Chapter 17
From Gene to Protein
AP Biology
Overview: The Flow of Genetic Information
•
The information content of 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
Fig. 17-1
•
Gene expression: the process by
which DNA directs protein synthesis
–
Includes two stages:
transcription and translation
Concept 17.1:
Genes specify proteins via
transcription and translation
Linking Genes to Enzymes
•
In 1909, 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
–
He referred to such errors as “inborn errors of metabolism”
• Ex) Alkaptonuria results from inability to make the enzyme
alkapton
•
Linking genes to enzymes required understanding that cells synthesize and
degrade molecules in a series of steps, a metabolic pathway
Nutritional Mutants in Neurospora
•
A breakthrough in the relationship between genes and enzymes came with the work
of George Beadle and Edward Tatum
–
Exposed the bread mold Neurospora crassa to X-rays
–
This created mutants that were unable to survive on a minimal medium (agar)
as a result of inability to synthesize certain molecules
Fig. 17-2a
•
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
EXPERIMENT
specific enzyme
Growth:
Wild-type
cells growing
and dividing
No growth:
Mutant cells
cannot grow
and divide
Minimal medium
Beadle and Tatum’s Experiment
Beadle and Tatum showed that the Neurospora crassa mutants fell into 3 classes,
each defective in a different gene
–
They grew these 3 classes of mutants under 4 different conditions
•
The wild-type strain was capable of growth under all experimental
conditions, requiring only minimal medium
•
The 3 classes of mutants each had a specific set of growth requirements
Fig. 17-2b
–
Ex) Class II mutants
RESULTS
could not grow when
Minimal
medium
(MM)
(control)
ornithine alone
was added but
could grow when
either citrulline
or arginine was
added
Classes of Neurospora crassa
Wild type
Condition
•
MM +
ornithine
MM +
citrulline
MM +
arginine
(control)
Class I mutants Class II mutantsClass III mutants
Beadle and Tatum’s Experiment Continued
•
Based on the growth requirements of these mutants, Beadle and Tatum deduced
that each class of mutants was unable to carry out one step in the pathway for
synthesizing arginine because it lacked the necessary enzyme
–
Because each mutant was mutated in only a single gene, they concluded that
each mutated gene must normally dictate the production of one enzyme
–
Fig. 17-2c
Their results supported the one gene-one enzyme hypothesis and also
confirmed the arginine pathway
CONCLUSION
Wild type
Precursor
Gene A
Gene B
Gene C
Class I mutants Class II mutantsClass III mutants
(mutation in
(mutation in
(mutation in
gene A)
gene B)
gene C)
Precursor
Precursor
Precursor
Enzyme A
Enzyme A
Enzyme A
Enzyme A
Ornithine
Ornithine
Ornithine
Ornithine
Enzyme B
Enzyme B
Enzyme B
Enzyme B
Citrulline
Citrulline
Citrulline
Citrulline
Enzyme C
Enzyme C
Enzyme C
Enzyme C
Arginine
Arginine
Arginine
Arginine
The Products of Gene Expression: A Developing Story
•
As researchers learned more about proteins, they made revisions to the one geneone enzyme hypothesis
–
Some proteins aren’t enzymes, so researchers later renamed the hypothesis to
read one gene–one protein
–
Many proteins are composed of several polypeptides, each of which has its
own gene
•
Ex) Hemoglobin is built from 2 polypeptides, and thus 2 genes code for this
single protein
–
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
more precisely as polypeptides
Basic Principles of Transcription and Translation
•
Though genes provide the instructions for making specific proteins, they do not build
proteins directly
–
•
Transcription is the synthesis of RNA under the direction of DNA
–
•
RNA is the intermediate between genes and the proteins for which they code
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
The central dogma is the concept that cells are governed by a cellular chain of
command: DNA RNA protein
Protein Synthesis in Prokaryotes
•
Though the basic mechanics of protein synthesis are similar for bacteria and
eukaryotes, there are important differences
•
Because prokaryotes lack nuclei, the lack of segregation between DNA
and ribosomes allows translation of mRNA to begin while transcription
Fig. 17-3a-2
is still in progress
• The mRNA produced by
transcription is immediately
translated without
TRANSCRIPTION
more processing
DNA
mRNA
Ribosome
TRANSLATION
Polypeptide
(a) Bacterial cell
Protein Synthesis in Eukaryotes
•
In eukaryotic cells, the nuclear envelope separates transcription and translation in
space and time
•
Transcription occurs in the nucleus, and the mRNA is then transported to the
cytoplasm, where translation occurs
•
Eukaryotic RNA transcripts are also modified through RNA processing to yield
Fig. 17-3b-3
finished mRNA
•
Nuclear
envelope
A primary transcript (or pre-mRNA)
is the initial RNA transcript from any
gene, including those coding for RNA
DNA
TRANSCRIPTION
Pre-mRNA
RNA PROCESSING
that are not translated into protein
mRNA
TRANSLATION
Ribosome
Polypeptide
(b) Eukaryotic cell
The Genetic Code
•
Biologists encountered a problem when they began to suspect that the
instructions for protein synthesis were encoded in DNA
–
There are 20 amino acids, but there are only four nucleotide bases in
DNA
–
•
Therefore, how many bases correspond to an amino acid?
