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CAMPBELL
BIOLOGY
TENTH
EDITION
Reece • Urry • Cain • Wasserman • Minorsky • Jackson
17
Gene
Expression:
From Gene to
Protein
Lecture Presentation by
Nicole Tunbridge and
Kathleen Fitzpatrick
© 2014 Pearson Education, Inc.
Overview: The Flow of Genetic Information
• The information in DNA is in the form of
specific sequences of nucleotides
• The DNA inherited by an organism leads to
specific traits by dictating the synthesis of
proteins
• Proteins are the links between genotype and
phenotype
• Gene expression, the process by which
DNA directs protein synthesis, includes two
stages: transcription and translation
© 2014 Pearson Education, Inc.
How does a single faulty gene result in the dramatic appearance of an
albino deer or racoon ?
Gene Expression: From Gene to
Protein
Figure 17.1
Evidence from the Study of Metabolic
Defects
• In 1902, British physician Archibald Garrod first
suggested that genes dictate phenotypes through
enzymes that catalyze specific chemical reactions
• He thought symptoms of an inherited disease
reflect an inability to synthesize a certain enzyme
• Linking genes to enzymes required understanding
that cells synthesize and degrade molecules in a
series of steps, a metabolic pathway
© 2014 Pearson Education, Inc.
Genes specify proteins via transcription
and translation
• How was the fundamental relationship between
genes and proteins discovered?
• The study of metabolic defects provided evidence that
genes specify proteins.
• Scientific experiments suggested that the symptoms
of an inherited disease reflect a person’s inability to
synthesize a particular enzyme.
– Such diseases are referred to as “inborn errors of
metabolism.”
• Several studies provided strong evidence for the one
gene–one enzyme hypothesis.
• It was found that not all proteins are enzymes.
• This tweaked the hypothesis to one gene–one protein.
© 2014 Pearson Education, Inc.
Nutritional Mutants in Neurospora:
Scientific Inquiry
• George Beadle and Edward Tatum exposed bread
mold to X-rays, creating mutants that were unable
to survive on minimal medium as a result of
inability to synthesize certain molecules
• Using crosses, they identified three classes of
arginine-deficient mutants, each lacking a different
enzyme necessary for synthesizing arginine
• They developed a one gene–one enzyme
hypothesis, which states that each gene dictates
production of a specific enzyme
© 2014 Pearson Education, Inc.
Figure 17.2
Precursor
Classes of Neurospora crassa
Results Table
Enzyme A
Wild type
Class I mutants
Class II mutants
Class III mutants
Can grow with
or without any
supplements
Can grow on
ornithine,
citrulline,
or arginine
Can grow only
on citrulline or
arginine
Require arginine
to grow
Class II mutants
(mutation in
gene B)
Class III mutants
(mutation in
gene C)
Minimal
medium
(MM)
(control)
Ornithine
Enzyme B
Citrulline
Enzyme C
MM +
ornithine
Condition
Arginine
Growth:
Wild-type
cells growing
and dividing
No growth:
Mutant cells
cannot grow
and divide
Control: Minimal medium
MM +
citrulline
MM +
arginine
(control)
Summary
of results
Gene
(codes for
enzyme)
Wild type
Precursor
Gene A
Enzyme A
Ornithine
Gene B
Enzyme B
Citrulline
Gene C
Enzyme C
Arginine
Class I mutants
(mutation in
gene A)
Precursor
Enzyme A
Ornithine
Enzyme B
Citrulline
Enzyme C
Arginine
Precursor
Enzyme A
Ornithine
Enzyme B
Citrulline
Enzyme C
Arginine
Precursor
Enzyme A
Ornithine
Enzyme B
Citrulline
Enzyme C
Arginine
The Products of Gene Expression: A
Developing Story
• Some proteins aren’t enzymes, so researchers
later revised the hypothesis: one gene–one protein
• Many proteins are composed of several
polypeptides, each of which has its own gene
• Therefore, Beadle and Tatum’s hypothesis is now
restated as the one gene–one polypeptide
hypothesis
• Note that it is common to refer to gene products as
proteins rather than polypeptides
© 2014 Pearson Education, Inc.
The language of DNA-gene produce
proteins
DNA and RNA, are polymers made from four
different nucleotides- A, T/U, G and C arranged in
specific orders.
Genes are made by thousands of nucleotides long
and each gene has a specific sequence of nucleotide
bases. Genes provide the instructions for making
specific proteins.
The bridge between DNA and protein synthesis is
the RNA.
Proteins are made from specific orders and types of
twenty amino acids.
Two processes ,Transcription and translation are the
two main processes linking gene to protein.
Transcription
DNA
→
mRNA
Translation
→
Protein
Basic Principles of Transcription and
Translation
• RNA is the intermediate between genes and
the proteins for which they code
• Transcription is the synthesis of RNA under
the direction of DNA. Transcription produces
messenger RNA (mRNA)
• Translation is the synthesis of a polypeptide,
which occurs under the direction of mRNA
• Ribosomes are the sites of translation
• Proteins are made from 2 or more polypeptide
chains and each polypeptide is specified by its
own gene.
