Transcript CHAPTER 12 - powerpoint

From DNA to Protein:
Genotype to Phenotype
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From DNA to Protein: Genotype to Phenotype
• One Gene, One Polypeptide
• DNA, RNA, and the Flow of Information
• Transcription: DNA-Directed RNA Synthesis
• The Genetic Code
• Preparation for Translation: Linking RNAs, Amino
Acids, and Ribosomes
• Translation: RNA-Directed Polypeptide Synthesis
• Regulation of Translation
• Posttranslational Events
• Mutations: Heritable Changes in Genes
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One Gene, One Polypeptide
• A gene is defined as a DNA sequence.
• There are many steps between genotype and
phenotype; genes cannot by themselves produce
a phenotype.
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One Gene, One Polypeptide
• In the 1940s, Beadle and Tatum showed that when
an altered gene resulted in an altered phenotype,
that altered phenotype always showed up as an
altered enzyme.
• They experimented with strains of the bread mold
Neurospora: a wild-type, and several mutant strains.
• Their results suggested that mutations cause a
defect in only one enzyme in a metabolic pathway.
• This lead to the one-gene, one-enzyme hypothesis.
Figure 12.1 One Gene, One Enzyme (Part 1)
Figure 12.1 One Gene, One Enzyme (Part 2)
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One Gene, One Polypeptide
• The gene–enzyme connection has undergone
several modifications. Some enzymes are
composed of different subunits coded for by
separate genes.
• This suggests, instead of the one-gene, one
enzyme hypothesis, a one-gene, onepolypeptide relationship.
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DNA, RNA, and the Flow of Information
• The expression of a gene takes place in two
steps:
 Transcription makes a single-stranded RNA
copy of a segment of the DNA.
 Translation uses information encoded in the
RNA to make a polypeptide.
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DNA, RNA, and the Flow of Information
• RNA (ribonucleic acid) differs from DNA in three
ways:
 RNA consists of only one polynucleotide
strand.
 The sugar in RNA is ribose, not deoxyribose.
 RNA has uracil instead of thymine.
• RNA can base-pair with single-stranded DNA
(adenine pairs with uracil instead of thymine) and
also can fold over and base-pair with itself.
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DNA, RNA, and the Flow of Information
• Francis Crick’s central dogma stated that DNA
codes for RNA, and RNA codes for protein.
• How does information get from the nucleus to the
cytoplasm?
• What is the relationship between a specific
nucleotide sequence in DNA and a specific amino
acid sequence in protein?
Figure 12.2 The Central Dogma
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DNA, RNA, and the Flow of Information
• Messenger RNA, or mRNA moves from the
nucleus of eukaryotic cells into the cytoplasm,
where it serves as a template for protein synthesis.
• Transfer RNA, or tRNA, is the link between the
code of the mRNA and the amino acids of the
polypeptide, specifying the correct amino acid
sequence in a protein.
Figure 12.3 From Gene to Protein
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DNA, RNA, and the Flow of Information
• Certain viruses use RNA rather than DNA as their
information molecule during transmission.
• These viruses transcribe from RNA to RNA; they
make a complementary RNA strand and then use
this “opposite” strand to make multiple copies of
the viral genome by transcription.
• HIV and certain tumor viruses (called retroviruses)
have RNA as their infectious information
molecule; they convert it to a DNA copy inside the
host cell and then use it to make more RNA.
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Transcription: DNA-Directed RNA Synthesis
• In normal prokaryotic and eukaryotic cells,
transcription requires the following:
 A DNA template for complementary base
pairing
 The appropriate ribonucleoside triphosphates
(ATP, GTP, CTP, and UTP) to act as substrates
 The enzyme RNA polymerase
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Transcription: DNA-Directed RNA Synthesis
• Just one DNA strand (the template strand) is used
to make the RNA.
• For different genes in the same DNA molecule,
the roles of these strands may be reversed.
• The DNA double helix partly unwinds to serve as
template.
• As the RNA transcript forms, it peels away,
allowing the already transcribed DNA to be
rewound into the double helix.
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Transcription: DNA-Directed RNA Synthesis
• The first step of transcription, initiation, begins at
a promoter, a special sequence of DNA.
• There is at least one promoter for each gene to be
transcribed.
• The RNA polymerase binds to the promoter
region when conditions allow.
• The promoter sequence directs the RNA
polymerase as to which of the double strands is
the template and in what direction the RNA
polymerase should move.