The flow of information from gene to protein is based on a triplet code: a
series of nonoverlapping, three-nucleotide words
–
These triplets are the smallest units of uniform length that can code for
all the amino acids (43 = 64)
• Ex) AGT = serine
Transcription of the DNA Template
•
For each gene, only one of the 2 DNA strands is transcribed
–
This strand is called the template strand because it provides the pattern (or
template) for the sequence of nucleotides in an RNA transcript
•
A given DNA strand is the template strand for some genes, while for other
genes, the complementary strand functions as a template
–
An mRNA is complementary rather than identical to its DNA template
•
RNA bases are assembled according to base-pairing rules
•
These pairs are similar to those that form during DNA replication, with one
exception
– The RNA substitute for thymine that pairs with adenine is called uracil
(U)
Codons
•
During translation, the mRNA base triplets, called codons, are read in the 5
to 3 direction
Fig. 17-4
–
Each codon specifies the
amino acid to be placed at the
DNA
molecule
Gene 2
Gene 1
Gene 3
corresponding position along a
polypeptide
DNA
template
strand
TRANSCRIPTION
mRNA
Codon
TRANSLATION
Protein
Amino acid
Cracking the Code
All 64 codons were deciphered by the mid-1960s
–
Of the 64 triplets, 61 code for amino acids
•
3 triplets are “stop” signals to end translation
Fig. 17-5
–
Second mRNA base
The genetic code is redundant but not
ambiguous
No codon specifies more than one
amino acid
–
Codons must be read in the correct
reading frame (correct groupings) in
order for the specified polypeptide
to be produced
Third mRNA base (3 end of codon)
•
First mRNA base (5 end of codon)
•
Evolution of the Genetic Code
•
The genetic code is nearly universal, shared by the simplest bacteria to the most
complex animals
–
•
Ex) CCG is translated into the amino acid proline in all organisms
In laboratory experiments, genes can be transcribed and translated after being
transplanted from one species to
Fig. 17-6
another
–
Ex) Bacteria can be
programmed to synthesize
human proteins (insulin) for
medical use by the insertion
of human genes
(a) Tobacco plant expressing
a firefly gene
(b) Pig expressing a
jellyfish gene
Concept Check 17.1
•
1) What polypeptide product would you expect from a poly-G mRNA that is
30 nucleotides long?
•
2) The template strand of a gene contains the sequence 3’-TTCAGTCGT-5’.
Draw the nontemplate sequence and the mRNA sequence, indicating the 5’
and 3’ ends of each. Compare the two sequences.
•
3) Imagine that the nontemplate sequence in question 2 was transcribed
instead of the template sequence. Draw the mRNA sequence and translate
it using Figure 17.5 (pp. 330). (Be sure to pay attention to the 5’ and 3’
ends.) Predict how well the protein synthesized from the nontemplate strand
would function, if at all.
