Download DNA to Protein

DNA to Protein
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DNA, RNA, and the Flow of Information
DNA is the genetic material but proteins are the
executors.
How is the genetic information translated into
function?
• 1958 -Francis Crick’s central dogma stated that
DNA codes for RNA, and RNA codes for protein.
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DNA to Protein Flow of Information
• DNA is an information molecule. The information is
stored in the order of the four different bases.
• DNA sequence codes for amino-acid sequence in
proteins
• A genetic code relates genes (DNA) to the amino acids
of proteins.
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Genes, the units of heredity
• Genes are the units of heredity
• A gene caries the information for a full polypeptide
(Typically, one gene=one polypeptide).
• Genes are segments of DNA
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DNA, RNA, and the Flow of Information
• The expression of a genetic information
takes place in two steps:
ƒ Transcription makes a singlestranded RNA copy of a segment of
the DNA.
ƒ Translation uses information
encoded in the RNA to make a
polypeptide.
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DNA, RNA, and the Flow of Information
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.
DNA
RNA
Deoxyribose
Ribose
Thymine
Uracil
Double strand
Single strand
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Transcription: DNA-Directed RNA Synthesis
Transcription
DNA
DNA
RNA
DNA
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RNA Polymerase
• Transcription requires:
ƒ A DNA template for complementary base pairing
ƒ The appropriate ribonucleoside triphosphates (ATP, GTP,
CTP, and UTP)
ƒ 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.
• The DNA double helix partly unwinds to serve as template.
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Transcription Initiation
• Three stages of transcription: Initiation, elongation,
termination.
• 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.
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Transcription: Elongation
• 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.
• 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: Termination
• Particular base sequences in the DNA specify
termination.
• Gene mechanisms for termination vary:
ƒ For some, the newly formed transcript simply falls
away from the DNA template.
ƒ For other genes, a helper protein pulls the transcript
away.
ƒ In prokaryotes, translation of the mRNA often begins
before transcription is complete.
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Promoter sequence and transcription initiation
• The first step of transcription, initiation, begins at
a promoter, a special sequence of DNA.
• 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.
Start signal
5’GCACTCTACTATATTCTCAATAGGTCCACG3’
3’CGTGAGATGATATAAGAGTTATCCAGGTGC5’
Template DNA
Start site
5’
3’ Transcription
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Promoter sequence and transcription initiation
• Pre-initiation complex
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TATA Binding protein
• DNA binding factors are proteins that bind DNA
through non-covalent interactions
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Transcription termination
• Particular base sequences in the DNA specify
termination. (Hairpin structure)
CCCACAGCCGCCAGTTCCGCTGGCGGCATTTTAA
GGGTGTCGGCGGTCAAGGCGACCGCCGTAAAATT
Stop signal
(dsDNA)
Transcription
CGGCGGUC
CCCACA
GCCGCCAG
CCCACAGCCGCCAGUUCCGCUGGCGGCAUUUUU
(RNAss)
(Hairpin RNA structure)
AUUUU
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Transcription results in amplification
• Many RNA molecules are produced in parallel from a
single DNA template
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Genome organization
• A gene should carry all the information for a full
polypeptide including information for defining initiation and
termination
• 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.
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Messenger RNA
• Gene transcription generates messenger RNA
(mRNA) which will be translated into protein
• mRNA ends are chemically modified
One RNA = one proteins
One RNA = several proteins
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Translation
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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.
AUGGGCAUGCCU
RNA
Translation
Met-Gly-Met-Pro
Protein
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The Genetic Code
• 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.
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DNA, RNA, and the Flow of Information
• The 64 possible codons code for only 20 amino acids and
the start and stop signals.
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The Genetic Code
• 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
• Three reading frames for genetic information
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mRNA structure
• 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.
• mRNA contains a coding sequence as well as
UnTranslated Regions (UTRs)
Start
(AUG)
UTR
Stop
UTR
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Breaking the Genetic Code
• In the early 1960s, molecular biologists used Invitro translation to brake 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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Translation machinery: Ribosomes
• Ribosomes are protein-RNA complexes that
translate RNA to protein.
Ribosome
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tRNA adaptor molecule
• 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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tRNA: adaptor molecules
• 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:
ƒ It carries an amino acid.
ƒ It associates with mRNA molecules.
ƒ It interacts with ribosomes.
tRNA
mRNA
Ribosome
Amino Acid
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tRNA: adaptor molecules
• A tRNA molecule has 75 to 80 nucleotides and a threedimensional 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.
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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.
mRNA
Ribosome
tRNA
Amino Acid
Protein
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Ribosome structure
• Each ribosome has two subunits: a large one and
a small one.
• In eukaryotes the ribosome has four different
associated rRNA molecules and 45 different
proteins.