© 2014 Pearson Education, Inc.
Transcription is synthesis of mRNA from
DNA
• To get from DNA, written in one chemical language
(nucleotides), to protein, written in another (amino
acids), requires two major stages: transcription and
translation.
• During transcription, a DNA strand provides a
template for the synthesis of a complementary RNA
strand. The information is transcribed, or copied
from DNA to mRNA.
– Just as a DNA strand provides a template for the
synthesis of each new complementary strand
during DNA replication, it provides a template for
assembling a sequence of RNA nucleotides.
© 2014 Pearson Education, Inc.
Translation is the synthesis of a
polypeptide
• mRNA carries the genetic message from the
DNA to the protein-synthesizing machinery
of the cell.
• Translation is the synthesis of a polypeptide
(via mRNA).
– During translation there is a change in
language: The cell must translate the
nucleotide sequence of mRNA molecules
into amino acid sequences of proteins.
• Ribosomes are the sites of translation
Transcription and translation occur in
prokaryotes
• A bacterial cell does not
have a nucleus, so the
mRNA produced by
transcription is
immediately translated
into protein by the
ribosomes.
– Ribosomes attach to the
leading end of an mRNA
molecule while
transcription is still in
progress.
© 2014 Pearson Education, Inc.
Figure 17.3
Transcription and translation occur in
eukaryotes
Figure 17.3
• The nucleus of a eukaryotic
cell provides a separate
compartment for
transcription, yielding premRNA, which is modified in
the nucleus making mRNA
through RNA processing to
yield finished mRNA.
• mRNA is then exported out
of the nucleus translation
occurs at ribosomes in the
cytoplasm, producing
polypeptides.
© 2014 Pearson Education, Inc.
Fig. 17.3 p329
• A primary transcript is the initial RNA
transcript from any gene
• The central dogma is the concept that
cells are governed by a cellular chain of
command:
DNA
© 2014 Pearson Education, Inc.
RNA
Protein
The Genetic Code
• How do the nucleotide bases of DNA provide the
instructions for making proteins that contain
specific amino acids?
• There are 20 amino acids, but there are only four
nucleotide bases in DNA:
C,G,A, and T
• How many nucleotide bases = an amino acid?
• How do nucleotide bases result in the production
of specific amino acids?
© 2014 Pearson Education, Inc.
Genetic codon is a three nucleotide sequence of
DNA→RNA that produces an amino acid
• The flow of information from gene to protein is based
on a triplet code: a series of nonoverlapping , 3
consecutive nucleotide bases.
• The genetic instructions for a polypeptide chain are
written in the DNA as a series of non-overlapping 3
nucleotide “words”.
• These triplets are the smallest units of uniform length
that can code for all the amino acids.
• Example: AGT at a particular position on a DNA strand
results in the transcription of AGU mRNA. An AGU
sequence in mRNA results in the placement of the
amino acid serine at the correct position of the
polypeptide that is produced at the ribosome.
© 2014 Pearson Education, Inc.
The Triplet Code
Figure 17.4
• During transcription, one of
the two DNA strands called the
template strand provides a
template for ordering the
sequence of nucleotides for
transcription of mRNA. The
base-pairing rules for DNA
replication also govern
transcription (but U is used
instead of T) of RNA.
• During translation, the mRNA
is read as a sequence of base
triplets (codons).
• Each codon specifies a
particular amino acid to be
added in the growing
polypeptide chain at the
ribosome.
© 2014 Pearson Education, Inc.
Codons are read in the 5′3′ direction
along the mRNA
• During translation, mRNA base
triplets, called the codons read in the
5′3′ direction along the mRNA
molecule is translated into a
sequence of amino acids making up
the polypeptide chain.
• 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.
• 300 nucleotides makes 100 triplet
codon which translate into 100
amino acids long protein.
© 2014 Pearson Education, Inc.
Cracking the Code
• There are 64 codons
– Of the 64 triplets, 61
code for amino acids
– 3 triplets are UAA,
UGA and UAG“stop”
signals to end
translation.
– AUG =methionine or
“start” signal
• The genetic code is
redundant but no codon
specifies more than one
amino acid
© 2014 Pearson Education, Inc.
Figure 17.5
Reading Frame
• Processing the message from the genetic code
requires specifying the correct starting point.
• The starting point establishes the reading
frame; subsequent codons are read in groups of
three nucleotides.
• The cell’s protein-synthesizing machinery reads
the message as a series of nonoverlapping
three-letter words (codons) each of which is
translated into a specific amino acid during
protein synthesis..
© 2014 Pearson Education, Inc.
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.
• Genes can be transcribed and translated after
being transplanted from one species to another.
• The evolutionary significance of the near
universality of the genetic code is clear: A
language shared by all living things arose very
early in the history of life—early enough to be
present in the common ancestors of all modern
organisms.