Figure 12.4 (Part 1) DNA is Transcribed in RNA
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Transcription: DNA-Directed RNA Synthesis
• After binding, RNA polymerase unwinds the DNA
about 20 base pairs at a time and reads the
template in the 3-to-5 direction (elongation).
• The new RNA elongates from its 5 end to its 3
end; thus the RNA transcript is antiparallel to the
DNA template strand.
• Transcription errors for RNA polymerases are high
relative to DNA polymerases.
Figure 12.4 (Part 2) DNA is Transcribed in RNA
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Transcription: DNA-Directed RNA Synthesis
• Particular base sequences in the DNA specify
termination.
• Gene mechanisms for termination vary:
 For some, the newly formed transcript simply
falls away from the DNA template.
 For other genes, a helper protein pulls the
transcript away.
 In prokaryotes, translation of the mRNA often
begins before transcription is complete.
Figure 12.4 (Part 3) DNA is Transcribed in RNA
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The Genetic Code
• A genetic code relates genes (DNA) to mRNA and
mRNA to the amino acids of proteins.
• mRNA is read in three-base segments called
codons.
• The number of different codons possible is 64
(43), because each position in the codon can be
occupied by one of four different bases.
• The 64 possible codons code for only 20 amino
acids and the start and stop signals.
Figure 12.5 The Universal Genetic Code
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The Genetic Code
• AUG, which codes for methionine, is called the
start codon, the initiation signal for translation.
• Three codons (UAA, UAG, and UGA) are stop
codons, which direct the ribosomes to end
translation.
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The Genetic Code
• After subtracting start and stop codons, the
remaining 60 codons code for 19 different amino
acids.
• This means that many amino acids have more
than one codon. Thus the code is redundant.
• However, the code is not ambiguous. Each codon
is assigned only one amino acid.
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The Genetic Code
• In the early 1960s, molecular biologists broke the
genetic code.
• Nirenberg prepared an artificial mRNA in which all
bases were uracil (poly U).
• When incubated with additional components, the
poly U mRNA led to synthesis of a polypeptide chain
consisting only of phenylalanine amino acids.
• UUU appeared to be the codon for phenylalanine.
• Other codons were deciphered from this starting
point.
Figure 12.6 Deciphering the Genetic Code
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Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
• The molecule tRNA is required to assure specificity
in the translation of mRNA into proteins.
• The tRNAs must read mRNA correctly.
• The tRNAs must carry the correct amino acids.
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Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
• The codon in mRNA and the amino acid in a
protein are related by way of an adapter—a
specific tRNA molecule.
• tRNA has three functions:
 It carries an amino acid.
 It associates with mRNA molecules.
 It interacts with ribosomes.
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Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
• A tRNA molecule has 75 to 80 nucleotides and a
three-dimensional shape (conformation).
• The shape is maintained by complementary base
pairing and hydrogen bonding.
• The three-dimensional shape of the tRNAs allows
them to combine with the binding sites of the
ribosome.
Figure 12.7 Transfer RNA
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Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
• At the 3 end of every tRNA molecule is a site to
which its specific amino acid binds covalently.
• Midpoint in the sequence are three bases called the
anticodon.
• The anticodon is the contact point between the
tRNA and the mRNA.
• The anticodon is complementary (and antiparallel)
to the mRNA codon.
• The codon and anticodon unite by complementary
base pairing.
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Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
• Amino acids are attached to the correct tRNAs by
activating enzymes called aminoacyl-tRNA
synthetases.
• The enzyme has a three-part active site that binds:
 A specific amino acid
 ATP
 A specific tRNA, charged with a high-energy
bond
• The high-energy bond provides the energy for
making the peptide bond.
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Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
• The reactions have two steps:
 Enzyme + ATP + AA  enzyme—AMP—AA + PPi
 Enzyme—AMP—AA + tRNA  enzyme + AMP +
tRNA—AA
Figure 12.8 Charging a tRNA Molecule
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Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
• Each ribosome has two subunits: a large one and
a small one.
• In eukaryotes the large one has three different
associated rRNA molecules and 45 different
proteins.
• The small subunit has one rRNA and 33 different
protein molecules.
• When they are not translating, the two subunits
are separate.
Figure 12.9 Ribosome Structure
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Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
• The proteins and rRNAs are held together by ionic
bonds and hydrophobic forces.
