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Chapter 17
From Gene to Protein
Beadle & Tatum
• Bread mold
• Neurospora crassa
• Created mutants using Xrays
• That differed from the
wild type in their
nutritional needs.
EXPERIMENT:
Control: minimal mediumwild type only- all grow
Class I: minimal medium +1
additional nutrient
Class II: minimal medium +1
additional nutrient
Class III: minimal medium +1
additional nutrient
The mediums the mutant
could grow in revealed the
enzyme pathway and the
defective enzyme.
The “one gene - one enzyme”
hypothesis states :
• The function of a gene is to dictate
the production of a specific
enzyme.
– Exception: Keratin and insulin
are not enzymes.
• Refined to the:
• “One gene - one polypeptide”
hypothesis.
The language of DNA
is universal.
This means that a gene
can be transplanted from
one species to another.
Ex.
Firefly gene in a tobacco
plant genome.
Ex.
Gene for human insulin in
a bacterium.
Question:
How exactly does a gene end up
making a protein?
Tell your friend in as much detail as
you can in the next 2 minutes. Then
switch… let your friend tell you what
else they know.
THE ANSWER IS CALLED
“The central dogma of modern biology”:
DNA --> RNA --> PROTEIN
1. DNA is used to construct 3 kinds of RNA.
2. 3 kinds of RNA work together to construct protein.
1st TRANSCRIPTION
DNA --> RNA
synthesis of RNA under the
direction of DNA (rewritten instructions)
3 steps:
1. Initiation
2. Elongation
3. termination
nd
2
TRANSLATION
RNA --> PROTEIN
synthesis of a polypeptide
under the direction of mRNA
(change in the language)
3 steps:
1. Initiation
2. Elongation
3. termination
If DNA & RNA are both
made of only 4 kinds of
nucleotides, how can they
“code” for PROTEINS, which
are made of 20 different
kinds of amino acids?
IT’S A TRIPLET CODE!
DNA”triplet”
corresponds to a
mRNA “codon”
corresponds to a
tRNA “anticodon”
Each tRNA carries a single
amino acid…
this “covers” the 20 aa
How does it work?
X NO 41=4
X NO 42=16
YES!!! 43=64
64 combinations of 3
nucleotides!!!! The genetic
code is “redundant”.
How are DNA and RNA different?
1) Sugar
• Deoxyribose in
DNA
• Ribose in RNA
2) Shape
• Double stranded
in DNA
• Single stranded
in RNA
3) Base
• Thymine in DNA
• Uracil in RNA
Different types of RNA:
1) Messenger RNA
(mRNA)
•
•
•
•
Single strand of RNA
Synthesized complementary
strand to a gene.
Provides the template used
for sequencing amino acids
into a polypeptide.
Triplet group of three
adjacent nucleotides on the
mRNA, called a codon,
codes for one specific
amino acid.
THE GENETIC CODE (mRNA CODONS)
If the gene sequence
Reads:
3’AAATTTCCCGGG5’
The mRNA Reads:
5’UUUAAAGGGCCC3’
Therefore,
Amino Acid seq is:
___ ___ ___ ___
THE GENETIC CODE (mRNA CODONS)
If the gene sequence
Reads:
3’AAATTTCCCGGG5’
The mRNA Reads:
5’UUUAAAGGGCCC3’
Therefore,
Amino Acid seq is:
Phe Lys Gly Pro
2) Transfer RNA (tRNA)
•
•
•
•
•
•
Short RNA molecules (about 80
nucleotides) used for transporting
amino acids to their proper place on
the mRNA template.
Arranged in a hairpin or clover-leaf
shape.
The 3’ end of the tRNA (-C-C-A-3’) is
the amino acid attachment site.
Another portion, specified by a triplet
combination of nucleotides, is the
anticodon.
Anticodon of tRNA is complementary
to the codon of mRNA during
translation.
Exact base pairing of 3rd nucleotide is
not always required- called “wobble”
effect.
TRANSFER RNA tRNA
Figure 17.13b The structure of transfer RNA (tRNA)
3) Ribosomal RNA (rRNA)
• Building blocks of ribosomes.
• Coordinates the activities of mRNA
and tRNA during translation.
