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12
From DNA to Protein:
Genotype to Phenotype
12 From DNA to Protein: Genotype to Phenotype
• Review
 12.1 What Is the Evidence that Genes
Code for Proteins?
 12.2 How Does Information Flow from
Genes to Proteins?
• 12.3 How Is the Information Content in DNA
Transcribed to Produce RNA?
• 12.4 How Is RNA Translated into Proteins?
• 12.5 What Happens to Polypeptides after
Translation?
• 12.6 What Are Mutations?
12-1 Recap:
• Beadle & Tatum’s studies of
mutations in bread molds led to
the one gene-one polypeptide
hypothesis:
• The function of a gene is to code
for a specific polypeptide
12-1 Recap ?s:
• What’s a “model organism”?
• Why was Neurospora a good model for
studying biochemical genetics?
• How were Beadle and Tatum’s expts set
up to determine, on the basis of
phenotypes of mutant strains, the order
of a biochemical pathway?
• What’s the distinction between
“protein” and “polypeptide”?
12.1 What Is the Evidence that Genes Code for Proteins?
The molecular basis of phenotypes was
known before it was known that DNA is
the genetic material.
Studies of many different organisms
showed that major phenotypic
differences were due to specific
proteins.
12.1 What Is the Evidence that Genes Code for Proteins?
Model organisms:
easy to grow or observe; show the
phenomenon to be studied
results from one organism applied to
others
Examples: pea plants, Drosophila, E. coli,
common bread mold Neurospora crassa
12.1 What Is the Evidence that Genes Code for Proteins?
Bread mold experiments:
Suggested the
one-gene, one-enzyme hypothesis
Figure 12.1 One Gene, One Enzyme (Part 1)
Figure 12.1 One Gene, One Enzyme (Part 2)
each mutant was missing a single enzyme in the pathway.
12.1 What Is the Evidence that Genes Code for Proteins?
The gene-enzyme relationship has been
revised to the one-gene, one-polypeptide
relationship.
Example: In hemoglobin, each polypeptide
chain is specified by a separate gene.
Other genes code for RNA that is not
translated to polypeptides; some genes are
involved in controlling other genes.
12-2 Recap:
• Central Dogma of molec bio:
 DNA codes for the production of RNA, RNA
codes for the production of polypeptides
(proteins)
• Proteins do NOT code for the production of
protein, DNA, or RNA
• Transcription = process that copies a DNA
sequence into mRNA
• Translation = process by which this info is
converted into protein
• tRNA recognizes the genetic info in mRNA and
brings the appropriate amino acid into
position in a growing polypeptide chain
12-2 Recap ?s:
• What’s that central dogma all
about?
• What are the roles of mRNA and
tRNA in gene expression?
12.2 How Does Information Flow from Genes to Proteins?
Expression of a gene to form a polypeptide:
• Transcription—copies information from
gene to a sequence of RNA.
• Translation—converts RNA sequence to
amino acid sequence.
12.2 How Does Information Flow from Genes to Proteins?
RNA, ribonucleic acid differs from DNA:
• Usually one strand
• The sugar is ribose
• Contains uracil (U) instead of thymine (T)
12.2 How Does Information Flow from Genes to Proteins?
RNA can pair with a single strand of
DNA, except that adenine pairs with
uracil instead of thymine.
RNA (CODONS)
Practice! DNA  RNA
Figure 12.2 The Central Dogma
The central dogma of molecular biology:
information flows in one direction when genes
are expressed (Francis Crick).
12.2 How Does Information Flow from Genes to Proteins?
The central dogma 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?
12.2 How Does Information Flow from Genes to Proteins?
One hypothesis—
messenger RNA (mRNA) forms as a
complementary copy of DNA and
carries information to the cytoplasm.
This process is transcription.
Figure 12.3 From Gene to Protein
12.2 How Does Information Flow from Genes to Proteins?
Other hypothesis—an adapter molecule
that can bind amino acids, and
recognize a nucleotide sequence—
transfer RNA (tRNA).
tRNA molecules carrying amino acids
line up on mRNA in proper sequence for
the polypeptide chain—translation.
