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General Biology (Bio107)
Chapter 10 – Molecular Biology
of the Gene
3D Structure of DNA
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Introduction
• In April 1953, James Watson and Francis Crick
shook the scientific world with an elegant doublehelical model for the structure of deoxyribonucleic
acid or DNA.
• Your genetic endowment is the DNA and its genetic
information you inherited from your parents.
• Nucleic acids are unique in their ability to direct
their own replication.
• To assure successful regeneration and
reproduction using mitosis and meiosis, parental
DNA has to be copied into two new strands of
daughter DNA.
• Life relies on a process called DNA replication to
achieve this.
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1. The search for genetic material lead to
DNA
• Once T.H. Morgan’s group showed that genes are
located on chromosomes, the two constituents of
chromosomes - proteins and DNA - were the
candidates for the genetic material.
• Until the 1940s, the great heterogeneity and
specificity of function of proteins seemed to indicate
that proteins were the genetic material.
• However, this was not consistent with experiments
with microorganisms, like bacteria and viruses.
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• The discovery of the genetic role of DNA began
with research by Frederick Griffith in 1928.
• He studied Streptococcus pneumoniae, a
bacterium that causes pneumonia in mammals.
– One strain, the R strain, was harmless.
– The other strain, the S strain, was pathogenic.
• In an experiment Griffith mixed heat-killed S
strain with live R strain bacteria and injected this
into a mouse.
• The mouse died and he recovered the
pathogenic strain from the mouse’s blood.
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• Griffith called this phenomenon transformation,
a change in genotype and phenotype due to the
assimilation of a foreign substance (now known
to be DNA) by a cell.
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• For the next 14 years scientists tried to identify
the transforming substance.
• Finally in 1944, Oswald Avery, Maclyn McCarty
and Colin MacLeod announced that the
transforming substance was DNA.
• Still, many biologists were skeptical.
– In part, this reflected a belief that the genes of
bacteria could not be similar in composition
and function to those of more complex
organisms.
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• Further evidence that DNA was the genetic
material was derived from studies that tracked
the infection of bacteria by viruses.
• Viruses consist of a DNA (sometimes RNA)
enclosed by a protective coat of protein.
• To replicate, a virus infects a host cell and takes
over the cell’s metabolic machinery.
• Viruses that specifically attack bacteria are called
bacteriophages or just phages.
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• In 1952, Alfred Hershey & Martha Chase showed
that DNA was the genetic material of the phage T2.
• The T2 phage, consisting almost entirely of DNA
and protein, attacks Escherichia coli (E. coli), a
common intestinal bacteria of mammals.
• This phage can quickly
turn an E. coli cell into
a T2-producing factory
that releases phages
when the cell ruptures.
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• The famous Hershey and Chase experiment.
• They radio-labeled protein (35S) and DNA (32P) of
T2 and then tracked which entered the E. coli cell
during infection.
– They grew one batch of T2 phage in the presence of
radioactive sulfur, marking the proteins but not DNA.
– They grew another batch in the presence of radioactive
phosphorus, marking the DNA but not proteins.
– They allowed each batch to infect separate E. coli
cultures.
– Shortly after the onset of infection, they spun the
cultured infected cells in a blender, shaking loose any
parts of the phage that remained outside the bacteria.
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– The mixtures were spun in a centrifuge which separated
the heavier bacterial cells in the pellet from lighter free
phages and parts of phage in the liquid supernatant.
– They then tested the pellet and supernatant of the
separate treatments for the presence of radioactivity.
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• Hershey and Chase found that when the bacteria
had been infected with T2 phages that contained
radio-labeled proteins, most of the radioactivity
was in the supernatant, not in the pellet.
• When they examined the bacterial cultures with T2
phage that had radio-labeled DNA, most of the
radioactivity was in the pellet with the bacteria.
• Hershey and Chase concluded that the injected
DNA of the phage provides the genetic information
that makes the infected cells produce new viral
DNA and proteins, which assemble into new
viruses.
