Download 1) From DNA to protein 2) Gene mutation

8.0 Gene expression and mutation
Related Sadava’s chapters:
1)  From DNA to protein
2) Gene mutation
8.1 From DNA to protein: gene expression
• What Is the Evidence that Genes Code for
Proteins?
• How Does Information Flow from Genes
to Proteins?
8.1 From DNA to protein: gene expression
• Identification of a gene product as a
protein began with a mutation.
• Garrod saw a disease phenotype—
alkaptonuria—occurring in children who
shared more alleles as first cousins.
• A substance in their blood (HA,
homogentisic acid) accumulated—was
not catalyzed—the gene for the enzyme
was mutated.
• Garrod correlated one gene to one
enzyme.
8.1 From DNA to protein: gene expression
•  Beadle and Tatum used Neurospora to test hypothesis that
specific gene expression → specific enzyme activity.
•  Neurospora is haploid for most of its life cycle—all alleles are
expressed as phenotypes.
•  Wild-type strains like Neurospora are prototrophs—have
enzymes to catalyze all reactions to make cell constituents.
•  Beadle and Tatum used X rays as mutagens to cause
mutations—inherited genotypic changes.
•  Mutants were auxotrophs—needed additional nutrients to grow.
•  For each auxotrophic mutant strain, the addition of just one
compound supported growth.
•  Results suggested that each mutation caused a defect in only
one enzyme in a metabolic pathway.
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
• The gene-enzyme relationship has since
been revised to the one-gene, onepolypeptide relationship.
• Example: In hemoglobin, each polypeptide
chain is specified by a separate gene.
• Other genes code for RNA are not
translated to polypeptides; some genes are
involved in controlling other genes.
8.1 From DNA to protein: gene expression
Gene expression to form a specific
polypeptide occurs in two steps:
• Transcription—copies information from
a DNA sequence (a gene) to a
complementary RNA sequence
• Translation—converts RNA sequence to
amino acid sequence of a polypeptide
“The central dogma of molecular
biology.”
8.1 From DNA to protein: gene expression
RNA (ribonucleic acid) differs from DNA:
• Usually one polynucleotide strand
• The sugar is ribose
• Contains uracil (U) instead of thymine (T)
8.1 From DNA to protein: gene expression
Bases in RNA can pair with a single
strand of DNA, except that adenine
pairs with uracil instead of thymine.
Single-strand RNA can fold into complex
shapes by internal base pairing.
8.1 From DNA to protein: gene expression
Three kinds of RNA in protein
synthesis:
• Messenger RNA (mRNA)—carries
copy of a DNA sequence to site of
protein synthesis at the ribosome
• Transfer RNA (tRNA)—carries amino
acids for polypeptide assembly
• Ribosomal RNA (rRNA)—catalyzes
peptide bonds and provides structure
8.1 From DNA to protein: gene expression
The central dogma suggested that
information flows from DNA to RNA to
protein, which raised two questions:
• How does genetic information get from
the nucleus to the cytoplasm?
• What is the relationship between a DNA
sequence and an amino acid sequence?
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
Exception to the central dogma:
•  Viruses: Non-cellular particles that reproduce inside cells; many have
RNA instead of DNA.
•  Viruses can replicate by transcribing from RNA to RNA, and then
making multiple copies by transcription.
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
• RNA polymerases catalyze synthesis
of RNA.
• RNA polymerases are processive—a
single enzyme-template binding results
in polymerization of hundreds of RNA
bases.
• Unlike DNA polymerases, RNA
polymerases do not need primers and
lack a proofreading function.
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
•  The genetic code: Specifies which amino
acids will be used to build a protein
•  Codon: A sequence of three bases—each
codon specifies a particular amino acid.
•  Start codon: AUG—initiation signal for
translation.
•  Stop codons: UAA, UAG, UGA—stop
translation and polypeptide is released.
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
•  For most amino acids, there is more than one codon; the
genetic code is redundant.
•  Wobble base pair
•  The genetic code is not ambiguous—each codon specifies only
one amino acid.
•  The genetic code is nearly universal: The codons that specify
amino acids are the same in all organisms.
•  Exceptions: within mitochondria and chloroplasts, and in one
group of protists, there are differences.
•  The frequency of synonymous codons varies between species.
•  Three reading frames coexist on the DNA/RNA coding
sequence.