© 2014 Pearson Education, Inc.
Genes can be transcribed and translated
after being transplanted from one species to another
(a) Tobacco plant expressing
a firefly gene
Figure 17-6
(b) Pig expressing a
jellyfish gene
Transcription is the DNA-directed
synthesis of RNA
• Transcription is the first stage of gene expression
• Messenger RNA is the carrier of information from DNA to
the cell’s protein-synthesizing machinery (ribosomes). It
is transcribed from the template strand of a gene.
• RNA synthesis is catalyzed by RNA polymerase, which
seprate the DNA strands apart and hooks together the
RNA nucleotides.
• RNA synthesis follows the same base-pairing rules as
DNA, except uracil substitutes for thymine.
• Like DNA polymerases, RNA polymerases can assemble
a polynucleotide only in a 5′3′ direction.
• Unlike DNA polymerases, RNA polymerases are able to
start a chain from scratch; they don’t need a primer.
© 2014 Pearson Education, Inc.
RNA polymerase attaches and initiates
transcription at the promoter
• Specific sequences of nucleotides along the DNA mark
where gene transcription begins and ends.
• RNA polymerase attaches and initiates transcription at
the promoter.
• The sequence that signals the end of transcription is
called the terminator.
• Molecular biologists refer to the direction of transcription
as “downstream” and the other direction as “upstream.”
• The terms downstream and upstream are also used to
describe the positions of nucleotide sequences within the
DNA or RNA.
• The promoter sequence in DNA is upstream from the
terminator.
• The stretch of DNA that is transcribed into an RNA
molecule is called a transcription unit.
© 2014 Pearson Education, Inc.
Transcription can be separated into three
stages: initiation, elongation, and termination
Figure 17.7
• The presence of a promoter sequence determines which strand of
the DNA helix is the template.
– Within the promoter is the starting point for the
transcription of a gene. A DNA sequence called a TATA box
is located near the start site.
– The promoter also includes a binding site for RNA
polymerase several dozen nucleotides from the start point.
• In eukaryotes, proteins called transcription factors regulate the
binding of RNA polymerase and the initiation of transcription.
• Only after certain transcription factors are attached to the promoter
does RNA polymerase II bind to it and start transcription.
• The completed assembly of transcription factors and RNA
polymerase II bound to a promoter is called a transcription
initiation complex.
© 2014 Pearson Education, Inc.
Transcription Initiation at the Promoter
• The DNA strand will contain a
TATA box (containing a
sequence of thymine/adenine
nucleotides) very close to the
transcription start point.
• One transcription factor binds
specifically to the TATA box,
then other transcription factors
bind together at this area to
form a complex.
• After all of the required
transcription factors have
bound to the DNA RNA
polymerase II can bind to the
DNA strand, forming the
transcription initiation complex.
• The DNA double helix unwinds
and RNA synthesis begins at
the start point.
Figure 17.8
Initiation
Promoter
Transcription unit
3′
5′
5′
3′
1 Initiation
Start point
RNA polymerase
3′
5′
5′
3′
Figure 17.7
Unwound
DNA
Template strand of DNA
RNA
transcript
After RNA polymerase II binds to the
promoter:
The DNA strands unwind.
RNA polymerase II initiates RNA synthesis at
the start point along the template DNA strand.
Elongation
Promoter
Transcription unit
3′
5′
5′
3′
1 Initiation
Figure 17.7
Start point
RNA polymerase
3′
5′
5′
3′
Unwound
DNA
2 Elongation
5′
3′
Template strand of DNA
RNA
transcript
Rewound
DNA
3′
5′
RNA
transcript
3′
5′
Direction of
transcription
(“downstream”)
The RNA polymerase II moves downstream both untwist the DNA 10 to 20 bases at
a time and synthesizing the RNA transcript in the 5’→3’ direction.
Nucleotides are added to the 3′ end of the growing RNA molecule
Several RNA polymerase molecules can work together to transcribe a gene.
The DNA strands simultaneously reform a double helix just after RNA polymerase
has made the transcript.
Transcription progresses at a rate of 40 nucleotides per second in eukaryotes.
Figure 17.9
Nontemplate
strand of DNA
RNA nucleotides
RNA
polymerase
A
3′
T
C
C
A
A
5′
3′ end
A
U
C
C
U
A
3′
5′
T
5′
A
G
G
T
T
Direction of transcription
Template
strand of DNA
Newly made
RNA
Termination
Promoter
Transcription unit
3′
5′
5′
3′
1 Initiation
Start point
RNA polymerase
3′
5′
5′
3′
Unwound
DNA
2 Elongation
5′
3′
Template strand of DNA
RNA
transcript
Rewound
DNA
5′
RNA
transcript
3 Termination
3′
5′
3′
Direction of
transcription
(“downstream”)
3′
5′
5′
3′
5′
Completed RNA transcript
Figure 17.7
3′
Termination
• The mechanisms of termination are different in bacteria
and eukaryotes.