12.2 How Does Information Flow from Genes to Proteins?
Exception to the central dogma:
Viruses: acellular particles that reproduce
inside cells; many have RNA instead of DNA.
12.2 How Does Information Flow from Genes to Proteins?
Synthesis of DNA from RNA is reverse
transcription.
Viruses that do this are retroviruses.
12-3 Recap:
• Transcription (catalyzed by an
RNA polymerase) proceeds in 3
steps:
• Initiation, elongation, termination
• Genetic code relates the
information in mRNA (linear
sequence of codons) to protein
(linear sequence of amino acids)
12-3 Recap ?s:
• What are the steps of gene
transcription (producing mRNA)?
• How do RNA polymerases work?
• How was the genetic code
deciphered?
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?
Within each gene, only one strand of
DNA is transcribed—the template
strand.
Transcription produces mRNA;
the same process is used to produce
tRNA and rRNA.
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?
RNA polymerases catalyze synthesis of
RNA.
single enzyme-template binding results in
polymerization of hundreds of RNA
bases.
Figure 12.4 RNA Polymerase
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?
Transcription occurs in three phases:
• Initiation
• Elongation
• Termination
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?
Initiation requires a promoter—a
special sequence of DNA.
RNA polymerase binds to it.
tells RNA polymerase where to start,
which direction to go in, and which
strand of DNA to transcribe.
Part of it is the initiation site.
Figure 12.5 DNA Is Transcribed to Form RNA
RNA polymerase binds to the
promoter and starts to
unwind the DNA strands.
RNA polymerase
Initiation site
Complementary strand
Termination site
3′
3′
5′
5′
Rewinding
of DNA
Template
strand
Promoter
Unwinding
of DNA
Figure 12.5 DNA Is Transcribed to Form RNA
RNA polymerase reads the DNA template strand
from 3′ to 5′ and produces the RNA transcript by
adding nucleotides to the 3′ end.
5′
3′
3′
3′
5′
5′
5′
Direction of transcription
Nucleoside triphosphates
(A, U, C, G)
3′
3′
3′
5′
5′
5′
RNA transcript
Figure 12.5 DNA Is Transcribed to Form RNA
When RNA polymerase reaches the
termination site, the RNA transcript is set
free from the template.
3′
3′
5′
5′
5′
3′
RNA
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?
Elongation: RNA polymerase unwinds
DNA about 10 base pairs at a time;
reads template in 3′ to 5′ direction.
The RNA transcript is antiparallel to the
DNA template strand.
RNA polymerases do not proofread and
correct mistakes.
Figure 12.5 DNA Is Transcribed to Form RNA (B)
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?
Termination: specified by a specific DNA
sequence.
Mechanisms of termination are complex
and varied.
Eukaryotes—first product is a pre-mRNA
that is longer than the final mRNA and
must undergo processing.
Figure 12.5 DNA Is Transcribed to Form RNA (C)
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?
The genetic code: specifies which amino
acids will be used to build a protein
Codon: a sequence of three mRNA
bases. Each specifies a particular
amino acid.
Start codon: AUG—initiation signal for
translation
Stop codons: stops translation and
polypeptide is released
Figure 12.6 The Genetic Code
AUG UAC CAU UUA GCC AUC AAC UUU UAC UAU AAU UGA
ANIMATIONS!
• Transcription
• Deciphering the Genetic Code
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?
For most amino acids, there is more than
one codon; the genetic code is
redundant.
But not ambiguous— each codon
specifies only one amino acid.
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?
The genetic code is nearly universal:
codons that specify amino acids are the
same in all organisms!!
Exceptions: within mitochondria and
chloroplasts, and in one group of
protists.
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?
This common genetic code is a common
language for evolution.
 Ancient; has remained intact
 also facilitates genetic engineering
12.3 How Is the Information Content in DNA Transcribed to
Produce RNA?
How was the code deciphered?
20 “code words” (amino acids) are written
with only four “letters.”
Triplet code seemed likely: could account
for 4 × 4 × 4 = 64 codons.
