Transcript Presentation

14
The Eukaryotic Genome and
Its Expression
14 The Eukaryotic Genome and Its Expression
• 14.1 What Are the Characteristics of the Eukaryotic
Genome?
• 14.2 What Are the Characteristics of Eukaryotic
Genes?
• 14.3 How Are Eukaryotic Gene Transcripts
Processed?
• 14.4 How Is Eukaryotic Gene Transcription
Regulated?
• 14.5 How Is Eukaryotic Gene Expression Regulated
After Transcription?
• 14.6 How Is Gene Expression Controlled During and
After Translation?
14.1 What Are the Characteristics of the Eukaryotic Genome?
Key differences between eukaryotic and
prokaryotic genomes:
• Eukaryotic genomes are larger.
• Eukaryotic genomes have more
regulatory sequences.
• Much of eukaryotic DNA is noncoding.
14.1 What Are the Characteristics of the Eukaryotic Genome?
• Eukaryotes have multiple chromosomes.
• In eukaryotes, translation and
transcription are physically separated
which allows many points of regulation
before translation begins.
Figure 14.1 Eukaryotic mRNA is Transcribed in the Nucleus but Translated in the Cytoplasm
Table 14.1
14.1 What Are the Characteristics of the Eukaryotic Genome?
Eukaryote model organisms:
• Yeast, Saccharomyces cerevisiae
• Nematode (roundworm), Caenorhabditis
elegans
• Fruit fly, Drosophila melanogaster
• Thale cress, Arabidopsis thaliana
14.1 What Are the Characteristics of the Eukaryotic Genome?
The yeast (Saccharomyces cerevisiae)
has 16 chromosomes; haploid content of
12 million base pairs (bp).
Compartmentalization into organelles
requires more genes than prokaryotes
have.
Table 14.2
14.1 What Are the Characteristics of the Eukaryotic Genome?
Some eukaryotic genes that have no
homologs in prokaryotes:
• Genes encoding histones
• Genes encoding cyclin-dependent
kinases that control cell division
• Genes encoding proteins involved in
processing of mRNA
14.1 What Are the Characteristics of the Eukaryotic Genome?
The soil nematode, Caenorhabditis
elegans, is only 1 mm long.
A model organism to study development:
the body is transparent, an adult has
about 1,000 cells
The genome is eight times larger than
yeasts.
Table 14.3
14.1 What Are the Characteristics of the Eukaryotic Genome?
Drosophila melanogaster has been used
extensively in genetic studies.
Genome is larger than C. elegans, but
has fewer genes
The genome codes for more proteins than
it has genes.
Figure 14.2 Functions of the Eukaryotic Genome
14.1 What Are the Characteristics of the Eukaryotic Genome?
Arabidopsis thaliana is in the mustard
family.
Has some genes that have homologs in
C. elegans and Drosophila
Also has genes that distinguish it as a
plant, such as genes for photosynthesis.
Table 14.4
14.1 What Are the Characteristics of the Eukaryotic Genome?
Rice (Oryza sativa) genome has also
been sequenced—two subspecies
Has many genes similar to Arabidopsis.
Table 14.5
14.1 What Are the Characteristics of the Eukaryotic Genome?
Eukaryote genomes have two types of
highly repetitive sequences that do not
code for proteins:
Minisatellites: 10–40 bp, repeated several
thousand times. Number of copies varies
among individuals—provides molecular
markers.
Microsatellites: 1–3 bp, 15–100 copies
14.1 What Are the Characteristics of the Eukaryotic Genome?
Moderately repetitive sequences (genes):
code for tRNA and rRNA
These molecules are needed in large
quantities; the genome has multiple
copies of the sequence.
14.1 What Are the Characteristics of the Eukaryotic Genome?
Mammals: Four different rRNAs
16S, 5.8S, 28S are transcribed as a
single precursor molecule. Humans have
280 copies of the sequence on five
different chromosomes;
and 5S.
(S = Svedberg unit)
Figure 14.3 A Moderately Repetitive Sequence Codes for rRNA
14.1 What Are the Characteristics of the Eukaryotic Genome?
Other moderately repetitive sequences
can move from place to place in the
genome—transposons.
Transposons make up 40 percent of
human genome, only 3–10 percent in
other sequenced eukaryotes.