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• By 1947, Erwin Chargaff had developed a series
of rules based on a survey of DNA composition in
organisms.
– He already knew that DNA was a polymer of
nucleotides consisting of a nitrogenous base,
deoxyribose, and a phosphate group.
– The bases could be adenine (A), thymine (T),
guanine (G), or cytosine (C).
• Chargaff noted that even though the DNA
composition varies from species to species, the
four bases are found in characteristic, but not
necessarily equal, ratios in different species.
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• He also found a peculiar regularity in the ratios of
nucleotide bases which are known as Chargaff’s
rule.
• The number of adenines was approximately equal
to the number of thymines (%T = %A).
• The number of guanines was approximately equal
to the number of cytosines (%G = %C).
– Human DNA is 30.9% adenine, 29.4% thymine,
19.9% guanine and 19.8% cytosine.
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2. Watson and Crick discovered the double
helix by building models to conform to Xray data
• By the beginnings of the 1950’s, the race was on
to move from the structure of a single DNA strand
to the three-dimensional structure of DNA.
– Among the scientists working on the problem
were Linus Pauling, in California, and Maurice
Wilkins and Rosalind Franklin, in London.
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• The phosphate
group of one
nucleotide is
attached to the
sugar of the next
nucleotide in line.
• The result is a
“backbone” of
alternating
phosphates and
sugars, from which
the bases project.
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• Maurice Wilkins & Rosalind Franklin used X-ray
crystallography to study the structure of DNA.
– In this technique, X-rays are diffracted as they
passed through aligned fibers of purified DNA.
– The diffraction pattern can be used to deduce
the three-dimensional shape of molecules.
• James Watson learned
from their research
that DNA was helical
in shape and he deduced
the width of the helix
and the spacing of bases.
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• Watson and his colleague Francis Crick
began to work on a model of DNA with two
strands, the DNA double helix.
• Using molecular models made of wire, they
first tried to place the sugar-phosphate
chains on the inside.
• However, this did not fit the X-ray
measurements and other information on the
chemistry of DNA.
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• The key breakthrough towards unraveling the 3D
structure of DNA came when Watson put the
sugar-phosphate chain on the outside and the
nitrogen bases on the inside of the double helix.
– The sugar-phosphate chains of each strand are
like the side ropes of a rope ladder.
– Pairs of nitrogen bases, one from each strand,
form rungs.
– The ladder forms a twist every ten bases.
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Watson-Crick Base-pairing Rule
• The nitrogenous bases are paired in specific
combinations: adenine with thymine and guanine
with cytosine.
• Pairing like nucleotides did not fit the uniform
diameter indicated by the X-ray data.
– A purine-purine pair would be too wide and a
pyrimidine-pyrimidine pairing would be too short.
– Only a pyrimidinepurine pairing would
produce the 2-nm
diameter indicated
by the X-ray data.
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• In addition, Watson and Crick determined that
chemical side groups off the nitrogen bases would
form hydrogen bonds, connecting the two
strands.
– Based on details of their
structure, adenine would
form two hydrogen bonds
only with thymine and
guanine would form three
hydrogen bonds only with
cytosine.
– This finding explained
Chargaff’s rules.
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• The base-pairing rules dictate the combinations of
nitrogenous bases that form the “rungs” of DNA.
• However, this does not restrict the sequence of
nucleotides along each DNA strand.
• The linear sequence of the four bases can be
varied in countless ways.
• Each gene has a unique order of nitrogen bases.
• In April 1953, Watson and Crick published a
succinct, one-page paper in Nature reporting their
double helix model of DNA.
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The DNA Molecule
 Polymer of nucleotides:
Adenine, thymine,
cytosine, and guanine
 Two polynucleotide strands
form double helix
associated with proteins
 "Backbone" is
deoxyribose-phosphate
 Center contains the
nitrogenous bases
1. During DNA replication, base pairing
enables existing DNA strands to serve as
templates for new complimentary strands
• In a second paper Watson and Crick published
their hypothesis for how DNA becomes copied
within cells by a process called DNA replication.