12-4 Recap:
• Key step in protein synthesis:
attachment of amino acid to proper
tRNA (activating enzyme)
• Translation of genetic info from
mRNA into protein occurs @
ribosome
• Multiple ribosomes may act on a
single mRNA to make multiple
copies of the protein for which it
codes
12-4 Recap ?s:
• How is an amino acid attached to a
specific tRNA?
• Why’s it called “second genetic
code”?
• Describe initiation, elongation, and
termination of translation.
12.4 How Is RNA Translated into Proteins?
tRNA: for each amino acid, there’s a
specific type
Functions:
• Carries an amino acid
• Associates with mRNA molecules
• Interacts with ribosomes
Figure 12.8 Transfer RNA
Video: tRNA
12.4 How Is RNA Translated into Proteins?
The conformation (3D shape) of tRNA
results from base pairing (H bonds)
within the molecule.
3′ end is the amino acid attachment
site—binds covalently. Always CCA.
Anticodon: site of base pairing with
mRNA. Unique for each tRNA.
Figure 12.9 Charging a tRNA Molecule
tRNA
The enzyme activates the amino acid,
catalyzing reaction with ATP to form high energy
AMP–amino acid and a pyrophosphate ion.
Activating
enzyme
Pi
Specific
amino acid
(e.g., alanine)
Amino acid site
Pyrophosphate (PPi)
ATP site
Activated
alanine
tRNA site
Alanine
Charged tRNA
Activating
enzyme (aminoacyltRNA synthase) for
a specific amino acid
tRNA bonded
to alanine
Alaninespecific
tRNA
The enzyme then catalyzes a
reaction of the activated amino acid
with the correct tRNA.
The charged tRNA will deliver the
appropriate amino acid to join the
elongating polypeptide product
of translation.
The specificity of the enzyme ensures that
the correct amino acid and tRNA have been
brought together.
Figure 12.9 Charging a tRNA Molecule (Part 2)
12.4 How Is RNA Translated into Proteins?
Amino acid is attached to the 3′ end of
tRNA by an energy-rich bond—
provides energy for synthesis of
peptide bond to join amino acids.
12.4 How Is RNA Translated into Proteins?
Ribosome: holds mRNA and tRNA in
correct positions to allow assembly of
polypeptide chain.
are not specific, they can make any type
of protein.
12.4 How Is RNA Translated into Proteins?
Ribosomes have two subunits:
large and small
In eukaryotes, the large subunit has three molecules of
ribosomal RNA (rRNA) and 45 different proteins in a
precise pattern.
The small subunit has one rRNA and 33 proteins.
12.4 How Is RNA Translated into Proteins?
Subunits are held together by ionic and
hydrophobic forces (not covalent
bonds).
When not active in translation, the
subunits exist separately.
Figure 12.10 Ribosome Structure
12.4 How Is RNA Translated into Proteins?
Large subunit has three tRNA binding
sites:
• A site binds with anticodon of charged
tRNA.
• P site is where tRNA adds its amino
acid to the growing chain.
• E site is where tRNA sits before being
released.
12.4 How Is RNA Translated into Proteins?
H bonds form between anticodon of
tRNA and codon of mRNA.
Small subunit rRNA validates the match—if hydrogen
bonds have not formed between all three base pairs,
it must be an incorrect match, and the tRNA is
rejected.
12.4 How Is RNA Translated into Proteins?
Translation also occurs in three steps:
• Initiation
• Elongation
• Termination
12.4 How Is RNA Translated into Proteins?
Initiation:
An initiation complex forms—
charged tRNA and small ribosomal
subunit, both bound to mRNA.
rRNA binds to recognition site on mRNA,
“upstream” from the start codon.
Figure 12.11 The Initiation of Translation (Part 1)
Figure 12.11 The Initiation of Translation (Part 2)
12.4 How Is RNA Translated into Proteins?
Start codon is AUG; first amino acid is
always methionine, which may be
removed after translation.
The large subunit joins the complex, the
charged tRNA is now in the P site of the
large subunit.
Initiation factors are responsible for
assembly of the initiation complex.
12.4 How Is RNA Translated into Proteins?
Elongation: 2nd charged tRNA enters A
site.