• Ribosomes have 2 subunits: large &
small
• Three/Four binding sites:
1) one for the mRNA,
2) one for a tRNA that carries a
growing polypeptide chain (P site),
3) one for a second tRNA that
delivers the next amino acid (A site).
4) ??? *Also (E site) or exit site… the
P site becomes the E site when
the ribosome moves down the
mRNA
Figure 17.16 Structure of the large ribosomal subunit at the atomic level
TRANSCRIPTION IS BUILDING MOLECULES OF RNA
BASED ON THE DNA TEMPLATE (GENES).
How is
transcription
different in
prokaryotic cells
and in eukaryotic
cells?
In prokaryotes:
1)
The promoter
sequence, segment of
DNA that the RNA
polymerase attaches
to, may be blocked by
proteins at its operator.
2) While RNA polymerase
transcribes mRNA the
mRNA is immediately
translated to a
polypeptide (without
additional processing)
SIMULTANEOUS
TRANSCRIPTION &
TRANSLATION…
3) Polyribosomes (multiple)
TRANSCRIPTION AND TRANSLATION
COUPLED IN BACTERIA
Transcription In Eukaryotes:
Initiation; Elongation; Termination
Transcription In Eukaryotes:
Initiation; Elongation; Termination
1) INITIATION
• The promoter sequence is
a region before the
actual gene. Within it, the
T-A-T-A (TATA box) binds
(a/or multiple)
transcription factor (s).
• NEXT, RNA polymerase
binds to this complex. It
unzips the DNA into two
strands temporarily.
2) ELONGATION
•
•
is synthesis of the mRNA molecule 5’ --> 3’
by RNA polymerase II at the start point
(on the 3’ end of the gene).
Transcription progresses at a rate of 60
nucleotides per second.
3) Termination
•
•
•
occurs when the RNA
polymerase reaches a
special sequence of DNA
nucleotides that serve as a
termination point- terminator.
The termination sequence is
an inverted repeat of GC-rich
sequence followed by 4 or
more adenosines
(AAAAAA)m
RNA Polymerase is released
from the DNA template.
IN EUKARYOTES the mRNA is MODIFIED before
leaving the nucleus (not in prokaryotes):
a.
b.
c.
The 5’ end: 5’ cap (P-P-P-G-5’) modified guanosine
tri-phosphate GTP provides stability to the mRNA
and a point of attachment for the ssu of the
ribosome
The 3’ end: poly-A-tail (150-200 adenosines)
provides stability and controls the movement of the
mRNA across the nuclear envelope.
Splicing (see next slide for details.)
RNA processing: 5’ cap & poly A tail
c) Sections of the primary transcript
are removed others are bonded
together! RNA splicing
• The primary mRNA transcript has
sections called: introns & exons
(unedited, called “heterogenous
nuclear RNA”)
• Introns are… intervening
sequences that are noncoding.
• Exons are… sequences that
express a code for a
polypeptide.
• A RNA/protein complex called a
snRNP “snurp” (small nuclear
ribonucleoprotein)
The spliceosome
deletes out the introns
• Several snRNP’s and additional
proteins create a spliceosome.
and splices the exons
together.
Figure 17.9 RNA processing: RNA splicing
Figure 17.11 Correspondence between exons and protein domains
A problem from
the HUMAN GENOME
PROJECT was that many
fewer genes were
discovered
than proteins.
So 1 gene - 1 protein is
wrong???
Alternative
splicing can
result in multiple
proteins created
by one gene.
Transcription:
initiation, elongation, termination
TRANSLATION
• Occurs… on
ribosomes outside of
the nucleus.
• Ribosomes consist of
rRNA and proteins.
• The information on
mRNA is read off in
3’s (codon).
• Polypeptides (amino
acid chains) are
assembled.
Translation
Initiation Stage
1)
2)
3)
4)
The 5’ end of mRNA (5’cap) attracts the small ribosomal
subunit. Attachment.
A molecule of tRNA, with the complementary
anticodon: UAC hydrogen bonds to the mRNA start
codon: AUG.
The 1st amino acid on the tRNA is methionine
The large sub-unit of the ribosome attaches forming a
complete ribosome with the methionine tRNA & mRNA
The 1st tRNA is now occupying the P site.