– Essentially, because each strand is
complementary to each other, each can form a
template when separated.
– The order of bases on one strand can be used to
add in complementary bases and therefore
duplicate the pairs of bases exactly.
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• When a cell copies a DNA molecule, each
parental strand serves as a template for ordering
nucleotides into a new complimentary strand.
– One at a time, nucleotides line up along the template
strand according to the base-pairing rules.
– The nucleotides are linked to form new strands.
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• Watson and Crick’s model, semiconservative
replication, predicts that when a double helix
replicates each of the daughter molecules will
have one old strand and one newly made strand.
• Other competing models, the conservative model
and the dispersive model, were also proposed.
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• Experiments in the late 1950s by Matthew
Meselson & Franklin Stahl supported the
semiconservative model, proposed by Watson
and Crick, over the other two models.
– In their experiments, they labeled the
nucleotides of the old strands with a heavy
isotope of nitrogen (15N) while any new
nucleotides would be indicated by a lighter
isotope (14N).
– Replicated strands could be separated by
density in a centrifuge.
– Each model: the semi-conservative model, the
conservative model, and the dispersive model,
made specific predictions on the density of
replicated DNA strands.
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• The first replication in the 14N medium produced a
band of hybrid (15N-14N) DNA, eliminating the
conservative model.
• A second replication produced both light and hybrid
DNA, eliminating the dispersive model and
supporting the semiconservative model.
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DNA Molecule
• The two strands are
held together by
hydrogen bonds
between AT and CG
• Strands are
antiparallel regarding
5’ and 3’ ends
Figure 8.3b
DNA Replication
• Sustainability of life is guaranteed due
copying of DNA by a process called DNA
replication
“DNA characters (= nucleotides) are copied with an accuracy
that rivals anything modern engineering can do.
They are just copied down to generations, with just enough
occasional errors to introduce variety
(into the genetic material) …”
R. Dawkins: River out of Eden
Semi-conservative DNA Replication
Figure 8.3a
Synthesis of New DNA
DNA Synthesis
• DNA replication starts at origin (‘ori” region)
• First event is unwinding by helicase enzyme
(forms two replication forks)
• DNA is copied by DNA polymerase
–
–
–
–
–
–
Proceeds in the 5'  3' direction
Initiated by single-stranded RNA primer
Leading strand is synthesized continuously
Lagging strand is synthesized discontinuously
Many Okazaki fragments on lagging strand
RNA primers are removed and Okazaki fragments
joined by a DNA polymerase and DNA ligase
Replication Fork Events
DNA Synthesis at Replication
Fork
Figure 8.5
The “Leading-Lagging Strand Problem”
• DNA polymerase reads parental DNA strand only in
3’ --> 5’ direction, it is only able to run with the
replication fork along the leading strand
• On the lagging strand, DNA polymerase
synthesizes in the opposite direction of the
movement of the replication fork
• Multiple primers have to be synthesized on the
lagging strand to generate multiple start sites for
the DNA polymerase
• Many Okazaki fragments temporarily appear on
the lagging strand
The “Leading-Lagging Strand Problem”
Ligase Action
• The enzyme Ligase forms
a continuous daugther
DNA strand by closing
the tiny 3'-5' gaps between
the different daugther DNA
fragments
(= Okazaki fragments)
Summary of DNA Replication
• It is a fast process
- the DNA polymerase adds about 50 nucleotides
per second to growing DNA daughter strand!
• It is a very accurate process
- only about one out of 1 million nucleotides,
incorporated into the newly formed daughter
strand is incorrectly build-in or wrongly paired
during “un-repaired” DNA replication!
- intrinsic 3’-exonuclease (“proof reading”)
activity of DNA polymerase reduces this low error
rate to one mistake per every 109 nucleotides
added
Proof Reading Activity of DNA
POL
Replication of Bacterial DNA
Figure 8.6
DNA Transcription is the DNA-directed
synthesis of RNA
• Messenger RNA is transcribed from the template
strand of a gene.