• In bacteria, the polymerase stops transcription at the end
of the terminator and the mRNA can be translated without
further modification
• In eukaryotes, the pre-mRNA is cleaved from the growing
RNA chain while RNA polymerase II continues to
transcribe the DNA.
• The RNA polymerase transcribes a DNA sequence called
the polyadenylation signal sequence, (AAUAAA) in the
pre-mRNA.
• At a point about 10 to 35 nucleotides past this sequence,
the pre-mRNA is cut from the enzyme.
• The completed single-stranded RNA transcript is released
and the RNA polymerase will detach from the DNA
strand.
Eukaryotic cells modify RNA after
transcription
• RNA processing-Enzymes in the
eukaryotic nucleus modify pre-mRNA
before it is released into the cytoplasm
• During RNA processing, both ends of the
mRNA transcript are usually altered
• Some interior parts of the molecule are
cut out, and the other parts spliced
together
© 2014 Pearson Education, Inc.
Alteration of mRNA Ends occurs in the
Nucleus
Figure 17.10
• Each end of a pre-mRNA molecule is modified in a particular way:
– The 5′ end receives a modified nucleotide 5′ cap consisting of
guanine
– The 3′ end gets a poly-A tail consisting of 50-250 adenine
nucleotides
• These modifications share several functions:
– They facilitate the export of mRNA into the cytoplasm
– They protect mRNA from hydrolytic enzymes
– They help ribosomes (in the cytoplasm) attach to the 5′ end
© 2014 Pearson Education, Inc.
Split Genes and RNA Splicing
Figure 17.11
• Most eukaryotic genes and their RNA transcripts have long
noncoding stretches of nucleotides that lie between coding regions
• These noncoding regions are called introns
• The other regions are called exons-they are eventually translated
into amino acid sequences
• RNA splicing removes introns and joins exons, creating an mRNA
molecule with a continuous coding sequence
The signal for RNA splicing is a short nucleotide
sequence at each end of an intron
• Particles called small nuclear ribonucleoproteins
(snRNPs) recognize the splice sites.
• snRNPs are located in the cell nucleus and are
composed of RNA and protein molecules.
• Each snRNP has several protein molecules and a small
nuclear RNA molecule (snRNA).
• Several different snRNPs join with a variety of proteins to
form a larger spliceosome.
• The spliceosome interacts with specific sites along an
intron, releasing the intron and joining together the two
exons that surrounded the intron.
© 2014 Pearson Education, Inc.
Figure 17-12
RNA transcript (pre-mRNA)
5′
Exon 1
Intron
Protein
snRNA
Exon 2
Other
proteins
snRNPs
Spliceosome
5′
Spliceosome
components
5′
mRNA
Exon 1
Exon 2
Cut-out
intron
Ribozymes
• Ribozymes are catalytic RNA molecules that function as
enzymes and can splice RNA
• The discovery of ribozymes rendered obsolete the belief
that all biological catalysts were proteins
• Three properties of RNA enable it to function as an
enzyme
– It can form a three-dimensional structure because of its
ability to base pair with itself
– Some bases in RNA contain functional groups
– RNA may hydrogen-bond with other nucleic acid
molecules
© 2014 Pearson Education, Inc.
Why is RNA splicing important?
The Functional and Evolutionary Importance of Introns
•
•
•
Split genes can encode for more than one polypeptide, depending on
which segments are treated as exons during RNA splicing.
Alternative RNA splicing gives rise to two or more different
polypeptides, depending on which segments are treated as exons.
Because of alternative splicing, the number of different protein
products an organism can produce is much greater than its number of
genes.
– The presence of introns increases the probability of crossing over
between genes.
– Introns increase the opportunity for recombination between two
alleles of a gene.
– Recombination raises the probability that a crossover will switch
one version of an exon for another version found on the
homologous chromosome.
– There may also be occasional mixing and matching of exons
between completely different genes.
– Either way, exon shuffling can lead to the production of new
proteins.
– Proteins often have a modular architecture consisting of discrete
regions called domains. In many cases, different exons code for
the different domains in a protein
© 2014 Pearson Education, Inc.
Figure 17.13
Gene
DNA
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 2
Domain 1
Polypeptide
Translation is the RNA-directed
synthesis of a protein
• Genetic information flows from mRNA to protein
through the process of translation.
• In the process of translation, a cell interprets a
series of codons along an mRNA molecule and
builds a protein.
• The interpreter is transfer RNA (tRNA), which
transfers amino acids from the cytosol to a
ribosome.
• The ribosome adds each amino acid carried by tRNA
to the growing end of the polypeptide chain.
• During translation, each type of tRNA links an mRNA
codon with the appropriate amino acid.
© 2014 Pearson Education, Inc.
tRNA
• The tRNA (transfer RNA), molecule is a translator
because it can read a the mRNA codon and translate it
into a protein.
• 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.
• Example: If the codon on mRNA is UUU, a tRNA with an
AAA anticodon and carrying phenylalanine amino acid
will bind to it.