Concept 17.2:
Transcription is the DNA-directed
synthesis of RNA: a closer look
Transcription: A Closer Look
•
RNA synthesis is catalyzed by an enzyme called RNA polymerase
–
This enzyme pries the DNA strands apart and hooks together the RNA
nucleotides
•
The DNA sequence where RNA polymerase attaches is called the promoter
–
In bacteria, the sequence signaling the end of transcription is called the
terminator
–
The stretch of DNA that is transcribed is called a transcription unit
–
RNA synthesis follows the same base-pairing rules as DNA, except
uracil substitutes for thymine
•
There are 3 stages of transcription: initiation, elongation, and termination
Stages of Transcription: Initiation
Step 1: Initiation
•
RNA polymerase binds to the promoter
•
DNA strands unwind
•
Polymerase initiates RNA synthesis at the start point on the template
strandFig. 17-7a-2
Promoter
Transcription unit
5
3
Start point
RNA polymerase
3
5
DNA
1
Initiation
5
3
Unwound
DNA
3
5
RNA
transcript
Template strand
of DNA
Stages of Transcription: Elongation
•
Step 2: Elongation
–
As RNA polymerase moves “downstream,” the enzyme unwinds DNA and
elongates the RNA transcript 5’3’
•
Biologists refer to the direction of transcription as “downstream” and the
Fig. 17-7a-3
other direction as “upstream”
•
These terms are also used to
describe positions of nucleotide
sequences within DNA and RNA
–
Ex) The promoter sequence in
Promoter
5
3
Start point
RNA polymerase
strands reform a double helix
DNA
5
3
3
5
Unwound
DNA
RNA
transcript
Template strand
of DNA
2 Elongation
terminator
In the wake of transcription, the DNA
3
5
1 Initiation
DNA is upstream from the
–
Transcription unit
Rewound
DNA
5
3
3
5
RNA
transcript
3
5
Stages of Transcription: Termination
•
Step 3: Termination
–
Fig. 17-7a-4
The RNA transcript is
eventually released
–
Promoter
Transcription unit
5
3
Start point
RNA polymerase
DNA
1
RNA polymerase
5
3
detaches from the DNA
Unwound
DNA
3
5
Initiation
3
5
RNA
transcript
Template strand
of DNA
2 Elongation
Rewound
DNA
5
3
3
5
3
5
RNA
transcript
3 Termination
5
3
3
5
5
Completed RNA transcript
3
RNA Polymerase Binding and Initiation of Transcription
•
•
Promoters signal the initiation of RNA synthesis
–
Include the transcription start point (the nucleotide where RNA synthesis
actually begins) and extends several dozen nucleotides pairs upstream from
start point
–
Determine which of the 2 DNA strands is used as a template
A collection of proteins called transcription factors mediate the binding of RNA
polymerase and the initiation of transcription in eukaryotes
–
In prokaryotes, RNA polymerase itself
recognizes and binds to the promoter
–
The completed assembly of transcription
factors and RNA polymerase II bound
to a promoter is called a transcription
initiation complex
–
A promoter called a TATA box is
crucial in forming the initiation complex
in eukaryotes
Elongation of the RNA Strand
• As RNA polymerase moves along the DNA, it untwists the
double helix, 10 to 20 bases at a time
– The enzyme adds nucleotides to the 3’ end of the new RNA
molecule at a rate of 40 nucleotides per second in
eukaryotes
• A gene can be transcribed simultaneously by several RNA
polymerases
– Congregation of many polymerase molecules increases the
amount of mRNA transcribed, allowing cells to make the
encoded proteins in large amounts
Termination of Transcription
•
The mechanisms of termination are different in bacteria and eukaryotes
–
In bacteria, the polymerase stops transcription at the end of the terminator
–
In eukaryotes, the polymerase continues transcription after the pre-mRNA is
cleaved from the growing RNA chain
•
RNA polymerase transcribes a sequence called the polyadenylation signal
sequence (AAUUAA) in pre-mRNA
•
Proteins associated with new RNA transcript cut it free from polymerase at
a point ~10-35 nucleotides downstream from polyadenylation signal
•
Polymerase continues transcribing DNA for 100s of nucleotides past the
site where pre-mRNA is released
•
The polymerase eventually falls off the DNA
Concept Check 17.2
•
1) Compare DNA polymerase and RNA polymerase in terms of how they
function, the requirement for a template and primer, the direction of
synthesis, and the type of nucleotides used.
•
2) What is a promoter, and is it located at the upstream or downstream end
of a transcription unit
•
3) What makes RNA polymerase start transcribing a gene at the right place
on the DNA in a bacterial cell? In a eukaryotic cell?
•
4) Suppose X-rays caused a sequence change in the TATA box of a
particular gene’s promoter. How would that affect transcription of the gene?
(See Figure 17.8, pp. 333).