• RNA polymerase separates the DNA strands at
the appropriate point and bonds the RNA
nucleotides as they base-pair along the DNA
template.
• Like DNA polymerases, RNA polymerases can add
nucleotides only to the 3’ end of the growing
polymer.
– Genes are read 3’->5’, creating a 5’->3’ RNA
molecule.
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• Specific sequences of nucleotides along the DNA
mark where gene transcription begins and ends.
– RNA polymerase attaches and initiates
transcription at the promotor, “upstream” of
the information contained in the gene, the
transcription unit.
– The terminator signals the end of
transcription.
• Bacteria have a single type of RNA polymerase
that synthesizes all RNA molecules.
• In contrast, eukaryotes have three RNA
polymerases (I, II, and III) in their nuclei.
– RNA polymerase II is used for mRNA
synthesis.
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DNA Transcription
• DNA sequence of a gene is transcribed to make
RNA (mRNA, tRNA, and rRNA)
• Transcription begins when RNA polymerase binds
to the promoter sequence located in front of gene
• Transcription is performed by RNA polymerase
enzyme
• Elongation of transcription proceeds in 5'  3'
direction
• Transcription stops when it reaches the
terminator sequence
• In a eukaryotic cell, almost all transcription occurs
in the nucleus and translation occurs mainly at
ribosomes in the cytoplasm.
• In addition, before the
primary transcript
can leave the nucleus
it is modified in
various ways during
RNA processing
before the finished
mRNA is exported
to the cytoplasm.
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Transcription
Figure 8.7
Organization of Typical Bacterial Gene
Sense
strand
ATG
TAC
Anti-Sense
strand
RNA Polymerase
• Adds new nucleotides
to growing RNA strand
during DNA transcription
• Synthesizes RNA in
5’  3’ direction)
• Requires Mg and Zn as
cofactors
• DNA
transcription
can be
separated
into three
stages:
1. Initiation,
2. Elongation,
and
3. Termination.
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Initiation of DNA Transcription
Bacteria
• Starts with binding of the RNA polymerase (POL) Sigma factor complex to promoter sequence of a
gene
• Prokaryotic promoter sequence is located about
35 bp before the transcription start site
- contains a 6 base sequence, usually TTGACA
- contains a TATAAT or Pribnow box sequence
about
10 bp before the transcription start site
• Bound RNA POL locally unwinds the DNA double
helix of the gene to form a "transcription bubble"
Initiation of Eukaryotic DNA Transcription
• Activated transcription factors plus RNA polymerase II
assemble and form the transcription-initiation complex, or
transcriptome
• Transcriptomes are large and include many components:
1. RNA polymerase I, II or III (= POL)
- RNA POL II synthesizes mRNA
- large multi-protein complex with about 500,000 Da
- inhibited by mushroom poison alpha-amanitin;
- RNA POL I and RNA POL III synthesize rRNA and tRNA
2. TBP (= TATA box-binding protein)
- binds to the TATA box of the promoter region
3. Many Transcription factors, e.g. TFII F, TFII E, TFII H
and TFII D
- important for regulation of transcription
The Process of Transcription
Initiation
Elongation
Figure 8.7
• As RNA polymerase moves along the DNA, it
untwists the double helix, 10 to 20 bases at time.
• The enzyme adds
nucleotides to the
3’ end of the
growing strand.
• Behind the point
of RNA synthesis,
the double helix
re-forms and the
RNA molecule
peels away.
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The Process of Transcription
Elongation
Termination
ANIMATION Transcription: Overview
ANIMATION Transcription: Process
Figure 8.7
Termination of DNA Transcription
• Terminator sequence on gene sets the stop signal
for DNA transcription
• Prokaryotic terminators often contain two types of
stop signals:
1. Poly-uridine-(poly-U) based termination
- consists of 6 uridine residues (UUUUUU)
following a hairpin structure
- no protein factors required
2. Rho-dependent termination
- lacks a poly-U region, and often the hairpin loop;
- requires the protein factor "rho"
Organization of Eukaryotic Gene
Eukaryotic cells modify mRNA after
transcription
• Enzymes in the eukaryotic nucleus modify premRNA before the genetic messages are dispatched
to the cytoplasm.