• The large subunit has four binding sites:
 The T site where the tRNA first lands
 The A site where the tRNA anticodon binds to
the mRNA codon
 The P site where the tRNA adds its amino acid to
the polypeptide chain
 The E site where the tRNA goes before leaving
the ribosome
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Preparation for Translation:
Linking RNAs, Amino Acids, and Ribosomes
• The small ribosomal subunit plays a role in
validating the three-base-pair match between the
mRNA and the tRNA.
• If hydrogen bonds have not formed between all
three base pairs, the tRNA is ejected from the
ribosome.
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Translation: RNA-Directed Polypeptide Synthesis
• Translation begins with an initiation complex: a
charged tRNA with its amino acid and a small
subunit, both bound to the mRNA.
• This complex is bound to a region upstream of
where the actual reading of the mRNA begins.
• The start codon (AUG) designates the first amino
acid in all proteins.
• The large subunit then joins the complex.
• The process is directed by proteins called
initiation factors.
Figure 12.10 The Initiation of Translation
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Translation: RNA-Directed Polypeptide Synthesis
• Ribosomes move in the 5-to-3 direction on the
mRNA.
• The peptide forms in the N–to–C direction.
• The large subunit catalyzes two reactions:
 Breaking the bond between the tRNA in the P
site and its amino acid
 Peptide bond formation between this amino acid
and the one attached to the tRNA in the A site
• This is called peptidyl transferase activity.
Figure 12.11 Translation: The Elongation Stage
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Translation: RNA-Directed Polypeptide Synthesis
• After the first tRNA releases methionine, it
dissociates from the ribosome and returns to the
cytosol.
• The second tRNA, now bearing a dipeptide,
moves to the P site.
• The next charged tRNA enters the open A site.
• The peptide chain is then transferred to the P site.
• These steps are assisted by proteins called
elongation factors.
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Translation: RNA-Directed Polypeptide Synthesis
• When a stop codon—UAA, UAG, or UGA—
enters the A site, a release factor and a water
molecule enter the A site, instead of an amino
acid.
• The newly completed protein then separates from
the ribosome.
Figure 12.12 The Termination of Translation
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Regulation of Translation
• Antibiotics are defensive molecules produced by
some fungi and bacteria, which often destroy
other microbes.
• Some antibiotics work by blocking the synthesis of
the bacterial cell walls, others by inhibiting protein
synthesis at various points.
• Because of differences between prokaryotic and
eukaryotic ribosomes, the human ribosomes are
unaffected.
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Regulation of Translation
• Polysomes are mRNA molecules with more than
one ribosome attached.
• These make protein more rapidly, producing
multiple copies of protein simultaneously.
Figure 12.13 A Polysome (Part 1)
Figure 12.13 A Polysome (Part 2)
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Posttranslational Events
• Two posttranslational events can occur after the
polypeptide has been synthesized:
 The polypeptide may be moved to another
location in the cell, or secreted.
 The polypeptide may be modified by the
addition of chemical groups, folding, or
trimming.
Figure 12.14 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell
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Posttranslational Events
• As the polypeptide chain forms, it folds into its 3-D
shape.
• The amino acid sequence also contains an
“address label” indicating where in the cell the
polypeptide belongs. It gives one of two sets of
instructions:
 Finish translation and be released to the
cytoplasm.
 Stall translation, go to the ER, and finish
synthesis at the ER surface.
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Posttranslational Events
• Polypeptides sent to the cytoplasm may contain
information (signal sequences) that specifies a
destination.
• The signal sequence binds to docking proteins
at the outer membrane of the appropriate
organelle.
• A channel opens in the membrane, allowing the
protein to pass through.
• In the process, the protein usually is unfolded by a
chaperonin so that it can pass through the
channel.
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Posttranslational Events
• Polypeptides destined for the ER have a 25amino-acid-long leader sequence.
• Before translation is finished, the leader sequence
binds to a signal recognition particle.
• This stalls protein synthesis until the ribosome
attaches to a specific receptor protein on the
surface of the ER.
• Translation continues with the protein moving
through a pore in the ER membrane.
Figure 12.15 A Signal Sequence Moves a Polypeptide into the ER (Part 1)
Figure 12.15 A Signal Sequence Moves a Polypeptide into the ER (Part 2)
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Posttranslational Events
• Other signals are needed to direct further protein
sorting:
 Sequences of amino acids that allow the
protein to stay in the ER
 Sugars added in the Golgi apparatus to form
glycoproteins, which go to lysosomes or the
plasma membrane
• Proteins with no signals from the ER go through
the Golgi apparatus and are secreted from the
cell.