Concept 17.3:
Eukaryotic cells modify RNA after
transcription
Eukaryotic cells modify RNA after transcription
• Enzymes in the eukaryotic nucleus modify pre-mRNA before
the genetic messages are dispatched to the cytoplasm
– During RNA processing, both ends of the primary transcript
are usually altered
– Also, usually some interior parts of the molecule are cut
out, and the other parts spliced together
– These modifications produce an mRNA molecule ready for
translation
Alteration of mRNA Ends
•
Each end of a pre-mRNA molecule is modified in a particular way:
–
The 5 end receives a modified nucleotide 5 cap consisting of a modified
guanine nucleotide
•
–
The 3 end gets a poly-A tail before mRNA exits the nucleus
Fig. 17-9
•
•
Occurs after transcription of the first 20-40 nucleotides
Consists of 50-250 additional adenine nucleotides following the
polyadenylation signal (AAUAAA)
These modifications share several functions:
–
They seem to facilitate the export of mRNA from nucleus
–
They protect mRNA from degradation by hydrolytic enzymes
–
They help ribosomes attach to the 5 end of mRNA once it reaches the
cytoplasm
Protein-coding segment Polyadenylation signal
5
3
G
P P P
5 Cap
AAUAAA
5 UTR Start codon
Stop codon
3 UTR
AAA…AAA
Poly-A tail
Split Genes and RNA Splicing
•
Most eukaryotic genes and their RNA transcripts have long noncoding stretches of
nucleotides that lie between coding regions
•
These noncoding regions are called intervening sequences, or introns
•
The other regions are called exons because they are eventually expressed,
usually translated into amino acid sequences
Fig. 17-10
•
RNA splicing removes introns and joins exons, creating an mRNA molecule
with a continuous coding sequence
5 Exon Intron
Exon
Exon
Intron
3
Pre-mRNA 5 Cap
Poly-A tail
1
30
31
Coding
segment
mRNA 5 Cap
1
5 UTR
104
105
146
Introns cut out and
exons spliced together
Poly-A tail
146
3 UTR
snRNPs and Spliceosomes
•
The signal for RNA splicing is a short nucleotide sequence at each end of an intron
–
Particles called small nuclear ribonucleoproteins (snRNPs) recognize these
splice sites
•
Fig. 17-11-3
The RNA in a snRNP particle is called
RNA transcript (pre-mRNA)
5
Exon 1
Intron
Exon 2
a small nuclear RNA (snRNA)
–
Several different snRNPs join with additional
Protein
snRNA
Other
proteins
snRNPs
proteins to form even larger assemblies
Spliceosome
called spliceosomes
5
•
Spliceosomes interact with certain sites along
an intron, releasing the intron and
joining together the 2 exons that
flanked the intron
Spliceosome
components
5
mRNA
Exon 1
Exon 2
Cut-out
intron
Ribozymes
•
•
In some organisms, RNA splicing can occur without proteins or even additional RNA
molecules
–
Ribozymes are catalytic RNA molecules that function as enzymes and can
splice RNA
–
Some intron RNA functions as a ribozyme and catalyze their own excision
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, as well as its single-stranded structure
•
Some bases in RNA contain functional groups that can participate in
reactions
•
RNA may hydrogen-bond with other nucleic acid molecules
The Functional and Evolutionary Importance of Introns
•
A direct consequence of the presence of introns in genes is that a single
gene can encode more than one kind of polypeptide
–
Many genes can give rise to 2+ different polypeptides depending on
which segments are treated as exons during RNA processing
• Such variations are called alternative RNA splicing
– Ex) Sex differences in fruit flies are largely due to differences in
how males and females splice their transcribed RNA
–
Because of alternative splicing, the number of different proteins an
organism can produce is much greater than its number of genes
Domains
•
Proteins often have a modular architecture consisting of discrete regions called
domains
Fig. 17-12
Gene
DNA
–
Ex) One domain may include the active site
Exon 1 Intron Exon 2 Intron Exon 3
Transcription
while another might attach the it to the
RNA processing
cell membrane
Translation
•
In many cases, different exons code for the different
Domain 3
domains in a protein
–
Exon shuffling may result in evolution of
Domain 2
Domain 1
new proteins
Polypeptide
•
Introns increase the probability of crossing over between exons of
alleles by providing more terrain for crossovers without interrupting coding
sequences
•
Occasional mixing and matching of exons between completely different
(nonallelic) genes may also occur
Concept Check 17.3
• 1) How does alteration of the 5’ and 3’ ends of pre-mRNA affect
the mRNA that exits the nucleus?