8.1 From DNA to protein: gene expression
•  Prokaryotes and eukaryotes differ in gene structure—in the
organization of nucleotide sequences.
•  In eukaryotes a nucleus separates transcription and translation.
•  Eukaryotic genes may have noncoding sequences—introns.
•  The coding sequences are exons.
•  Introns and exons appear in the primary mRNA transcript—premRNA; introns are removed from the final mRNA.
Figure 14.7 Transcription of a Eukaryotic Gene (1)
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
• 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.
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
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8.1 From DNA to protein: gene expression
WT1
24 isoforms
CD44
1024 isoforms
DSCAM
38016 isoforms !!!
Roberts & Smith (2002)
8.1 From DNA to protein: gene expression
• In the disease β-thalassemia, a
mutation may occur at an intron
consensus sequence in the β-globin
gene—the pre-mRNA can not be
spliced correctly.
• Non-functional β-globin mRNA is
produced.
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
• Mature mRNA leaves the nucleus
through nuclear pores.
• TAP protein binds to the 5′ end; this
binds to other proteins that are
recognized by receptors at the nuclear
pore.
• These proteins lead the mRNA through
the pore—unused pre-mRNAs stay in
the nucleus.
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
•  tRNA, the adapter molecule, links information in mRNA codons
with specific amino acids.
•  For each amino acid, there is a specific type or “species” of
tRNA.
Three functions of tRNA:
•  It binds to an amino acid, and is then “charged”
•  It associates with mRNA molecules
•  It interacts with ribosomes
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
• Wobble: Specificity for the base at the 3′
end of the codon is not always
observed.
• Example: Codons for alanine—GCA,
GCC, and GCU—are recognized by the
same tRNA.
• Wobble allows cells to produce fewer
tRNA species, but does not allow the
genetic code to be ambiguous.
8.1 From DNA to protein: gene expression
• Activating enzymes—aminoacyl-tRNA
synthetases—charge tRNA with the
correct amino acids.
• Each enzyme is highly specific for one
amino acid and its corresponding tRNA;
the process of tRNA charging is called
the second genetic code.
• The enzymes have three-part active
sites: They bind a specific amino acid, a
specific tRNA, and ATP.
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
Experiment by Benzer and others:
• Cysteine already bound to tRNA was
chemically changed to alanine.
• Which would be recognized—the amino
acid or the tRNA in protein synthesis?
• Answer: Protein synthesis machinery
recognizes the anticodon, not the amino
acid.
8.1 From DNA to protein: gene expression
•  Ribosome: the workbench—holds mRNA and
charged tRNAs in the correct positions to allow
assembly of polypeptide chain.
•  Ribosomes are not specific, they can make any type
of protein.
•  Ribosomes have two subunits, large and small.
•  In eukaryotes, the large subunit has three molecules
of ribosomal RNA (rRNA) and 49 different proteins in
a precise pattern.
•  The small subunit has one rRNA and 33 proteins.
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
Figure 14.17 The Termination of Translation (Part 1)
Figure 14.17 The Termination of Translation (Part 2)
Figure 14.17 The Termination of Translation (Part 3)
8.1 From DNA to protein: gene expression
• The large subunit has peptidyl
transferase activity—if rRNA is
destroyed, the activity stops
• Therefore rRNA is the catalyst in
peptidyl transferase activity.
• This supports the idea that catalytic
RNA evolved before DNA.
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
• Several ribosomes can work together to
translate the same mRNA, producing
multiple copies of the polypeptide.
• A strand of mRNA with associated
ribosomes is called a polyribosome, or
polysome.
Figure 14.18 A Polysome (A)
Figure 14.18 A Polysome (B)
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
• Posttranslational aspects of protein
synthesis:
• Polypeptide emerges from the ribosome
and folds into its 3-D shape.
• Its conformation allows it to interact with
other molecules—it may contain a
signal sequence indicating where in
the cell it belongs.
8.1 From DNA to protein: gene expression
8.1 From DNA to protein: gene expression
Figure 14.21 A Signal Sequence Moves a Polypeptide into the ER (1)
8.1 From DNA to protein: gene expression
14.6 What Happens to Polypeptides after Translation?
If finished protein enters ER lumen, it
receives signals of two types:
• Sequences of amino acids allow protein
to stay in ER
• Sugars are added—glycoproteins end
up at the plasma membrane, lysosome,
(vacuole in plants), or are secreted
14.6 What Happens to Polypeptides after Translation?
Protein modifications:
• Proteolysis: Cutting of a long polypeptide
chain into final products, by proteases
• Glycosylation: Addition of sugars to form
glycoproteins
• Phosphorylation: Addition of phosphate
groups catalyzed by protein kinases—
charged phosphate groups change the
conformation
15.1 What Are Mutations?
Genetic mutations are changes in the
nucleotide sequences of DNA that are
passed on to the next generation.