• Codon by codon, tRNAs deposit amino acids in the
prescribed order, and the ribosome joins them into a
polypeptide chain.
© 2014 Pearson Education, Inc.
Translation
• As a molecule of mRNA
moves through a
ribosome, codons are
translated into amino
acids one at a time.
• tRNA have a specific
anticodon of 3 nucleotide
complementary sequence
to the mRNA on one end
and the corresponding
amino acid at the other
end.
• When H bonding between
the anticodon and mRNA
occurs, tRNA adds the
amino acid to the growing
polypeptide chain.
Figure 17.14
tRNA- Structure and Function
•
•
•
•
•
tRNA is transcribed in the nucleus from a
DNA template and must enter the
cytoplasm, where the ribosomes are
located.
Each tRNA is used repeatedly, picking up
its designated amino acid in the cytosol,
depositing the amino acid at the ribosome,
and returning to the cytosol to pick up
another copy of that amino acid.
A tRNA molecule consists of a single RNA
strand that is only about 80 nucleotides
long that folds back on itself to form a
three-dimensional structure. Flattened into
one plane to reveal its base pairing, a
tRNA molecule looks like a cloverleaf.
tRNA includes a loop containing the
anticodon and an attachment site at the 3′
end for an amino acid. Because of
hydrogen bonds, tRNA actually twists and
folds into a three-dimensional molecule
tRNA is roughly L-shaped
Figure 17.15
• Accurate translation requires two steps:
– First: a correct match between a tRNA and an
amino acid, done by the enzyme aminoacyltRNA synthetase
– Second: a correct match between the tRNA
anticodon and an mRNA codon
• Flexible pairing at the third base of a
codon is called wobble and allows some
tRNAs to bind to more than one codon
© 2014 Pearson Education, Inc.
Amino acids joined to the mRNA
• Each amino acid is joined to the correct tRNA by
aminoacyl-tRNA synthetase.
• The 20 different synthetases match the 20 different
amino acids.
• Each has active sites for only a specific tRNA–amino
acid combination.
• The synthetase catalyzes a covalent bond between them
in a process driven by ATP hydrolysis.
• The result is an aminoacyl-tRNA or charged amino acid.
• The second recognition process involves a correct match
between the tRNA anticodon and an mRNA codon.
© 2014 Pearson Education, Inc.
1 Amino acid
and tRNA
enter active
site.
Tyr-tRNA
A U A
Complementary
tRNA anticodon
Figure 17.16-1
Tyrosine (Tyr)
(amino acid)
Tyrosyl-tRNA
synthetase
1 Amino acid
and tRNA
enter active
site.
Tyrosine (Tyr)
(amino acid)
Tyrosyl-tRNA
synthetase
Tyr-tRNA
A U A
Complementary
tRNA anticodon
Figure 17.16-2
Aminoacyl-tRNA
synthetase
ATP
AMP + 2 P i
2 Using ATP,
synthetase
catalyzes
covalent
bonding.
tRNA
Amino
acid
Computer model
1 Amino acid
and tRNA
enter active
site.
Figure 17.16-3
Tyrosine (Tyr)
(amino acid)
Tyrosyl-tRNA
synthetase
Tyr-tRNA
A U A
Complementary
tRNA anticodon
3 Aminoacyl
tRNA
released.
Aminoacyl-tRNA
synthetase
ATP
AMP + 2 P i
2 Using ATP,
synthetase
catalyzes
covalent
bonding.
tRNA
Amino
acid
Computer model
The Ribosome is the site of translation
• Ribosomes facilitate specific coupling of tRNA
anticodons with mRNA codons in protein synthesis.
• Each ribosome is made up of a large and a small
subunit.
• The subunits are composed of proteins and ribosomal
RNA (rRNA).
• After rRNA genes are transcribed to rRNA in the
nucleus, the rRNA and proteins are assembled to form
the subunits with proteins from the cytoplasm.
• The large and small subunits join to form a functional
ribosome only when they attach to an mRNA molecule.
• Bacterial and eukaryotic ribosomes are somewhat
similar but have significant differences: some antibiotic
drugs specifically target bacterial ribosomes without
harming eukaryotic ribosomes
© 2014 Pearson Education, Inc.
Ribosomes
•
•
•
•
•
•
•
Each ribosome has a binding site
for mRNA and three binding sites
for tRNA molecules.
The P site (peptidyl-tRNA site) holds
the tRNA carrying the growing
polypeptide chain.
The A site (aminoacyl-tRNA site)
holds the tRNA with the next amino
acid to be added to the chain.
Discharged tRNAs leave the
ribosome at the E (exit) site.
The ribosome holds the tRNA and
mRNA in close proximity and
positions the new amino acid for
addition to the carboxyl end of the
growing polypeptide.
The ribosome then catalyzes the
formation of the peptide bond (via
rRNA).
As the polypeptide becomes longer,
it passes through an exit tunnel in
the ribosome’s large unit and is
released to the cytosol.