Large subunit catalyzes two reactions:
• Breaks bond between tRNA in P site
and its amino acid.
• Peptide bond forms between that amino
acid and amino acid on tRNA in A site.
Figure 12.12 The Elongation of Translation (Part 1)
Figure 12.12 The Elongation of Translation (Part 2)
12.4 How Is RNA Translated into Proteins?
1st tRNA releases its methionine,
moves to E site
dissociates from ribosome—can then
become charged again.
Elongation occurs as the steps are
repeated, assisted by proteins called
elongation factors.
12.4 How Is RNA Translated into Proteins?
Termination: translation ends when a
stop codon enters the A site.
binds a protein release factor—allows
hydrolysis of bond between polypeptide chain
and tRNA on P site.
Polypeptide chain—C terminus is last amino
acid added.
Figure 12.13 The Termination of Translation (Part 1)
Figure 12.13 The Termination of Translation (Part 2)
Figure 12.13 The Termination of Translation (Part 3)
Table 12.1
Last Animation!
• Protein Synthesis
12.4 How Is RNA Translated into Proteins?
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 12.14 A Polysome (Part 1)
Figure 12.14 A Polysome (Part 2)
12-5 Recap:
12-5 Recap ?s:
12.5 What Happens to Polypeptides after Translation?
Posttranslational aspects of protein
synthesis:
Polypeptide may be moved from
synthesis site to an organelle, or out of
the cell.
Polypeptides are often modified with
more chemical groups.
12.5 What Happens to Polypeptides after Translation?
Polypeptide folds as it emerges from the
ribosome.
The amino acid sequence determines the
pattern of folding.
Amino acid sequence also contains a
signal sequence—an “address label.”
12.5 What Happens to Polypeptides after Translation?
Amino acid sequence gives a set of
instructions:
“Finish translation and send to an
organelle.”
OR
“Stop translation, go to the ER, finish
synthesis there.”
Figure 12.15 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell
12.5 What Happens to Polypeptides after Translation?
If the protein is sent to the ER:
• Signal sequence binds to a signal
receptor particle, before translation is
done.
• Ribosome attaches to a receptor on the
ER, the growing polypeptide chain
passes through the channel.
• An enzyme removes the signal
sequence.
Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER
Rough endoplasmic reticulum
Inside of cell
Ribosome
5′
3′
mRNA
1
Signal sequence
Protein synthesis begins
on free ribosomes in the cytosol.
The signal sequence
is at the N-terminal end of
the polypeptide chain.
Signal
recognition
particle
Receptor
protein
ER
membrane
Interior of RER
Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER
Rough endoplasmic reticulum
5′
3′
2
The polypeptide binds to a
signal recognition particle, and
both bind to a receptor protein
in the membrane of the ER.
Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER
Rough endoplasmic reticulum
3′
3
The signal recognition particle
is released. The signal
sequence passes through a
channel in the receptor.
Enzyme for
removal
Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER
Rough endoplasmic reticulum
3′
4
The signal sequence is
removed by an enzyme
in the ER.
Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER
Rough endoplasmic reticulum
3′
5
The polypeptide continues to
elongate.
Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER
Rough endoplasmic reticulum
3′
6
Translation terminates.
Figure 12.16 A Signal Sequence Moves a Polypeptide into the ER
Rough endoplasmic reticulum
7
The ribosome is released.
The protein folds inside
the ER.
12.5 What Happens to Polypeptides after Translation?
Sugars may be added in Golgi apparatus—
the resulting glycoproteins end up in the
plasma membrane, lysosomes, or
vacuoles.
12.5 What Happens to Polypeptides after Translation?
Protein modifications:
Proteolysis: cutting the polypeptide chain,
by proteases.
Glycosylation: addition of sugars to form
glycoproteins.
Phosphorylation: addition of phosphate
groups by kinases. Charged phosphate
groups change the conformation.
Figure 12.17 Posttranslational Modifications of Proteins
12-6 Recap:
12-6 Recap ?s:
12.6 What Are Mutations?
Somatic mutations occur in somatic
(body) cells. Mutation is passed to
daughter cells, but not to sexually
produced offspring.
