

(1)

(2)

• Overview: How Eukaryotic Genomes Work and Evolve

• In eukaryotes, the DNA-protein complex, called

chromatin

– _{Is ordered into higher structural levels than the}

DNA-protein complex in prokaryotes

(3)

• _{Both prokaryotes and eukaryotes}

– Must alter their patterns of gene expression in

(4)

• _{Concept 19.1: Chromatin structure is based on}

successive levels of DNA packing

• Eukaryotic DNA

– Is precisely combined with a large amount of

protein

• Eukaryotic chromosomes

– Contain an enormous amount of DNA relative

(5)

Nucleosomes, or “Beads on a String”

• _{Proteins called histones}

– Are responsible for the first level of DNA

packing in chromatin

– Bind tightly to DNA

• The association of DNA and histones

– Seems to remain intact throughout the cell

(6)

• In electron micrographs

– _{Unfolded chromatin has the appearance of beads}

on a string

• Each “bead” is a nucleosome

– The basic unit of DNA packing

Figure 19.2 a

2 nm

10 nm DNA double helix

Histone tails

His-tones

Linker DNA (“string”)

Nucleosome (“bad”)

Histone H1

(7)

Nucleosome

30 nm

(b) 30-nm fiber

Higher Levels of DNA Packing

• _{The next level of packing}

– Forms the 30-nm chromatin fiber

(8)

• _{The 30-nm fiber, in turn}

– Forms looped domains, making up a 300-nm

fiber

Figure 19.2 c

Protein scaffold

300 nm

(c) Looped domains (300-nm fiber)

Loops

(9)

• _{In a mitotic chromosome}

– The looped domains themselves coil and fold

forming the characteristic metaphase chromosome

Figure 19.2 d

700 nm

1,400 nm

(10)

• _{In interphase cells}

– Most chromatin is in the highly extended form

(11)

• _{Concept 19.2: Gene expression can be}

regulated at any stage, but the key step is

transcription

• All organisms

– Must regulate which genes are expressed at

any given time

• During development of a multicellular organism

– Its cells undergo a process of specialization in

(12)

Differential Gene Expression

• _{Each cell of a multicellular eukaryote}

– Expresses only a fraction of its genes

• _{In each type of differentiated cell}

(13)

• _{Many key stages of gene expression}

– Can be regulated in eukaryotic cells

Figure 19.3

Signal

NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and

DNA demethlation Gene DNA Gene available for transcription RNA Exon Transcription Primary transcript RNA processing

Transport to cytoplasm Intron

Cap mRNA in nucleus Tail

CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypetide Cleavage Chemical modification

Transport to cellular destination Active protein

(14)

Regulation of Chromatin Structure

• _{Genes within highly packed heterochromatin}

(15)

Histone Modification

• _{Chemical modification of histone tails}

– Can affect the configuration of chromatin and

thus gene expression

Figure 19.4a (a) Histone tails protrude outward from a nucleosome

(16)

• _{Histone acetylation}

– Seems to loosen chromatin structure and

thereby enhance transcription

Figure 19.4 b (b) Acetylation of histone tails promotes loose chromatin structure that _{permits transcription}

(17)

DNA Methylation

• _{Addition of methyl groups to certain bases}

in DNA

– Is associated with reduced transcription in

(18)

Epigenetic Inheritance

• _{Epigenetic inheritance}

– Is the inheritance of traits transmitted by

(19)

Regulation of Transcription Initiation

• _{Chromatin-modifying enzymes provide initial}

control of gene expression

– By making a region of DNA either more or less

(20)

Organization of a Typical Eukaryotic Gene

• Associated with most eukaryotic genes are

multiple control elements

– _{Segments of noncoding DNA that help regulate}

transcription by binding certain proteins

Figure 19.5

Enhancer (distal control elements)

Proximal control elements

DNA

Upstream

Promoter

Exon Intron Exon Intron

Poly-A signal sequence Exon Termination region Transcription Downstream Poly-A signal Exon Intron Exon Intron Exon Primary RNA transcript (pre-mRNA) 5 Intron RNA RNA processing:

Cap and tail added; introns excised and exons spliced together

Coding segment

P P P G

mRNA

5 Cap _{5 UTR}_

(untranslated region)

Start codon

Stop

codon _{(untranslated}3 UTR region) Poly-A tail Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation

Cleared 3 end

(21)

The Roles of Transcription Factors

• _{To initiate transcription}

– Eukaryotic RNA polymerase requires the

(22)

Enhancers and Specific Transcription Factors

• _{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 in

(23)

Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Distal control element Activators Enhancer Promoter Gene TATA box _General transcription factors DNA-bending protein Group of Mediator proteins RNA Polymerase II RNA Polymerase II RNA synthesis Transcription Initiation complex Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation

A DNA-bending protein brings the bound activators closer to the promoter. Other transcription factors, mediator proteins, and RNA polymerase are nearby.