14.1 What Are the Characteristics of the Eukaryotic Genome?
Four types of transposons:
• SINEs
• LINEs
• Retrotransposons
• DNA transposons
14.1 What Are the Characteristics of the Eukaryotic Genome?
SINEs (short interspersed elements)—500
bp; 15 percent of human DNA. One, Alu,
is present in a million copies
LINEs (long interspersed elements)—
7,000 bp; about 17 percent of human
DNA; some code for proteins
14.1 What Are the Characteristics of the Eukaryotic Genome?
SINEs and LINEs make an RNA copy of
themselves that is a template for new
DNA inserted somewhere else—“copy
and paste” mechanism.
14.1 What Are the Characteristics of the Eukaryotic Genome?
Retrotransposons: about 8 percent of
human genome; also make an RNA
copy of themselves.
DNA transposons move to a new place in
the genome without replicating.
Figure 14.4 DNA Transposons and Transposition
14.1 What Are the Characteristics of the Eukaryotic Genome?
The function of the transposons is
unclear.
They may be cellular parasites.
If a transposon is inserted into a coding
region, a mutation results. If it’s in a
somatic cell, cancer can result.
Transposons can carry genes to new
locations—adding to genetic variation.
14.1 What Are the Characteristics of the Eukaryotic Genome?
Transposons may have played a role in
endosymbiosis:
Genes from the once-independent
prokaryotes may have moved to the
nucleus by DNA transposons.
14.2 What Are the Characteristics of Eukaryotic Genes?
Gene characteristics not found in
prokaryotes:
• Eukaryote genes contain noncoding
internal sequences.
• Form gene families—groups of
structurally and functionally related
genes
14.2 What Are the Characteristics of Eukaryotic Genes?
Eukaryote genes have a promoter to
which RNA polymerase binds and a
terminator sequence to signal end of
transcription.
Terminator sequence comes after the stop
codon.
Stop codon is transcribed into mRNA and
signals the end of translation at the
ribosome.
Figure 14.5 Transcription of a Eukaryotic Gene (Part 1)
Figure 14.5 Transcription of a Eukaryotic Gene (Part 2)
14.2 What Are the Characteristics of Eukaryotic Genes?
Protein-coding genes have noncoding
sequences—introns.
The coding sequences are extrons.
Transcripts of introns appear in the premRNA, they are removed from the final
mRNA.
14.2 What Are the Characteristics of Eukaryotic Genes?
Nucleic acid hybridization reveals
introns.
Target DNA is denatured; then incubated
with a probe—a nucleic acid strand from
another source.
If the probe has a complementary
sequence, base pairing forms a hybrid.
Figure 14.6 Nucleic Acid Hybridization
14.2 What Are the Characteristics of Eukaryotic Genes?
If researchers used mature mRNA as the
probe, the DNA-RNA hybrid would have
loops where base pairing did not occur—
the introns.
If pre-mRNA was used, resulted in
complete hybridization
Figure 14.7 Nucleic Acid Hybridization Revealed the Existence of Introns (Part 1)
Figure 14.7 Nucleic Acid Hybridization Revealed the Existence of Introns (Part 2)
14.2 What Are the Characteristics of Eukaryotic Genes?
Introns interrupt, but do not scramble, the
DNA sequence that encodes a
polypeptide.
Sometimes, the separated exons code for
different domains (functional regions) of
the protein.
14.2 What Are the Characteristics of Eukaryotic Genes?
About half of the eukaryote genes are
present in multiple copies.
Different mutations can occur in copies,
giving rise to gene families.
Family that encodes for immunoglobulins
have hundreds of members.
14.2 What Are the Characteristics of Eukaryotic Genes?
As long as one member of a gene family
retains the original sequence, copies can
mutate without losing original function.
This is important in evolution.
14.2 What Are the Characteristics of Eukaryotic Genes?
The globin gene family arose from a common
ancestor gene.
In humans:
• Alpha-globin (α-globin)—three functional
genes
• Beta-globin (β-globin)—five functional genes
• Hemoglobin is a tetramer of two α units and
two β units.
Figure 14.8 The Globin Gene Family
Figure 3.9 Quaternary Structure of a Protein
14.2 What Are the Characteristics of Eukaryotic Genes?
During development, different globin
genes are expressed at different times:
differential gene expression.
γ-globin is in hemoglobin of human
fetus—it binds oxygen more tightly than
adult hemoglobin.
Figure 14.9 Differential Expression in the Globin Gene Family
14.2 What Are the Characteristics of Eukaryotic Genes?
Some gene families have
pseudogenes—result from a mutation
that results in loss of function.
Pseudogenes may lack a promoter, or
recognition sites for removal of introns.