Mutations may or may not have a
phenotypic effect.
15.1 What Are Mutations?
Mutations occur in two types:
• Somatic mutations occur in somatic
(body) cells—passed on by mitosis but
not to sexually produced offspring
• Germ line mutations—occur in germ
line cells, the cells that give rise to
gametes. A gamete passes a mutation
on at fertilization
15.1 What Are Mutations?
Mutations have different phenotypic
effects:
• Silent mutations do not affect protein
function
• Loss of function mutations affect
protein function and may lead to
structural proteins or enzymes that no
longer work—almost always recessive
15.1 What Are Mutations?
Mutations have different phenotypic
effects:
• Gain of function mutations lead to a
protein with altered function
• Conditional mutations cause
phenotypes under restrictive conditions
but are not detectable under permissive
conditions
15.1 What Are Mutations?
At the molecular level, mutations or
alterations in the nucleotide sequence
are in two categories:
• A point mutation—results from the
gain, loss, or substitution of a single
nucleotide
• Chromosomal mutations are more
extensive—may change the position or
cause a DNA segment to be duplicated
or lost
8.1 From DNA to protein: gene expression
8.2 Gene mutation and molecular medicine
Mutations occur in two types:
• Somatic mutations occur in somatic
(body) cells—passed on by mitosis but
not to sexually produced offspring
• Germ line mutations—occur in germ
line cells, the cells that give rise to
gametes. A gamete passes a mutation
on at fertilization
8.2 Gene mutation and molecular medicine
8.2 Gene mutation and molecular medicine
Figure 15.4 Chromosomal Mutations (A,B)
8.2 Gene mutation and molecular medicine
Figure 15.5 Spontaneous and Induced Mutations (B,C)
8.2 Gene mutation and molecular medicine
8.2 Gene mutation and molecular medicine
8.2 Gene mutation and molecular medicine
• New methods of human genetic
analysis include reverse genetics.
• A clinical phenotype is related to a DNA
variation—then the protein is identified.
• Previously, as in sickle-cell anemia:
• Clinical phenotype→ protein
phenotype→ gene
8.2 Gene mutation and molecular medicine
Mutations may have benefits:
• Provide the raw material for evolution in
the form of genetic diversity
• Diversity may benefit the organism
immediately—if mutation is in somatic
cells
• Or may cause an advantageous change
in offspring
8.2 Gene mutation and molecular medicine
• Most human diseases are
multifactorial—caused by interactions
of many genes and proteins and the
environment.
• Susceptibility to disease is determined
by these complex interactions.
• 60 percent of people are affected by
diseases that are genetically influenced.
8.2 Gene mutation and molecular medicine
• DNA testing is direct analysis of DNA
for mutation; the most accurate way of
detecting an abnormal allele.
• Preimplantation screening of a zygote
can be used for parents of a child with a
disease like cystic fibrosis.
• Fetal cells and newborns can be tested
for sickle-cell disease and others.
8.2 Gene mutation and molecular medicine
Two main approaches to treating genetic
diseases:
• Modifying the disease phenotype
• Replacing the defective gene
8.2 Gene mutation and molecular medicine
Modifying the disease phenotype can be
done in three ways:
• Restricting the substrate—as in PKU,
reducing phenylalanine in the diet
• Metabolic inhibitors, such as drugs
that can target specific proteins
• Supplying the missing protein—blood
factor VIII in hemophilia
8.2 Gene mutation and molecular medicine
•  In gene therapy, the aim is to supply the missing
allele(s) by inserting a new gene that will be
expressed in the host.
•  The challenges: Must find appropriate vector, ensure
precise insertion into host DNA, ensure appropriate
expression, and select cells to target.
•  The nonfunctional alleles cannot be replaced in every
cell of the body.
•  Ex vivo techniques—cells are removed from the
body, new genes inserted in the laboratory, cells
returned to the body so that correct gene products
are made.