© 2014 Pearson Education, Inc.
Figure 17.17
Building a Polypeptide
• The three stages of translation:
– Initiation
– Elongation
– Termination
• All three stages require protein “factors”
that aid in the translation process
© 2014 Pearson Education, Inc.
Initiation of the Polypeptide Chain
• Initiation brings together mRNA, a tRNA, the
first amino acid, and the two ribosomal subunits.
• First, the small ribosomal subunit binds with
mRNA and a special initiator tRNA, which
carries methionine.
• The small subunit binds to the 5′ cap of the
mRNA and then moves, or scans, downstream
along the mRNA until it reaches the start codon
AUG, which signals the start of translation.
• This establishes the reading frame for the
mRNA.
• The initiator tRNA, already associated with the
complex, then hydrogen-bonds with the start
codon.
• Initiation factors (proteins) and GTP are required
for complex formation.
© 2014 Pearson Education, Inc.
Figure 17.18
Large
ribosomal
subunit
3′ U A C 5′
5′ A U G 3′
P site
Pi
Initiator
tRNA
+
GTP
GDP
E
mRNA
5′
Start codon
mRNA binding site
© 2014 Pearson Education, Inc.
3′
Small
ribosomal
subunit
5′
A
3′
Translation initiation complex
Elongation of the Polypeptide Chain
• During the elongation stage, amino acids
are added one by one to the preceding
amino acid at the C-terminus of the growing
chain
• Each addition involves proteins called
elongation factors and occurs in three steps:
codon recognition, peptide bond formation,
and translocation
• Translation proceeds along the mRNA in a
5′ to 3′ direction
© 2014 Pearson Education, Inc.
Figure 17.19-1
Amino end
of polypeptide
E
5′
3′
mRNA
P A
site site
1 Codon
recognition
GTP
GDP + P i
STEP-1
Codon Recognition-The anticodon of an
incoming aminoacyl tRNA (containing an amino
acid at one end) base pairs with the
complementary mRNA codon in the A site. GTP
is needed for this step of elongation.
E
P A
Figure 17.19-2
Amino end
of polypeptide
E
5′
3′
mRNA
P A
site site
STEP-2
Peptide Bond Formation- During
peptide bond formation, an rRNA
molecule catalyzes the formation of
a peptide bond between the
polypeptide in the P site and 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.
1 Codon
recognition
GTP
GDP + P i
E
P A
2 Peptide bond
formation
E
P A
STEP-3
Translocation - 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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Figure 17.19-3
Amino end
of polypeptide
E
Ribosome ready for
next aminoacyl tRNA
3′
mRNA
P A
site site
5′
1 Codon
recognition
GTP
GDP + P i
E
E
P A
P A
GDP + P i
3 Translocation
2 Peptide bond
formation
GTP
E
These three steps of elongation
continue to add amino acids
codon by codon until the
polypeptide chain is completed.
P A
Figure 17.20
Termination
Release
factor
Free
polypeptide
5′
3′
3′
5′
5′
Stop codon
(UAG, UAA, or UGA)
3′
GTP
2
2 GDP + 2 P i
2 Release factor
promotes hydrolysis.
3 Ribosomal subunits
and other components
dissociate.
Termination occurs when one of the three stop codons (UAG, UAA, or UGA).
When a ribosome reaches a stop codon along the mRNA strand, the A site accepts a
“release factor”- a protein shaped like a tRNA.
The release factor hydrolyzes the bond between the tRNA in the P site and the last amino
acid of the polypeptide chain by the addition of a water molecule . The polypeptide is then
released from the chain.
The two ribosomal machinery subunit disassembles-this requires GTP.
This reaction releases the polypeptide, and the translation assembly then comes apart
1 Ribosome reaches a
stop codon on mRNA.
Completing and Targeting the Functional
Protein
• Often translation is not sufficient to make a
functional protein
• Polypeptide chains are modified after
translation or targeted to specific sites in
the cell
Protein folding and post-translational
modifications
• During and after synthesis, a polypeptide coils and folds to its
three-dimensional shape spontaneously.
• The primary structure, the order of amino acids, determines the
secondary and tertiary structure.
• Chaperone proteins may aid correct folding.
• In addition, proteins may require post-translational
modifications before doing their particular job.
– These modifications may require additions such as sugars,
lipids, or phosphate groups to amino acids.
– Enzymes may remove some amino acids from the leading
(amino) end of the polypeptide chain.
– In some cases, a single polypeptide chain may be
enzymatically cleaved into two or more pieces.
– In other cases, two or more polypeptides may join to form a
protein with quaternary structure.
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Targeting polypeptides to specific
locations in the cell
• Two populations of ribosomes are evident in cells:
free ribsomes and bound ribosomes
• Free ribosomes are suspended in the cytosol and
synthesize proteins that reside in the cytosol.
• Bound ribosomes are attached to the cytosolic side of
the endoplasmic reticulum (ER) or to the nuclear
envelope.