Elongation Stage
5) The next codon is read off as
another tRNA (bearing an
amino acid) binds to the A
site of the ribosome.
6) The amino acids attached
to the the P-site and A-site
tRNA’s are peptide
bonded together.
7) The ribosome translocates
(moves) the tRNA in the A
site (containing the
polypeptide chain) to the
8)The ribosome shifts the
P site. The P site tRNA
mRNA through, one codon
moves to the E site and
at a time… translocation.
exits. The A site is now open
again for a new tRNA to bring
the next AA.
Termination Stage
9) Translation continues until a ribosome
encounters one of three “stop” codons:
UAA, UAG, UGA (cave man talk?)
• Which all bind RELEASE FACTOR instead of an
actual tRNA.
• The completed polypeptide, the last tRNA, and
the two ribosomal subunits are released.
Translation:
initiation, elongation,termination
WHAT HAPPENS WHEN WE HAVE
ERRORS IN THE DNA CODE????
MUTATIONS!!!!
POINT MUTATIONS
1) Substitution- switched pair of
nucleotides for the right pair.
Can switch the identity of a
single amino acid.
• Since most amino acids
have multiple codons, they
don’t always result in a
mutation!!!
• ie: GAU and GAC both
code for Asparagine
• GCU, GCC, GCG, and GCA
all code for Alanine!
DNA Mutations That Alter Translation:
Missense mutation
• base-pair substitution of one
nucleotide and its partner for
another pair.
• altered codon still codes for
an amino acid (still makes
sense) but not necessarily the
right sense.
• Does not always alter the ex. Sickle Cell Anemia
protein.
is caused by a substitution
of A for T,
therefore Valine instead of
Glutamic Acid,
in the polypeptide chain
alters the shape of hemoglobin.
Nonsense Mutation
• Alterations that change
an amino acid codon to
a stop signal.
• Translation will be
terminated prematurely.
• The polypeptide will be
shorter than it should be.
• Always leads to a
nonfunctional protein.
2) Insertion/Deletion
•
•
•
Frameshift mutations are
caused by insertions or
deletions of nucleotide
pairs in a gene.
since the mRNA is read as
a series of nucleotide
triplets- insertions or
deletions of more than or
fewer than 3 nucleotide
pairs results in a change of
the reading frame.
This changes the identity
of all the amino acids
downstream of the
insertion/deletion.
Figure 17.25 A summary of transcription and translation in a eukaryotic cell
The processing of genetic information is
imperfect and is a source of genetic variation
Changes in genotype can result in changes in phenotype.
1. Alterations in a DNA sequence can lead to changes in the type or
amount of the protein produced and the consequent phenotype.
2. DNA mutations can be positive, negative or neutral based on the
effect or the lack of effect they have on the resulting nucleic acid
or protein and the phenotypes that are conferred by the protein.
3. Errors in DNA replication or DNA repair mechanisms, and external
factors, including radiation and reactive chemicals, can cause
random changes, e.g., mutations in the DNA.
4. Changes in genotype may affect phenotypes that are subject to
natural selection. Genetic changes that enhance survival and
reproduction can be selected by environmental conditions.
Evidence:
• Whether or not a mutation is detrimental, beneficial or neutral
depends on the environmental context. Mutations are the primary
source of genetic variation.
• Errors in mitosis or meiosis can result in changes in phenotype.
• Changes in chromosome number often result in new phenotypes,
including sterility caused by triploidy and increased vigor of other
polyploids.
• Changes in chromosome number often result in human disorders
with developmental limitations, including Trisomy 21 (Down
syndrome) and XO (Turner syndrome).
• Antibiotic resistance mutations
• Pesticide resistance mutations
• Sickle cell disorder and heterozygote advantage
• Selection results in evolutionary change.
Learning Objectives:
• LO 3.24 The student is able to predict how a change in
genotype, when expressed as a phenotype, provides
a variation that can be subject to natural selection.
[See SP 6.4, 7.2]
• LO 3.25 The student can create a visual representation
to illustrate how changes in a DNA nucleotide
sequence can result in a change in the polypeptide
produced. [See SP 1.1]
• LO 3.26 The student is able to explain the connection
between genetic variations in organisms and
phenotypic variations in populations. [See SP 7.2]