Designated by ψ (psi)
14.3 How Are Eukaryotic Gene Transcripts Processed?
In the nucleus, pre-mRNA is modified at
both ends:
G-cap added at the 5′ end (modified
guanosine triphosphate)—facilitates
binding to ribosome.
Protects it from being digested by
ribonucleases.
14.3 How Are Eukaryotic Gene Transcripts Processed?
Poly A tail added at 3′ end.
AAUAAA sequence after last codon is a
signal for an enzyme to cut the premRNA; then another enzyme adds 100
to 300 adenines—the “tail.”
May assist in export from nucleus;
important for stability of mRNA.
Figure 14.10 Processing the Ends of Eukaryotic Pre-mRNA (Part 1)
Figure 14.10 Processing the Ends of Eukaryotic Pre-mRNA (Part 2)
14.3 How Are Eukaryotic Gene Transcripts Processed?
RNA splicing removes introns and splices
exons together.
Pre-mRNA is bound by small nuclear
ribonucleoprotein particles (snRNPs).
Consensus sequences are short
sequences between exons and introns.
snRNP binds here, and also near the 3′
end of the intron.
14.3 How Are Eukaryotic Gene Transcripts Processed?
With energy from ATP, proteins are added
to form an RNA-protein complex, the
spliceosome.
The complex cuts pre-mRNA, releases
introns, and splices exons together.
Figure 14.11 The Spliceosome: An RNA Splicing Machine
14.3 How Are Eukaryotic Gene Transcripts Processed?
In the disease beta thalassemia, a
mutation occurs at the consensus
sequence in the β-globin gene—the premRNA can not be spliced correctly.
Non-functional β-globin mRNA is
produced.
14.3 How Are Eukaryotic Gene Transcripts Processed?
Mature mRNA leaves the nucleus through
nuclear pores.
TAP protein binds to the 5′ end, TAP binds
to other proteins that are recognized by
receptors at the nuclear pore.
14.4 How Is Eukaryotic Gene Transcription Regulated?
Expression of genes must be precisely
regulated during development.
Gene expression can be regulated at
several points in the transcription and
translation processes.
Figure 14.12 Potential Points for the Regulation of Gene Expression (Part 1)
Figure 14.12 Potential Points for the Regulation of Gene Expression (Part 2)
Figure 14.12 Potential Points for the Regulation of Gene Expression (Part 3)
14.4 How Is Eukaryotic Gene Transcription Regulated?
Transcriptional regulation and
posttranscriptional regulation can be
determined by examining mRNA
sequences made in different cell types.
14.4 How Is Eukaryotic Gene Transcription Regulated?
Eukaryote genes are not organized into
operons.
Regulation of several genes at once
requires common control elements.
Eukaryotes have three RNA polymerases:
• I codes for rRNA; III codes for tRNA
• II transcribes protein-coding genes
14.4 How Is Eukaryotic Gene Transcription Regulated?
Most eukaryotic genes have sequences
that regulate rate of transcription.
Initiation of transcription involves many
proteins (in contrast to prokaryotes in
which RNA polymerase directly
recognized the promoter).
14.4 How Is Eukaryotic Gene Transcription Regulated?
In prokaryotes, promoter has two
sequences:
The recognition sequence is recognized
by RNA polymerase.
The TATA box, where DNA begins to
denature.
14.4 How Is Eukaryotic Gene Transcription Regulated?
In eukaryotes, transcription factors
(regulatory proteins) must assemble on
the chromosome before RNA
polymerase can bind to the promoter.
TFIID binds to the TATA box; then other
transcription factors bind, forming a
transcription complex.
Figure 14.13 The Initiation of Transcription in Eukaryotes (Part 1)
Figure 14.13 The Initiation of Transcription in Eukaryotes (Part 2)
14.4 How Is Eukaryotic Gene Transcription Regulated?
Some sequences are common to
promoters of many genes; recognized by
transcription factors in all cells.
Some sequences are specific to a few
genes and are recognized by
transcription factors found only in certain
tissues. These play an important role in
differentiation.
14.4 How Is Eukaryotic Gene Transcription Regulated?
Regulator sequences are located
upstream of the promoter.
Regulator proteins bind to these
sequences. Resulting complex binds to
the transcription complex to activate
transcription.
14.4 How Is Eukaryotic Gene Transcription Regulated?
Enhancer sequences are farther away—
up to 20,000 bp.
Activator proteins bind to enhancer
sequences, which stimulates
transcription complex. Mechanism not
known; perhaps by DNA bending.