• 2) How is RNA splicing similar to editing a video?
• 3) In nematode worms, a gene that codes for an ATPase has
two alternatives for exon 4 and three alternatives for exon 7.
How many different forms of the protein could be made from
this gene?
Concept 17.4:
Translation is the RNA-directed
synthesis of a polypeptide: a closer
look
Transfer RNA
The translation of mRNA to protein can be examined in more detail
A cell translates an mRNA message into protein with the help of transfer RNA
(tRNA)
•
Molecules of tRNA are not identical:
Fig. 17-13
–
Each carries a specific amino acid on one end
– Each has an anticodon on the other end
•
Amino
acids
Polypeptide
The anticodon base-pairs with a
tRNA with
amino acid
attached
complementary codon on mRNA
Ribosome
p
–
Tr
•
Phe
Gly
tRNA
Anticodon
Codons
5
mRNA
3
The Structure and Function of Transfer RNA
•
A tRNA molecule consists of a single RNA strand that is only about 80
nucleotides long
–
Fig. 17-14
A
C
C
3
Amino acid
attachment site
5
Flattened into one plane to reveal its base pairing,
a tRNA molecule looks like a cloverleaf
–
Because of hydrogen bonds, tRNA actually twists
Hydrogen
bonds
and folds into a three-dimensional molecule
• tRNA is roughly L-shaped
Anticodon
(a) Two-dimensional structure
Amino acid
attachment site
5
3
Hydrogen
bonds
3
Anticodon
(b) Three-dimensional structure
5
Anticodon
(c) Symbol used
in this book
Steps Required for Accurate Translation
•
Accurate translation requires two steps:
–
First: a correct match between a tRNA and an amino acid, done by the enzyme
aminoacyl-tRNA synthetase
–
Second: a correct match between the tRNA anticodon and an mRNA codon
•
Some tRNAs can bond to more than one codon because the rules for
base-pairing between the 3rd base of a codon and its corresponding
anticodon are more relaxed than those of the 1st two bases
–
Ex) Uracil at the 5’ end of a tRNA anticodon can pair with either
adenine or guanine in the 3rd position of an mRNA codon
–
This flexible pairing at the third base of a codon is called wobble
•
Wobble explains why codon synonymous for an amino acid can
differ in their 3rd base
Matching tRNA and Amino Acids
•
Aminoacyl-tRNA sythetases catalyze the covalent attachment of amino
acids to their appropriate tRNA in a process driven by the hydrolysis of ATP
–
Fig. 17-15-4
1) The active site binds the amino acid
and ATP
Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P P P
Adenosine
ATP
–
2) ATP loses 2 phosphate groups and
joins the amino acid as AMP
P
P Pi
–
Pi
3)The appropriate tRNA covalently binds to
Adenosine
tRNA
Aminoacyl-tRNA
synthetase
Pi
tRNA
the amino acid, displacing AMP
–
4)The tRNA and its bound amino acid is
released by the enzyme
P
Adenosine
AMP
Computer model
Aminoacyl-tRNA
(“charged tRNA”)
Ribosomes
•
Ribosomes facilitate specific coupling of tRNA anticodons with mRNA codons in
protein synthesis
–
Fig. 17-16
Growing
polypeptide
Exit tunnel
tRNA
molecules
Ribosomes are made up of 2 subunits, called the
EP
large and small subunits
•
Large
subunit
A
Small
subunit
5
These two ribosomal subunits are made of
mRNA 3
(a) Computer model of functioning ribosome
proteins and ribosomal RNA (rRNA)
–
A ribosome has three binding sites for tRNA:
•
The P site holds the tRNA that carries the
growing polypeptide chain
•
The A site holds the tRNA that carries the
P site (Peptidyl-tRNA
binding site)
E site
(Exit site)
A site (AminoacyltRNA binding site)
E P A
mRNA
binding site
The E site is the exit site, where discharged
tRNAs leave the ribosome
carries
Small
subunit
(b) Schematic model showing binding sites
Growing polypeptide
Amino end
Next amino acid
to be added to
polypeptide chain
next amino acid to be added to the chain
•
Large
subunit
E
mRNA
5
tRNA
3
Codons
(c) Schematic model with mRNA and tRNA
Building a Polypeptide
•
•
The three stages of translation:
–
Initiation
–
Elongation
–
Termination
All three stages require protein “factors” that aid in the translation process
–
Energy is also required for certain aspects of chain initiation and
elongation