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Preparation for Translation:
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Linking RNAs, Amino Acids, and Ribosomes
• The large subunit has four binding sites:
ƒ The T site where the tRNA first lands
ƒ The A site where the tRNA anticodon binds to
the mRNA codon
ƒ The P site where the tRNA adds its amino acid to
the polypeptide chain
ƒ The E site where the tRNA goes before leaving
the ribosome
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Preparation for Translation:
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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:
ƒ Breaking the bond between the tRNA in the P
site and its amino acid
ƒ 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
(Directions)
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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Polysomes
• Polysomes are mRNA molecules with more than
one ribosome attached.
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Polysomes
• Polysomes make protein more rapidly, producing
multiple copies of protein simultaneously.
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Classes of RNA
• 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.
• Ribosomal RNA, or rRNA, Functional component of
Ribosomes
rRNA
mRNA
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Inhibition of translation
• Some antibiotics work by inhibiting protein
synthesis at various points.
• Because of differences between prokaryotic and
eukaryotic ribosomes, the human ribosomes are
unaffected.
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DNA and the Flow of Information
• DNA stores information and is used to transmit
heritable information
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DNA, RNA, and the Flow of Information
• The expression of a genetic
information takes place in two
steps:
• Transcription makes a singlestranded 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
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One Gene, One Polypeptide
• A gene is defined as a DNA sequence.
• A one-gene, one-polypeptide relationship.
Figure 12.5 The Universal Genetic Code
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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:
ƒ Somatic mutations are passed on during
mitosis, but not to subsequent generations.
ƒ Germ-line mutations are mutations that occur
in cells that give rise to gametes.
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Mutations: Heritable Changes in Genes
• Changes in DNA sequence can lead to changes in
Protein level or function and lead to change in cell
function.
• Mutations are alterations of the DNA nucleotide
sequence and are of two types:
ƒ Point mutations are mutations of single sites.
ƒ Chromosomal mutations are changes in the
arrangements of chromosomal DNA segments.
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Point Mutations
• 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.
Figure 12.5 The Universal Genetic Code
Silent Mutation
• Because of redundancy in the genetic code, some
point mutations, called silent mutations, result in no
change in the amino acids in the protein.
Wild type
DNA template 3′
strand 5′
5′
3′
mRNA 5′
3′
Protein
Stop
Amino end
Carboxyl end
A instead of G
5′
3′
3′
5′
U instead of C
5′
3′
Stop
Silent (no effect on amino acid sequence)
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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 β-globin subunits of hemoglobin.
• The β-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
Wild type
DNA template 3′
strand 5′
5′
3′
mRNA 5′
3′
Protein
Stop
Amino end
Carboxyl end
T instead of C
5′
3′
3′
5′
A instead of G
3′
5′
Stop
Missense
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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.
Wild type
DNA template 3′
strand 5′
5′
3′
mRNA 5′
3′
Protein
Stop
Amino end
Carboxyl end
A instead of T
3′
5′
5′
3′
U instead of A
5′
3′
Stop
Nonsense
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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
Wild type
DNA template 3′
strand 5′
5′
3′
mRNA 5′
3′
Protein
Stop
Amino end
Carboxyl end
Extra A
5′
3′
3′
5′
Extra U
5′
3′
Stop
Frameshift causing immediate nonsense (1 base-pair insertion)
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Chromosomal mutations
• DNA molecules can break and re-form, causing four
different types of mutations:
ƒ Deletions are a loss of a chromosomal segment.
ƒ Duplications are a repeat of a segment.
ƒ Inversions result from breaking and rejoining
when segments get reattached in the opposite
orientation.
ƒ Translocations result when a portion of one
chromosome attaches to another.
Figure 12.18 Chromosomal Mutations (Part 1)
DNA molecules can break and re-form, causing four different
types of mutations:
ƒDeletions are a loss of a chromosomal segment.
ƒDuplications are a repeat of a segment.
Figure 12.18 Chromosomal Mutations (Part 2)
ƒInversions result from breaking and rejoining when
segments get reattached in the opposite orientation.
ƒTranslocations result when a portion of one
chromosome attaches to another.
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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:
ƒ Altering covalent bonds in nucleotides
ƒ Adding groups to the bases
ƒ 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)
Guanine
Benzo(a)pyrene
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T
T
U.V
5’--C C G A ATTC
tt A G--3’
3’--G G CTTA A GT C --5’
Frame Shift
Thymine Dimer
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Mutations: Heritable Changes in Genes
• Most mutations are repaired by cellular mechanisms.
1012Events/min
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Mutations: Heritable Changes in Genes
• DNA replication is a mechanism for introducing spontaneous
mutations and for fixing mutations.
Figure 12.19 Spontaneous and Induced Mutations (Part 2)
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Nucleic Acids: Informational Macromolecules
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That Can Be Catalytic
• Closely related living species have DNA base
sequences that are more similar than distantly
related species.
• The comparative study of base sequences has
confirmed many of the traditional classifications of
organisms.
• DNA comparisons confirm that our closest living
relatives are chimpanzees: We share more than
98 percent of our DNA base sequences.
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06_27_humans_whales.jpg
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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.
• 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.