Figure 14.14 Transcription Factors, Regulators, and Activators (Part 1)
Figure 14.14 Transcription Factors, Regulators, and Activators (Part 2)
14.4 How Is Eukaryotic Gene Transcription Regulated?
Negative regulatory sequences or
silencer sequences turn off
transcription by binding repressor
proteins.
14.4 How Is Eukaryotic Gene Transcription Regulated?
DNA-binding proteins have four structural
themes or motifs:
• Helix-turn-helix
• Zinc finger
• Leucine zipper
• Helix-loop-helix
Figure 14.15 Protein–DNA Interactions (Part 1)
Figure 14.15 Protein–DNA Interactions (Part 2)
Figure 14.15 Protein–DNA Interactions (Part 3)
Figure 14.15 Protein–DNA Interactions (Part 4)
14.4 How Is Eukaryotic Gene Transcription Regulated?
Bases in DNA can form hydrogen bonds
with proteins, especially in major and
minor grooves.
Many repressor proteins have helix-turnhelix configuration—binding of repressor
prevents other proteins from binding and
initiating transcription.
14.4 How Is Eukaryotic Gene Transcription Regulated?
Regulation of genes that are far apart or
on different chromosomes—genes must
have same regulator sequences.
Example: Some plant genes have a
regulatory sequence called stress
response element (SRE).
Genes with this sequence encode for
proteins needed to cope with drought.
Figure 14.16 Coordinating Gene Expression (Part 1)
Figure 14.16 Coordinating Gene Expression (Part 2)
14.4 How Is Eukaryotic Gene Transcription Regulated?
Transcription can also be regulated by
changes in chromatin and
chromosomes.
14.4 How Is Eukaryotic Gene Transcription Regulated?
Chromatin remodeling:
DNA is wound around histones to form
nucleosomes, which block initiation and
elongation.
One remodeling protein disaggregates the
nucleosome to allow initiation.
The second remodeling protein binds to
the nucleosomes to allow elongation to
proceed.
Figure 14.17 Local Remodeling of Chromatin for Transcription (Part 1)
Figure 14.17 Local Remodeling of Chromatin for Transcription (Part 2)
14.4 How Is Eukaryotic Gene Transcription Regulated?
Histone proteins have “tails” with
positively charged amino acids—
enzymes add acetyl groups:
14.4 How Is Eukaryotic Gene Transcription Regulated?
This reduces positive charges, and
decreases affinity of histones for
negatively charged DNA.
Allows chromatin remodeling
14.4 How Is Eukaryotic Gene Transcription Regulated?
Gene activation requires histone acetyl
transferases to add acetyl groups.
Gene repression requires histone
deacetylases to remove the acetyl
groups.
14.4 How Is Eukaryotic Gene Transcription Regulated?
The “histone code”—histone modifications
affect gene activation and repression.
Example: Methylation of histones is
associated with gene inactivation.
Whether a gene becomes activated by
chromatin remodeling may be
determined by histone modification.
14.4 How Is Eukaryotic Gene Transcription Regulated?
Two types of chromatin:
Euchromatin contains DNA that is
transcribed into mRNA.
Heterochromatin: genes it contains are
usually not transcribed.
14.4 How Is Eukaryotic Gene Transcription Regulated?
Example of heterochromatin: inactive X
chromosome in mammals.
Each female has two copies of genes on
the X chromosome.
Y chromosome gradually lost most of the
genes it once shared with its X homolog.
Female has potential to produce twice as
much protein from the X-linked genes.
14.4 How Is Eukaryotic Gene Transcription Regulated?
One X chromosome remains inactive in
female cells.
Can be seen under a light microscope as
a clump of heterochromatin—called a
Barr body
Thus, dosage of expressed X
chromosome is the same in males and
females.
Figure 14.18 A Barr Body in the Nucleus of a Female Cell
14.4 How Is Eukaryotic Gene Transcription Regulated?
Methylation of cystosines contributes to
condensation and inactivation of the
DNA.
One gene is active: Xist (X inactivationspecific transcript). RNA that is
transcribed binds to the chromosome
and inactivates it—interference RNA.
Figure 14.19 A Model for X Chromosome Inactivation
14.4 How Is Eukaryotic Gene Transcription Regulated?
An anti-Xist gene, Tsix, codes for RNA
that binds to the Xist site on the active X
chromosome.
14.4 How Is Eukaryotic Gene Transcription Regulated?
Transcription can be increased by making
more copies of a gene—gene
amplification.
Example: The genes that code for three of
the rRNAs in humans are linked and
there are several hundred copies in the
genome.
14.4 How Is Eukaryotic Gene Transcription Regulated?
Fish and frog eggs have up to a trillion
ribosomes.