• At the 5’ end of the pre-mRNA molecule, a modified
form of guanine is added, the 5’ cap.
– This helps protect mRNA from hydrolytic
enzymes.
– It also functions as an “attach here” signal for
ribosomes.
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• At the 3’ end, an enzyme adds 50 to 250 adenine
nucleotides, the poly(A) tail.
– In addition to inhibiting hydrolysis and
facilitating ribosome attachment, the poly(A)
tail also seems to facilitate the export of mRNA
from the nucleus.
• The mRNA molecule also includes nontranslated
leader and trailer segments.
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• The most remarkable stage of RNA processing
occurs during the removal of a large portion of
the RNA molecule during RNA splicing.
• Most eukaryotic genes and their RNA transcripts
have long noncoding stretches of nucleotides.
– Noncoding segments, introns, lie between
coding regions.
– The final mRNA transcript includes coding
regions, exons, that are translated into amino
acid sequences, plus the leader and trailer
sequences.
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Splicing of mRNA in Eukaryotes
• RNA splicing removes introns and joins exons to
create an mRNA molecule with a continuous
coding sequence.
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mRNA Splicing
• Splicing = cutting out of the non-coding intron
regions from nascent mRNA
• Two different splicing mechanisms are known:
1. protein-independent self-splicing
- requires catalytically active snRNA
2. spliceosome-dependent splicing
- requires several RNA and protein components,
e.g. U1, U2, U4, U5, U6 snRNA and hnRNPs
- most introns are spliced by this mechanism
• RNA splicing appears to have several functions.
– First, at least some introns contain sequences
that control gene activity in some way.
– Splicing itself may regulate the passage of
mRNA from the nucleus to the cytoplasm.
– One clear benefit of split genes is to enable a
one gene to encode for more than one
polypeptide.
• Alternative RNA splicing gives rise to two or
more different polypeptides, depending on which
segments are treated as exons.
– Early results of the Human Genome Project
indicate that this phenomenon may be
common in humans.
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• Split genes may also facilitate the evolution of
new proteins.
• Proteins often have a
modular architecture
with discrete structural
and functional regions
called domains.
• In many cases,
different exons
code for different
domains of a
protein.
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RNA Processing Steps
• 3 major mRNA processing steps have been
identified in cells
1. CAPing of the 5’-end of the pre-pro-mRNA
- covalent attachment of a chemical group(m7GpppNmpN)
- done by capping enzyme and methyl transferase
2. Attachment of Poly-A tail to 3’ end of mRNA
3. Splicing of introns (eukaryotic mRNA only)
- ATP-dep. polymerization of >200 adenine residues to
the 3’-end of nascent mRNA molecule
- catalyzed by Poly-A polymerase (PAP)
Poisons & Inhibitors of DNA Transcription
Protein Translation
• Nucleotide sequence
of mRNA is translated
at the ribosome in
codons (three
nucleotides) steps
• Translation of mRNA
begins at the start
codon: AUG
• Translation ends at
noncoding codons:
UAA, UAG, UGA
• During transcription, one DNA strand, the template
strand, provides a template for ordering the
sequence of nucleotides in an RNA transcript.
• During translation, blocks
of three nucleotides,
codons, are decoded into
a linear sequence of
linked amino acids.
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• During translation, each type of tRNA links a
mRNA codon with the appropriate amino acid.
• Each tRNA arriving at the ribosome carries a
specific amino acid at one end and has a specific
nucleotide triplet, an anticodon, at the other.
• The anticodon base-pairs with a complementary
codon on mRNA.
– If the codon on mRNA is UUU, a tRNA with an
AAA anticodon and carrying phenyalanine will
bind to it.
• Codon by codon, tRNAs deposit amino acids in
the prescribed order and the ribosome joins them
into a polypeptide chain.
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• A tRNA molecule consists of a strand of about 80
nucleotides that folds back on itself to form a
three-dimensional structure.