• This energy is provided by the hydrolysis of GTP
Ribosome Association and Initiation of Translation
•
The initiation stage of translation brings together mRNA, a tRNA with the first amino
acid, and the two ribosomal subunits
–
First, a small ribosomal subunit binds with mRNA and a special initiator tRNA
carrying the amino acid methionine
–
Then the small subunit moves along the mRNA until it reaches the start codon
(AUG)
–
Proteins called initiation factors bring in the large subunit that completes the
translation initiation complex
•
–
Fig. 17-17
The cell must expend energy
in the form of a GTP molecule to form this
initiation complex
At the completion of the initiation process, the initiator tRNA sits in the P site of
Large
the ribosome
ribosomal
•
The A site is vacant and ready
for the next aminoacyl
Initiator
tRNA
tRNA
3 U A C 5
t
e
5 A U G 3
M
P site
Met
subunit
GTP GDP
E
mRNA
5
Start codon
mRNA binding site
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
–
Each addition involves several proteins called elongation factors and
occurs in three steps
• 1) Codon recognition
• 2) Peptide bond formation
• 3) Translocation
–
Two molecules are GTP are required, one for codon recognition and
the other for translocation
Steps in the Elongation of a Polypeptide Chain
•
Step 1 - Codon recognition: the anticodon of an incoming aminoacyl tRNA basepairs with the complementary mRNA codon in the A site
–
•
Hydrolysis of GTP increases the accuracy and efficiency of this step
Step 2 - Peptide bond formation: an rRNA molecule of the large ribosomal subunit
catalyzes the formation of a peptide bond between the new amino acid in the A site
and the carboxyl end of the growing polypeptide in the P site
Fig. 17-18-2
–
This step removes the polypeptide from
Amino end
of polypeptide
the tRNA in the P site and attaches
it to the amino acid on the tRNA
E
5
in the A site
3
mRNA
P A
site site
GTP
GDP
E
P A
Steps in the Elongation of a Polypeptide Chain
•
Step 3 – Translocation: the ribosome translocates the tRNA in the A site to the P
site
–
At the same time, the empty tRNA in the P site is moved to the E site, where it
is released
–
Fig. 17-18-4
Amino end
of polypeptide
The mRNA then moves along with
its bound tRNAs, bringing the next
E
3
mRNA
codon to be translated into
Ribosome ready for
next aminoacyl tRNA
P A
site site
5
GTP
GDP
the A site
E
E
P A
P A
GDP
GTP
E
P A
Termination of Translation
•
Termination occurs when a stop codon in the mRNA reaches the A site of the
ribosome
–
The base triplets UAG, UAA, and UGA act as signals to stop translation
–
A protein called a release factor binds directly to the stop codon in the A site
•
•
The release factor causes the addition of a water molecule instead of an
amino acid
This reaction breaks (hydrolyzes) the bond between the completed polypeptide and
the tRNA in the P site
–
This releases the polypeptide
through the exit tunnel of the ribosome’s large
Fig. 17-19-3
subunit
–
The remainder of the translation assembly then comes apart in a multistep
process, aided by other protein factors
•
This breakdown requires the hydrolysis of 2 more GTP molecules
Release
factor
Free
polypeptide
3
5
Animation: Translation
5
Stop codon
(UAG, UAA, or UGA)
5
3
2 GTP
2 GDP
3
Polyribosomes
•
A number of ribosomes can translate a single mRNA simultaneously, forming a
polyribosome (or polysome)
–
Fig. 17-20
Once a ribosome moves past the start
Growing
polypeptides
codon, a second ribosome can attach
to the mRNA
•
Incoming
ribosomal
subunits
Polyribosomes enable a cell to make
Start of
mRNA
(5 end)
many copies of a polypeptide very
quickly
Completed
polypeptide
(a)
Polyribosom
e
End of
mRNA
(3 end)
Ribosomes
mRNA
(b)
0.1 µm
Completing and Targeting the Functional Protein
• Often translation is not sufficient to make a
functional protein
– Polypeptide chains are modified after
translation
– Completed proteins are then targeted to
specific sites in the cell
Protein Folding and Post-Translational Modifications
•
During and after synthesis, a polypeptide chain spontaneously coils and folds into its