Germ line mutations occur in cells that
produce gametes. Can be passed to
next generation.
12.6 What Are Mutations?
Conditional mutants: express
phenotype only under restrictive
conditions.
Example: the allele may code for an
enzyme that is unstable at certain
temperatures.
12.6 What Are Mutations?
All mutations are alterations of the
nucleotide sequence.
Point mutations: change in a single
base pair—loss, gain, or substitution of
a base.
Chromosomal mutations: change in
segments of DNA—loss, duplication, or
rearrangement.
12.6 What Are Mutations?
Point mutations can result from
replication and proofreading errors, or
from environmental mutagens.
Silent mutations have no effect on the
protein because of the redundancy of
the genetic code.
Silent mutations result in genetic diversity
not expressed as phenotype
differences.
12.6 What Are Mutations?
12.6 What Are Mutations?
Missense mutations: base substitution
results in amino acid substitution.
12.6 What Are Mutations?
Sickle allele for human β-globin is a
missense mutation.
Sickle allele differs from normal by only
one base—the polypeptide differs by
only one amino acid.
Individuals that are homozygous have
sickle-cell disease.
Figure 12.18 Sickled and Normal Red Blood Cells
Photo 12.1 Normal human erythrocytes. LM, differential interference contrast.
Photo 12.2 Sickled human erythrocytes (sickle-cell anemia). LM, differential interference contrast.
Photo 12.3 Computer-simulated space-filling model of phenylalanine transfer RNA (tRNA).
12.6 What Are Mutations?
Nonsense mutations: base substitution
results in a stop codon.
12.6 What Are Mutations?
Frame-shift mutations: single bases
inserted or deleted—usually leads to
nonfunctional proteins.
12.6 What Are Mutations?
Chromosomal mutations:
Deletions—severe consequences unless
it affects unnecessary genes or is
masked by normal alleles.
Duplications—if homologous
chromosomes break in different places
and recombine with the wrong partners.
Figure 12.19 Chromosomal Mutations (A, B)
12.6 What Are Mutations?
Chromosomal mutations:
Inversions—breaking and rejoining, but
segment is “flipped.”
Translocations—segment of DNA
breaks off and is inserted into another
chromosome. Can cause duplications
and deletions. Meiosis can be
prevented if chromosome pairing is
impossible.
Figure 12.19 Chromosomal Mutations (C, D)
12.6 What Are Mutations?
Spontaneous mutations—occur with no
outside influence. Several mechanisms:
• Bases can form tautomers—different
forms; rare tautomer can pair with the
wrong base.
• Chemical reactions may change bases
(e.g., loss of amino group).
12.6 What Are Mutations?
• Replication errors—some escape
detection and repair.
• Nondisjunction in meiosis.
12.6 What Are Mutations?
Induced mutation—due to an outside
agent, a mutagen.
Chemicals can alter bases (e.g., nitrous
acid can cause deamination).
Some chemicals add other groups to
bases (e.g., benzpyrene adds a group
to guanine and prevents base pairing).
DNA polymerase will then add any base
there.
12.6 What Are Mutations?
Ionizing radiation such as X-rays create
free radicals—highly reactive—can
change bases, break sugar phosphate
bonds.
UV radiation is absorbed by thymine,
causing it to form covalent bonds with
adjacent nucleotides—disrupts DNA
replication.
Figure 12.20 Spontaneous and Induced Mutations (Part 1)
Figure 12.20 Spontaneous and Induced Mutations (Part 2)
12.6 What Are Mutations?
Mutation provides the raw material for
evolution in the form of genetic diversity.
Mutations can harm the organism, or be
neutral.
Occasionally, a mutation can improve an
organism’s adaptation to its
environment, or become favorable as
conditions change.
12.6 What Are Mutations?
Complex organisms tend to have more
genes than simple organisms.
If whole genes are duplicated, the new
genes would be surplus genetic
information.
Extra copies could lead to the production of
new proteins.
New genes can also arise from
transposable elements (see Chapters 13
and 14).