2

Activator proteins bind to distal control elements grouped as an enhancer in the DNA. This enhancer has three binding sites.

1

The activators bind to certain general transcription factors and mediator proteins, helping them form an active transcription initiation complex on the promoter.

3

• _{An activator}

– Is a protein that binds to an enhancer and

stimulates transcription of a gene

(24)

• _{Some specific transcription factors function as}

repressors

– To inhibit expression of a particular gene

• Some activators and repressors

– Act indirectly by influencing chromatin

(25)

Coordinately Controlled Genes

• _{Unlike the genes of a prokaryotic operon}

– Coordinately controlled eukaryotic genes each

have a promoter and control elements

• _{The same regulatory sequences}

– Are common to all the genes of a group,

(26)

Mechanisms of Post-Transcriptional Regulation

• _{An increasing number of examples}

– Are being found of regulatory mechanisms that

(27)

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 Figure 19.8 Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Exons DNA Primary RNA transcript mRNA

(28)

mRNA Degradation

• _{The life span of mRNA molecules in the}

cytoplasm

– Is an important factor in determining the

protein synthesis in a cell

– Is determined in part by sequences in the

(29)

• RNA interference by single-stranded microRNAs

(miRNAs)

– _{Can lead to degradation of an mRNA or block its}

translation Figure 19.9 5 Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation

Degradation of mRNA OR

Blockage of translation Target mRNA miRNA Protein complex Dicer Hydrogen bond The micro-RNA (mimicro-RNA) precursor folds back on itself, held together by hydrogen bonds. 1 2 An enzyme called Dicer moves along the double-stranded RNA, cutting it into shorter segments.

2 One strand of

each short double-stranded RNA is degraded; the other strand (miRNA) then associates with a complex of proteins.

3 The bound miRNA can base-pair with any target mRNA that contains the complementary sequence.

4 _{The miRNA-protein}

complex prevents gene expression either by degrading the target mRNA or by blocking its translation.

(30)

Initiation of Translation

• _{The initiation of translation of selected}

mRNAs

– Can be blocked by regulatory proteins that

bind to specific sequences or structures of the mRNA

• Alternatively, translation of all the mRNAs

in a cell

(31)

Protein Processing and Degradation

• _{After translation}

– Various types of protein processing, including

(32)

• Proteasomes

– _{Are giant protein complexes that bind protein}

molecules and degrade them

Figure 19.10 Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Ubiquitin Protein to be degraded Ubiquinated protein Proteasome Proteasome and ubiquitin to be recycled

Protein fragments (peptides) Protein entering a

proteasome

Multiple ubiquitin mol-ecules are attached to a protein by enzymes in the cytosol.

1 The ubiquitin-tagged proteinis recognized by a proteasome, which unfolds the protein and sequesters it within a central cavity.

2

Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol.

(33)

• _{Concept 19.3: Cancer results from genetic}

changes that affect cell cycle control

• The gene regulation systems that go wrong

during cancer

– Turn out to be the very same systems that play

(34)

Types of Genes Associated with Cancer

• _{The genes that normally regulate cell growth}

and division during the cell cycle

– Include genes for growth factors, their

(35)

Oncogenes and Proto-Oncogenes

• _Oncogenes

– Are cancer-causing genes

• Proto-oncogenes

– Are normal cellular genes that code for

(36)

• A DNA change that makes a proto-oncogene excessively active

– _{Converts it to an oncogene, which may promote}

excessive cell division and cancer

Figure 19.11

Proto-oncogene

DNA

Translocation or transposition: gene moved to new locus, under new controls

Gene amplification: multiple copies of the gene

Point mutation within a control element

Point mutation within the gene

Oncogene Oncogene

Normal growth-stimulating

protein in excess Hyperactive or degradation-resistant protein Normal growth-stimulating

protein in excess Normal growth-stimulating

protein in excess New

(37)