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Posttranslational Events
• Most proteins are modified after translation.
• These modifications are often essential to the
functioning of the protein.
• Three types of modifications:
 Proteolysis (cleaving)
 Glycosylation (adding sugars)
 Phosphorylation (adding phosphate groups)
Figure 12.16 Posttranslational Modifications to Proteins
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Mutations: Heritable Changes in Genes
• Mutations are heritable changes in DNA—
changes that are passed on to daughter cells.
• Multicellular organisms have two types of
mutations:
 Somatic mutations are passed on during
mitosis, but not to subsequent generations.
 Germ-line mutations are mutations that occur
in cells that give rise to gametes.
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Mutations: Heritable Changes in Genes
• Some mutations, called conditional mutants,
exert their effect only under certain restrictive
conditions.
• A temperature-sensitive mutant allele, for
example, may code for an enzyme that is altered
at the restrictive temperature.
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Mutations: Heritable Changes in Genes
• All mutations are alterations of the DNA nucleotide
sequence and are of two types:
 Point mutations are mutations of single genes.
 Chromosomal mutations are changes in the
arrangements of chromosomal DNA segments.
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Mutations: Heritable Changes in Genes
• Point mutations result from the addition or
subtraction of a base or the substitution of one base
for another.
• Point mutations can occur as a result of mistakes
during DNA replication or can be caused by
environmental mutagens.
• Because of redundancy in the genetic code, some
point mutations, called silent mutations, result in
no change in the amino acids in the protein.
Silent Mutation
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Mutations: Heritable Changes in Genes
• Some mutations, called missense mutations,
cause an amino acid substitution.
• An example in humans is sickle-cell anemia, a
defect in the b-globin subunits of hemoglobin.
• The b-globin in sickle-cell differs from the normal
by only one amino acid.
• Missense mutations may reduce the functioning of
a protein or disable it completely.
Missense mutation
Figure 12.17 Sickled and Normal Red Blood Cells
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Mutations: Heritable Changes in Genes
• Nonsense mutations are base substitutions that
substitute a stop codon.
• The shortened proteins are usually not functional.
Nonsense mutation
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Mutations: Heritable Changes in Genes
• A frame-shift mutation consists of the insertion
or deletion of a single base in a gene.
• This type of mutation shifts the code, changing
many of the codons to different codons.
• These shifts almost always lead to the production
of nonfunctional proteins.
Frame-shift mutation
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Mutations: Heritable Changes in Genes
• DNA molecules can break and re-form, causing four
different types of mutations:
 Deletions are a loss of a chromosomal segment.
 Duplications are a repeat of a segment.
 Inversions result from breaking and rejoining
when segments get reattached in the opposite
orientation.
 Translocations result when a portion of one
chromosome attaches to another.
Figure 12.18 Chromosomal Mutations (Part 1)
Figure 12.18 Chromosomal Mutations (Part 2)
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Mutations: Heritable Changes in Genes
• Spontaneous mutations are permanent
changes, caused by any of several mechanisms:
 Nucleotides occasionally change their
structure (called a tautomeric shift).
 Bases may change because of a chemical
reaction.
 DNA polymerase sometimes makes errors in
replication which can escape being repaired.
 Meiosis is imperfect. Nondisjunction and
translocations can occur.
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Mutations: Heritable Changes in Genes
• Induced mutations are permanent changes caused
by some outside agent (mutagen).
• Mutagens can alter DNA in several ways:
 Altering covalent bonds in nucleotides
 Adding groups to the bases
 Radiation damages DNA:

Ionizing radiation (X rays) produces free
radicals.

Ultraviolet radiation is absorbed by thymine
and causes interbase covalent bonds to form.
Figure 12.19 Spontaneous and Induced Mutations (Part 1)
Figure 12.19 Spontaneous and Induced Mutations (Part 2)
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Mutations: Heritable Changes in Genes
• Mutations have both benefits and costs.
• Germ line mutations provide genetic diversity for
evolution, but usually produce an organism that
does poorly in its environment.
• Somatic mutations do not affect offspring, but can
cause cancer.
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Mutations: Heritable Changes in Genes
• Mutations are rare events and most of them are
point mutations involving one nucleotide.
• Different organisms vary in mutation frequency.
• Mutations can be detrimental, neutral, or
occasionally beneficial.
• Random accumulation of mutations in the extra
copies of genes can lead to the production of new
useful proteins.