– It includes a loop containing the anticodon and
an attachment site at the 3’ end for an amino
acid.
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Transfer RNA (tRNA)
• Has complex 3-dimensional structure with two
major interaction domains
1. Amino acid
attachment site
- site where corresponding
amino acid is covalently
attached to tRNA
2. Anti-codon site
- Watson-Crick base pairs
with complementary
codon on mRNA strand
• If each anticodon had to be a perfect match to
each codon, we would expect to find 61 types of
tRNA, but the actual number is about 45.
• The anticodons of some tRNAs recognize more
than one codon.
• This is possible because the rules for base
pairing between the third base of the codon and
anticodon are relaxed (called wobble).
– At the wobble position, U on the anticodon can
bind with A or G in the third position of a
codon.
– Some tRNA anticodons include a modified
form of adenine, inosine, which can hydrogen
bond with U, C, or A on the codon.
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In the genetic code, nucleotide triplets
specify amino acids
• If the genetic code consisted of a single nucleotide
or even pairs of nucleotides per amino acid, there
would not be enough combinations (4 and 16
respectively) to code for all 20 amino acids.
• Triplets of nucleotide bases are the smallest units
of uniform length that can code for all the amino
acids.
• In the triplet code, three consecutive bases specify
an amino acid, creating 43 (64) possible code
words.
• The genetic instructions for a polypeptide chain are
written in DNA as a series of three-nucleotide
words.
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• During protein translation at the ribosome, the
codons are read in the 5’->3’ direction along the
mRNA.
• Each codon specifies which one of the 20 amino
acids will be incorporated at the corresponding
position along a polypeptide.
• Because codons are base triplets, the number of
nucleotides making up a genetic message must be
three times the number of amino acids making up
the protein product.
– It would take at least 300 nucleotides to code for
a polypeptide that is 100 amino acids long.
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• Ribosomes facilitate the specific coupling of the
tRNA anticodons with mRNA codons.
– Each ribosome has a large and a small subunit.
– These are composed of proteins and ribosomal RNA
(rRNA), the most abundant RNA in the cell.
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• Each ribosome has a binding site for mRNA and
three binding sites for tRNA molecules.
– The P site holds the tRNA carrying the
growing polypeptide chain.
– The A site carries the tRNA with the next
amino acid.
– Discharged tRNAs leave the ribosome at the E
site.
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Protein Translation
• Requires the following cell components:
1. mature mRNA
2. amino-acyl tRNA
3. large and small ribosome units
4. rRNA
5. protein factors, e.g. IFs, EFs and RFs
6. Cell energy, in form of ATP, GTP
Ribosomal RNAs (rRNA)
• Structural and functional part of the ribosomes.
• Three forms of rRNA each with different length
and function exist:
1. Prokaryotic organisms:
5S-rRNA; 16S-rRNA & 23S-rRNA
2. Eukaryotic organisms:
7S-rRNA; 18S-rRNA & 28S-rRNA
• Synthesized (= transcribed) in high amount in
the nucleolus.
The Process of Translation
1. Initiation
+ Initiation factors (IFs)
+ GTP
Figure 8.9
• Elongation consists of a series of three step
cycles as each amino acid is added to the
proceeding one.
• During codon recognition, an elongation factor
assists hydrogen bonding between the mRNA
codon under the A site with the corresonding
anticodon of tRNA carrying the appropriate
amino acid.
– This step requires the hydrolysis of two GTP.
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2. Transamination
• During peptide bond formation
(“transpeptidylation” or “transamination”), an
rRNA molecule catalyzes the formation of a
peptide bond between the polypeptide in the P
site with the new amino acid in the A site.
• This step separates the tRNA at the P site from
the growing polypeptide chain and transfers the
chain, now one amino acid longer, to the tRNA at
the A site.
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3. Translocation & Elongation
+ Elongation factors (EFs)
+ GTP
• During translocation, the ribosome moves the
tRNA with the attached polypeptide from the A
site to the P site.