• Bound ribosomes synthesize proteins of the
endomembrane system as well as proteins secreted
from the cell.
• Although bound and free ribosomes are identical in
structure, their location depends on the type of protein
they are synthesizing.
• Polypeptide synthesis always begins in the cytosol.
– The polypeptide that is being synthesized will cause
the ribosome to attach to the ER if it contains a signal
peptide sequence. Polypeptides of membrane
proteins and proteins that are to be secreted contain
signal peptide sequences.
– Signal peptides are around 20 amino acids long and
are located near the leading end of the polypeptide
chain.
• The signal peptide is recognized by a protein-RNA
complex called a signal-recognition complex.
© 2014 Pearson Education, Inc.
SRP’s
• A signal recognition particle (SRP) binds to the signal
peptide and attaches it and its ribosome to a receptor
protein in the ER membrane.
• The SRP, which consists of a protein–RNA complex,
functions as an adapter to bring the ribosome to a
receptor protein built into the ER membrane.
• The receptor is part of a multiprotein translocation
complex.
• Protein synthesis resumes, with the growing polypeptide
snaking across the membrane into the ER lumen via a
protein pore.
• An enzyme usually cleaves the signal polypeptide.
• Secretory proteins are released into solution within the
ER lumen, but membrane proteins remain partially
embedded in the ER membrane.
© 2014 Pearson Education, Inc.
Figure 17.21
2
1
3
Polypeptide SRP
SRP
synthesis
binds to binds to
begins.
signal
receptor
peptide. protein.
4
5
SRP
detaches
and
polypeptide
synthesis
resumes.
Signalcleaving
enzyme cuts
off signal
peptide.
6
Completed
polypeptide
folds into
final
conformation.
Ribosome
mRNA
Signal
peptide
Signal
peptide
removed
SRP
CYTOSOL
SRP receptor
protein
ER LUMEN
Translocation complex
ER
membrane
Protein
Targeting Proteins to the ER
1. Polypeptide synthesis begins on a free ribosome in the cytosol.
2. An SRP binds to the signal peptide and temporarily stops polypeptide
synthesis.
3. The SRP binds to a receptor protein in the ER membrane.
4. The SRP leaves and polypeptide synthesis resumes and translocation
across the ER membrane occurs.
5. The signal-cleaving enzyme cuts off the signal peptide.
6. The rest of the completed polypeptide leaves the ribosome and folds.
Making Multiple Polypeptides in Bacteria
and Eukaryote- Polyribosomes
• Typically a single mRNA
is used to make many
copies of a polypeptide
simultaneously.
• Multiple ribosomes,
polyribosomes or
polysomes, may trail
along the same mRNA.
• Polyribosomes can be
found in prokaryotic and
eukaryotic cells.
Figure 17.23
• A bacterial cell ensures a streamlined process
by coupling transcription and translation
• In this case the newly made protein can quickly
diffuse to its site of function
• In eukaryotes, the nuclear envelop separates
the processes of transcription and translation
• RNA undergoes processes before leaving
the nucleus
Figure 17.24
DNA
TRANSCRIPTION
3′
5′
RNA
polymerase
RNA
transcript
Exon
RNA
PROCESSING
RNA transcript
(pre-mRNA)
Intron
Aminoacyl-tRNA
synthetase
NUCLEUS
Amino
acid
CYTOPLASM
AMINO ACID
ACTIVATION
tRNA
mRNA
3′
E
P
A
Aminoacyl
(charged)
tRNA
Ribosomal
subunits
TRANSLATION
E
A
A A A
U G G U U U A U G
Codon
Ribosome
Anticodon
Point mutations (in DNA) can affect
protein structure and function
• Mutations result in changes in genetic material of a cell
or virus.
• Mutations can be either large-scale, in which long
segments of DNA are affected (for example,
translocations, duplications, and inversions), or point
mutations- changes in just one base pair of a gene.
• If a point mutation occurs in a gamete or in a cell that
produces gametes, it may be transmitted to future
generations.
• If the mutation has an adverse effect on the phenotype
of an organism, the mutant condition is referred to as a
genetic disorder or hereditary disease- sickle-cell
disease .
• A change in a single nucleotide in the DNA’s
template strand can lead to an abnormal protein.
Types of Point Mutations
• Point mutations within a gene can be
divided into two general categories
– Base-pair substitutions
– Base-pair insertions or deletions
© 2014 Pearson Education, Inc.
Substitutions
• A point mutation that results in the replacement of a pair
of complementary nucleotides with another nucleotide
pair is called a base-pair substitution.
• Some base-pair substitutions have little or no impact on
protein function.
• In silent mutations, altered nucleotides still code for the
same amino acids because of redundancy in the genetic
code.
• Missense mutations change an amino acid but have
little effect on protein function but not necessarily the
right amino acid.
• Nonsense mutations change an amino acid codon into
a stop codon, causing premature termination of
translation nearly always leading to a nonfunctional
protein.
• In some cases, the mutation switches one amino acid for
another with similar properties.