Tumor-Suppressor Genes

• _{Tumor-suppressor genes}

– Encode proteins that inhibit abnormal cell

(38)

Interference with Normal Cell-Signaling Pathways

• _{Many proto-oncogenes and tumor suppressor}

genes

– Encode components of growth-stimulating and

(39)

Figure 19.12a

(a) Cell cycle–stimulating pathway. This pathway is triggered by a growth factor that binds to its receptor in the plasma membrane. The signal is relayed to a G protein called Ras. Like all G proteins, Ras is active when GTP is bound to it. Ras passes the signal to a series of protein kinases. The last kinase activates a transcription activator that turns on one or more genes for proteins that stimulate the cell cycle. If a mutation makes Ras or any other pathway component abnormally active, excessive cell division and cancer may result.

1 2 4 3 5 GTP Ras Ras GTP Hyperactive Ras protein (product of oncogene) issues signals on its own

NUCLEUS

Gene expression

Protein that stimulates the cell cycle

P P P P MUTATION P DNA P

• _{The Ras protein, encoded by the}

_ras

_gene

– Is a G protein that relays a signal from a

growth factor receptor on the plasma

membrane to a cascade of protein kinases

2 Receptor Transcription factor (activator) 5 G protein 3 Protein kinases (phosphorylation cascade) 4

1 Growth

(40)

• The p53 gene encodes a tumor-suppressor protein

– _{That is a specific transcription factor that}

promotes the synthesis of cell cycle–inhibiting proteins Figure 19.12b UV light DNA Defective or missing transcription factor, such as p53, cannot activate transcription MUTATION Protein that inhibits the cell cycle pathway, DNA damage is an intracellular

signal that is passed via protein kinases and leads to activation of p53. Activated p53 promotes transcription of the gene for a protein that inhibits the cell cycle. The resulting suppression of cell division ensures that the damaged DNA is not replicated. Mutations causing deficiencies in any pathway component can contribute to the development of cancer.

(b) Cell cycle–inhibiting pathway. In this

(41)

• _{Mutations that knock out the}

_p53

_gene

– Can lead to excessive cell growth and cancer

Figure 19.12c

EFFECTS OF MUTATIONS

Protein overexpressed

Cell cycle

overstimulated Increased celldivision

Cell cycle not inhibited Protein absent Effects of mutations. Increased

cell division, possibly leading to cancer, can result if the cell cycle is overstimulated, as in (a), or not inhibited when it normally would be, as in (b).

(42)

The Multistep Model of Cancer Development

• _{Normal cells are converted to cancer cells}

– By the accumulation of multiple mutations

(43)

• _{A multistep model for the development of}

colorectal cancer

Figure 19.13 Colon Colon wall Normal colon epithelial cells Small benign growth (polyp) Larger benign growth (adenoma) Malignant tumor (carcinoma)

2 Activation of

ras oncogene

3 Loss of tumor-suppressor gene DCC

4 Loss of

tumor-suppressor gene p53

5 Additional mutations

(44)

• _{Certain viruses}

– Promote cancer by integration of viral DNA into

(45)

Inherited Predisposition to Cancer

• _{Individuals who inherit a mutant oncogene or}

tumor-suppressor allele

– Have an increased risk of developing certain

(46)

• _{Concept 19.4: Eukaryotic genomes can have}

many noncoding DNA sequences in addition to

genes

• The bulk of most eukaryotic genomes

– Consists of noncoding DNA sequences, often

described in the past as “junk DNA”

• However, much evidence is accumulating

– That noncoding DNA plays important roles in

(47)

The Relationship Between Genomic Composition

and Organismal Complexity

• Compared with prokaryotic genomes, the

genomes of eukaryotes

– Generally are larger

– Have longer genes

– _{Contain a much greater amount of noncoding}

(48)

• Now that the complete sequence of the human genome is available

– _{We know what makes up most of the 98.5% that}

does not code for proteins, rRNAs, or tRNAs

Figure 19.14

Exons (regions of genes coding for protein, rRNA, tRNA) (1.5%)

Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (about 15%)

Alu elements (10%)