– Because the anticodon remains bonded to the
mRNA codon, the mRNA moves along with it.
– The next codon is now available at the A site.
– The tRNA that had been in the P site is moved
to the E site and then leaves the ribosome.
– Translocation is fueled by the hydrolysis of
GTP.
– Effectively, translocation ensures that the
mRNA is “read” 5’ -> 3’ codon by codon.
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• The three steps of elongation continue
codon by codon to add amino acids until
the polypeptide chain is completed.
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4. Polypeptide Chain Elongation
The Process of Translation
5. Termination
+ Releasing Factor (RF)
+ Water
RF
The Process of Translation
The Genetic Code
• 61 sense codons on
mRNA encode the 20
amino acids
• The genetic code is
degenerate
• tRNA carries the
complementary
anticodon
• The task of matching each codon to its amino acid
counterpart began in the early 1960s.
• Marshall Nirenberg determined the first match,
that UUU coded for the amino acid phenylalanine.
– He created an artificial mRNA molecule entirely
of uracil and added it to a test tube mixture of
amino acids, ribosomes, and other components
for protein synthesis.
– This “poly(U)” translated into a polypeptide
containing a single amino acid, phenyalanine, in
a long chain.
• Other more elaborate techniques were required to
decode mixed triplets such a AUA and CGA.
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The Genetic Code
The genetic code of life
is redundant; i.e. more
than one codon codes
for one amino acid
The genetic code must have evolved very
early in the history of life
• The genetic code is nearly universal, shared by
organisms from the simplest bacteria to the most
complex plants and animals.
• This enables genetic engineering.
• In laboratory experiments,
genes can be transcribed and
translated after they are
transplanted from one species
to another.
– This tobacco plant is expressing
a transpired firefly gene.
• The near universality of the genetic code
must have been operating very early in the
history of life.
• A shared genetic vocabulary is a reminder
of the kinship that bonds all life on Earth.
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Comparing protein synthesis in prokaryotes
and eukaryotes: a review
• Although bacteria and eukaryotes carry out
transcription and translation in very similar ways,
they do have differences in cellular machinery and
in details of the processes.
– Eukaryotic RNA polymerases differ from those of
prokaryotes and require transcription factors.
– They differ in how transcription is terminated.
– Their ribosomes are also different.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• In one big
differences,
prokaryotes can
transcribe and
translate the
same gene
simultaneously.
• The new protein
quickly diffuses to
its operating site.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
• In eukaryotes, the nuclear envelope
segregates transcription from translation.
• In addition, extensive RNA processing is
inserted between these processes.
– This provides additional steps whose
regulation helps coordinate the elaborate
activities of a eukaryotic cell.
• In addition, eukaryotic cells have
complicated mechanisms for targeting
proteins to the appropriate organelle.
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• Transcription,
RNA processing,
and translation are
the processes that
link DNA
sequences to the
synthesis of a
specific
polypeptide chain.
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Regulation of Protein Translation
• Gene expression is also regulated at the protein translation level at
the ribosome
• Regulation of protein translation happens via:
1. Control by protein phosphorylation of initiation factors
- phosphorylation of eIF-2 by heme-controlled inhibitor (HCI) blocks
exchange of the bound GDP for GTP
2. Control by Interferons (IFs)
- IFs are cellular signaling molecules that activate RNA-dependent
protein kinases (= PKRs)
- PKRs phosphorylate and inactivate eIF-2 therefore preventing
initiation of protein translation
- most IFs are released from white blood cells after viral attack
- 3 different classes of interferons are known
3. Control by post-translational protein modification
1. Glycosylation (= attachment of sugar residues to proteins)
2. Isoprenylation/Acylation (= attachment of isoprenoid or fatty acids)
3. Ubiquitination & Proteolytic Digest
(Proteasome, Apoptosis --> Caspases)
Regulation in Bacteria: Operon Model
Polycistronic mRNA
ANIMATION Operons: Overview
Figure 8.12
Induction – Lac Operon
1. Lactose absent
No Transcription!