Cells selectively amplify the rRNA gene
clusters to more than a million copies.
Transcribed at maximum rate, these
genes produce the ribosomes for a
mature egg in a few days.
Figure 14.20 Transcription from Multiple Genes for rRNA
14.4 How Is Eukaryotic Gene Transcription Regulated?
In some cancers, a cancer-causing
oncogene is amplified.
The mechanism of amplification is not
well understood.
14.5 How Is Eukaryotic Gene Expression Regulated After
Transcription?
Alternative splicing: some exons are
selectively deleted
Different proteins can be generated from
the same gene.
Example: The pre-mRNA for tropomyosin
is spliced five different ways to produce
five different forms of tropomyosin.
Figure 14.21 Alternative Splicing Results in Different Mature mRNAs and Proteins
14.5 How Is Eukaryotic Gene Expression Regulated After
Transcription?
In humans, there are many more mRNAs
than genes—mostly from alternative
splicing.
14.5 How Is Eukaryotic Gene Expression Regulated After
Transcription?
RNA has no repair mechanisms.
mRNA can be catabolyzed by
ribonucleases in the cytoplasm and
lysosomes.
mRNAs have different stabilities—a
mechanism for posttranscriptional
regulation.
14.5 How Is Eukaryotic Gene Expression Regulated After
Transcription?
Specific AU sequences on mRNA can
mark them for breakdown by a
ribonuclease complex called an
exosome.
Signaling molecules such as growth factor
are only synthesized when needed and
break down rapidly. Their mRNAs have
an AU sequence and are unstable.
14.5 How Is Eukaryotic Gene Expression Regulated After
Transcription?
Micro RNAs (about 20 bases long) bind
to mRNA before it reaches a ribosome.
Causes the mRNA to break down, or
inhibits translation.
14.5 How Is Eukaryotic Gene Expression Regulated After
Transcription?
The micro RNAs start as a 70 base-pair
double strand.
The protein complex called dicer cuts the
RNA strand.
Small RNAs are under development as
drugs to block gene expression of
certain genes in human diseases.
Figure 14.22 mRNA Inhibition by Small RNAs
14.5 How Is Eukaryotic Gene Expression Regulated After
Transcription?
RNA editing: change in sequence after
transcription and splicing
Insertion of nucleotides—stretches of
uracil are added
Alteration of nucleotides—an enzyme
catalyzes the deamination of cytosine to
from uracil.
Figure 14.23 RNA Editing
14.6 How Is Gene Expression Controlled During and After
Translation?
Translation can be modified by the G cap.
If the cap is an unmodified GTP, the
mRNA is not translated.
Example: The stored mRNA in egg cells
of tobacco hornworm moth: After the egg
is fertilized, the cap is modified, and
translation proceeds.
14.6 How Is Gene Expression Controlled During and After
Translation?
Cellular conditions can control translation.
Example: free iron (Fe2+) in cells is bound
by ferritin
When Fe2+ is low, a repressor binds to
ferritin mRNA and prevents translation.
As Fe2+ levels rise, Fe2+ binds to the
repressor, which detaches from the
mRNA.
14.6 How Is Gene Expression Controlled During and After
Translation?
Translational control can keep a balance
in the amount of subunits of proteins.
Example: Hemoglobin has four globin and
four heme units.
If there are more heme than globin units,
heme increases rate of translation of
globin by removing a block to initiation of
translation at ribosome.
14.6 How Is Gene Expression Controlled During and After
Translation?
Most proteins are modified after
translation.
A protein can be regulated by controlling
its lifetime in the cell.
In many cases, an enzyme attaches a
protein called ubiquitin to a lysine in a
protein targeted for breakdown.
14.6 How Is Gene Expression Controlled During and After
Translation?
Other ubiquitin chains attach to the first
one, forming a polyubiquitin complex.
The whole complex then binds to a
proteasome.
Ubiquitin is cut off for recycling; the
protein passes by three proteases that
digest it.
Figure 14.24 A Proteasome Breaks Down Proteins
14.6 How Is Gene Expression Controlled During and After
Translation?
Concentrations of many proteins are
determined by their degradation in
proteasomes.
Cyclins are degraded at the correct time
in the cell cycle.
Transcriptional regulators are broken
down after use; to prevent gene to be
always “on.”
14.6 How Is Gene Expression Controlled During and After
Translation?
Some viruses can take advantage of this
system.
Human papillomavirus (causes cervical
cancer) marks protein p53 for
degradation by proteasomes. p53
normally inhibits cell division.