Simple sequence

(49)

Transposable Elements and Related Sequences

• _{The first evidence for wandering DNA}

segments

– Came from geneticist Barbara McClintock’s

breeding experiments with Indian corn

(50)

Transposon

New copy of transposon

Transposon is copied DNA of genome

Insertion

Mobile transposon

(a) Transposon movement (“copy-and-paste” mechanism)

Retrotransposon

New copy of retrotransposon

DNA of genome

RNA Reverse

transcriptase

(b) Retrotransposon movement

Insertion

Movement of Transposons and Retrotransposons

• Eukaryotic transposable elements are of two types

– _{Transposons, which move within a genome by}

means of a DNA intermediate

– _{Retrotransposons, which move by means of an}

RNA intermediate

(51)

Sequences Related to Transposable Elements

• _{Multiple copies of transposable elements and}

sequences related to them

– Are scattered throughout the eukaryotic

genome

• _{In humans and other primates}

– A large portion of transposable element–

related DNA consists of a family of similar

(52)

Other Repetitive DNA, Including Simple Sequence DNA

• _{Simple sequence DNA}

– Contains many copies of tandemly repeated

short sequences

– Is common in centromeres and telomeres,

(53)

Genes and Multigene Families

• _{Most eukaryotic genes}

– Are present in one copy per haploid set of

chromosomes

• _{The rest of the genome}

– Occurs in multigene families, collections of

(54)

DNA RNA transcripts

Non-transcribed

spacer Transcription unit

DNA

18S 5.8S 28S

rRNA

5.8S 28S

18S

• _{Some multigene families}

– Consist of identical DNA sequences, usually

clustered tandemly, such as those that code for RNA products

(55)

• _{The classic examples of multigene families of}

nonidentical genes

– Are two related families of genes that encode

globins

Figure 19.17b The human -globin and -globin gene

  families -Globin Heme Hemoglobin -Globin

-Globin gene family -Globin gene family Chromosome 16 Chromosome 11

Embryo

Fetus

and adult Embryo Fetus Adult

 G A   

  _

2

_

(56)

• _{Concept 19.5: Duplications, rearrangements,}

and mutations of DNA contribute to genome

evolution

• The basis of change at the genomic level is

mutation

(57)

Duplication of Chromosome Sets

• _{Accidents in cell division}

– Can lead to extra copies of all or part of a

(58)

Duplication and Divergence of DNA Segments

• Unequal crossing over during prophase I of

meiosis

– _{Can result in one chromosome with a deletion and}

another with a duplication of a particular gene

Figure 19.18

Nonsister chromatids

Transposable

element Gene

Incorrect pairing of two homologues during meiosis

Crossover

(59)

Evolution of Genes with Related Functions: The

Human Globin Genes

• The genes encoding the various globin proteins

– _{Evolved from one common ancestral globin gene,}

which duplicated and diverged

Figure 19.19

Ancestral globin gene

 

 



  ₂ ₁ 2 1   G A    -Globin gene family

on chromosome 16 on chromosome 11-Globin gene family

(60)

• _{Subsequent duplications of these genes and}

random mutations

– Gave rise to the present globin genes, all of

(61)

• The similarity in the amino acid sequences of the

various globin proteins

– _{Supports this model of gene duplication and}

mutation

(62)

Evolution of Genes with Novel Functions

• _{The copies of some duplicated genes}

– Have diverged so much during evolutionary

(63)

Rearrangements of Parts of Genes: Exon

Duplication and Exon Shuffling

• A particular exon within a gene

– Could be duplicated on one chromosome and

(64)

• In exon shuffling

– _{Errors in meiotic recombination lead to the}

occasional mixing and matching of different exons either within a gene or between two nonallelic

genes

Figure 19.20

EGF EGF EGF EGF

Epidermal growth factor gene with multiple EGF exons (green)

F F F F

Fibronectin gene with multiple “finger” exons (orange)

Exon

shuffling Exon_duplication

Exon shuffling

K

F EGF K K

Plasminogen gene with a “kfingle” exon (blue)

(65)

How Transposable Elements Contribute to Genome Evolution

• _{Movement of transposable elements or}

recombination between copies of the same

element

– Occasionally generates new sequence

combinations that are beneficial to the organism

• Some mechanisms

– Can alter the functions of genes or their