Figure 8.12
Induction – Lac Operon
2. Lactose present
DNA Transcription
Figure 8.12
Mutation
• A change in the genetic material.
• Mutations change the DNA sequence and
therefore the genetic information.
• Mutations may be neutral, beneficial, or harmful
• Mutagen: Agent that causes mutations
• Spontaneous mutations: Occur in the absence
of a mutagen
Types of Mutations
• Base substitution
(point mutation)
• Missense mutation
• Change in one
base
• Result in change in
amino acid
Figure 8.17a, b
• For example, sickle-cell disease is caused by a
mutation of a single base pair in the gene that
codes for one of the polypeptides of hemoglobin.
– A change in a single nucleotide from T to A in
the DNA template leads to an abnormal
protein.
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• Nonsense mutation • Results in a
nonsense codon
Figure 8.17a, c
• Frameshift mutation • Insertion or
deletion of one or
more nucleotide
pairs
Figure 8.17a, d
The Frequency of Mutation
• Spontaneous mutation rate = 1 in 109
replicated base pairs or 1 in 106 replicated
genes
• Mutagens increase to 10–5 or 10–3 per
replicated gene
• Mutation rate vastly responsible for “pace
of evolutionary change” of life forms
ANIMATION Mutations: Types
Chemical Mutagens
Figure 8.19a
Chemical Mutagens
ANIMATION Mutagens
Figure 8.19b
Chemical mutagens & Carcinogens
•the molecules shown below act as mutagens and can cause cancers
•cancer-causing chemicals are called carcinogens
•all chemicals carcinogens are mutagens, i.e. are able to interact with DNA
Require prior “metabolic activation”
by
(Cyt P450) liver enzymes
Radiation
• Ionizing radiation (X rays and gamma
rays) causes the formation of ions that can
react with nucleotides and the
deoxyribose-phosphate backbone
- often cause DNA double strand breaks
(DSBs) with dangerous consequences
Radiation
• UV radiation
causes thymine
dimer formation
of neighboring
thymines (T) in
DNA.
Figure 8.20
Mutations & DNA Repair
• Photolyases separate thymine dimers
• Nucleotide excision repair (NER)
ANIMATION Mutations: Repair
• In mismatch repair, special enzymes fix incorrectly
paired nucleotides.
– A hereditary defect in
one of these enzymes
is associated with a
form of colon cancer.
• In nucleotide excision
repair, a nuclease cuts
out a segment of a
damaged strand.
– The gap is filled in by
DNA polymerase and
ligase.
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• The importance of proper function of repair
enzymes is clear from the inherited
disorder xeroderma pigmentosum.
– These individuals are hypersensitive to
sunlight.
– In particular, ultraviolet light can produce
thymine dimers between adjacent thymine
nucleotides.
– This buckles the DNA double helix and
interferes with DNA replication.
– In individuals with this disorder, mutations in
their skin cells are left uncorrected and cause
skin cancer.
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UV
irradiation
Mutagenic
Chemicals,
ROS
Incorrect base
incorporation
during DNA replication
Ionizing radiation (X-ray)
Oxygen radicals (ROS)
Bleomycin, Cisplatin
DNA POL III
Stop of
DNA Polymerase
T- dimer
formation
Base (N)
modification
(O6- ethyl-G, 8-OxoG)
+
Double-strand
break (DSB)
Mis-matched pairing
DNA POL
3’-exonuclease
UvrABC
XPC
Ku
heterodimer
mutHLS,
OGG1
DNA POL
Proof reading
Graphics©E.Schmid/2002
Nucleotide
Excision repair
(NER)
Mismatch
repair
Homologous
recombination (HR)
or
NH end joining
Ames Test for Chemical Carcinogens
Ames Test for Chemical Carcinogens
Figure 8.22
• The ends of eukaryotic chromosomal DNA
molecules, the telomeres, have special nucleotide
sequences.
– In human telomeres, this sequence is typically
TTAGGG, repeated between 100 and 1,000
times.
• Telomeres protect genes from being eroded
through multiple rounds of DNA replication.
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