© 2014 Pearson Education, Inc.
Figure 17.25
Sickle cell Disease is caused by a point
mutation
Wild-type β-globin
Sickle-cell β-globin
Wild-type β-globin DNA
3′
5′
C T C
G A G
Mutant β-globin DNA
5′
3′
3′
5′
mRNA
5′
C A C
G T G
5′
3′
G U G
3′
mRNA
G A G
Normal hemoglobin
Glu
3′
5′
Sickle-cell hemoglobin
Val
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.
Insertions and Deletions
• Insertions and deletions are additions or losses of
nucleotide pairs in a gene.
• Insertions and deletions have a disastrous effect on the
resulting protein more often than substitutions do.
• Unless insertion or deletion mutations occur in multiples
of 3, they cause a frameshift mutation.
• All the nucleotides downstream of the deletion or
insertion will be improperly grouped into codons.
• The result will be extensive missense, ending sooner or
later in nonsense—premature termination.
• Mutations can occur during DNA replication, DNA repair, or DNA
recombination
© 2014 Pearson Education, Inc.
Figure 17.26
Wild type
Types of Point
Mutations
DNA template strand 3′ T A C T T C A A A C C G A T T 5′
5′ A T G A A G T T T G G C T A A 3′
mRNA5′ A U G A A G U U U G G C U A A 3′
Protein
Met
Lys
Phe
Gly
Stop
Carboxyl end
Amino end
(b) Nucleotide-pair insertion or deletion
(a) Nucleotide-pair substitution
Extra A
A instead of G
3′ T A C T T C A A A C C A A T T 5′
5′ A T G A A G T T T G G T T A A 3′
3′ T A C A T T C A A A C C G A T T 5′
5′ A T G T A A G T T T G G C T A A 3′
Extra U
U instead of C
5′ A U G A A G U U U G G U U A A 3′
Met
Lys
Phe
Gly
Stop
Silent (no effect on amino acid sequence)
5′ A U G U A A G U U U G G C U A A 3′
Met
Stop
Frameshift causing immediate nonsense
(1 nucleotide-pair insertion)
T instead of C
A missing
3′ T A C T T C A A A T C G A T T 5′
5′ A T G A A G T T T A G C T A A 3′
3′ T A C T T C A A C C G A T T 5′T
′
5′ A T G A A G T T G G C T A A 3A
A instead of G
U missing
5′ A U G A A G U U U A G C U A A 3′
Met
Lys
Phe
Ser
Stop
Missense
5′ A U G A A G U U G G C U A A
Met
Lys
Leu
3′
Ala
Frameshift causing extensive missense
(1 nucleotide-pair deletion)
A instead of T
3′ T A C A T C A A A C C G A T T 5′
5′ A T G T A G T T T G G C T A A 3′
U instead of A
5′ A U G U A G U U U G G C U A A 3′
Met
Nonsense
Stop
T T C missing
3′ T A C A A A C C G A T T 5′
5′ A T G T T T G G C T A A 3′
A A G missing
′ A A
5′ A U G U U U G G C U A A 3U
Met
Phe
Gly
Stop
No frameshift, but one amino acid missing
(3 nucleotide-pair deletion)
Mutagens
• Mutagens are chemical or physical agents that interact
with DNA to cause mutations.
• Physical agents include high-energy radiation like X-rays
and ultraviolet light.
– Ultraviolet light can cause disruptive thymine dimers in DNA.
• Chemical mutagens cause mutations in different ways.
– Some chemicals may be substituted into DNA, but they pair
incorrectly during DNA replication.
– Other mutagens interfere with DNA replication by inserting into
DNA and distorting the double helix.
– Still others cause chemical changes in nucleotide bases that
change their pairing properties.
© 2014 Pearson Education, Inc.
While gene expression differs among the domains
of life, the concept of a gene is universal
• Archaea are prokaryotes, but share many features of gene
expression with eukaryotes
• Bacteria and eukarya differ in their RNA polymerases,
termination of transcription and ribosomes; archaea tend to
resemble eukarya in these respects
• Bacteria can simultaneously transcribe and translate the
same gene
• In eukarya, transcription and translation are separated by
the nuclear envelope
• In archaea, transcription and translation are likely coupled
© 2014 Pearson Education, Inc.
What Is a Gene? Revisiting the Question
• The idea of the gene itself is a unifying concept
of life
• We have considered a gene as:
– A discrete unit of inheritance
– A region of specific nucleotide sequence in a
chromosome
– A DNA sequence that codes for a specific polypeptide
chain
• In summary, a gene can be defined as a
region of DNA that can be expressed to
produce a final functional product, either a
polypeptide or an RNA molecule
© 2014 Pearson Education, Inc.
Figure 17.UN09
Type of RNA
Functions
Messenger RNA (mRNA)
Transfer RNA (tRNA)
Plays catalytic (ribozyme) roles and
structural roles in ribosomes
Primary transcript
Small RNAs in the spliceosome