three-dimensional shape
•
Proteins may also require post-translational modifications before doing their job
–
Certain amino acids may be chemically modified by the attachment of sugars,
lipids, phosphate groups, or other additions
–
Enzymes may remove one or more amino acids from the leading (amino) end
of the polypeptide chain
–
A polypeptide chain may also be cleaved by enzymes into two or more pieces
(insulin)
•
In other cases, two or more polypeptides that are synthesized separately
may come together, becoming subunits of a protein with a quaternary
structure (hemoglobin)
Targeting Polypeptides to Specific Locations
• Two populations of ribosomes are evident in cells: free
ribsomes (in the cytosol) and bound ribosomes (attached to the
ER)
– Free ribosomes mostly synthesize proteins that function in
the cytosol
– Bound ribosomes make proteins of the endomembrane
system and proteins that are secreted from the cell
• Ribosomes are identical and can switch from free to
bound
Targeting Polypeptides to Specific Locations
•
What determines whether a ribosome will be free in the cytosol or bound to rough ER
at any given time?
•
Polypeptide synthesis always begins in the cytosol
•
The process continues there until completion unless the growing polypeptide
itself cues the ribosome to attach to the ER
•
Polypeptides destined for the ER or for secretion are marked by a signal
peptide
that targets the protein to the ER
Fig. 17-21
•
The signal peptide is a sequence of ~20 amino acids at or near the
leading (amino) end of the polypeptide
Ribosome
mRNA
Signal
peptide
Signal
peptide
removed
Signalrecognition
particle (SRP)
CYTOSOL
ER LUMEN
Translocation
complex
SRP
receptor
protein
ER
membrane
Protein
SRPs
•
The signal peptide is recognized as it emerges from the ribosome by a protein-RNA
complex called a signal-recognition particle (SRP)
•
The SRP brings the signal peptide and its ribosome to a receptor protein built
into the ER membrane
•
Polypeptide synthesis continues here, and the growing polypeptide moves
into the ER lumen via a protein pore
•
The signal peptide is usually removed by an enzyme
•
If it is to be secreted from the cell, the rest of the completed polypeptide in
17-21
releasedFig.into
solution in within the ER lumen
•
If the polypeptide is to be a membrane protein, it remains partially
embedded in the ER membrane
Ribosome
mRNA
Signal
peptide
Signal
peptide
removed
Signalrecognition
particle (SRP)
CYTOSOL
ER LUMEN
Translocation
complex
SRP
receptor
protein
ER
membrane
Protein
Concept Check 17.4
• 1) What two processes ensure that the correct amino acid is
added to a growing polypeptide chain?
• 2) Describe how the formation of polyribosomes can benefit the
cell.
• 3) Describe how a polypeptide to be secreted is transported to
the endomembrane system.
• 4) Discuss the ways in which rRNA structure likely contributes
to ribosomal function.
Concept 17.5:
Point mutations can affect protein
structure and function
Point Mutations
•
Mutations are changes in the genetic material of a cell or virus
–
Point mutations are chemical changes in just one base pair of a gene
–
The change of a single nucleotide in a DNA template strand can lead to the
production of an abnormal protein
•
Ex) The genetic basis of sickle cell disease is the mutation of a single base
pair in the gene that encodes one of the polypeptides of hemoglobin
Fig. 17-22
Wild-type hemoglobin DNA
Mutant hemoglobin DNA
C T T
C A T
3
5 3
5
G T A
G A A
3 5
mRNA
5
5
3
mRNA
G A A
Normal hemoglobin
Glu
3 5
G U A
Sickle-cell hemoglobin
Val
3
Types of Point Mutations
• Point mutations within a gene can be divided
into two general categories
– Base-pair substitutions
– Base-pair insertions or deletions
Substitutions
•
A base-pair substitution replaces one nucleotide and its partner with
another pair of nucleotides
–
Some substitutions are called silent mutations
• These substitutions have no effect on the amino acid produced by
a codon because of redundancy in the genetic code
–
Missense mutations still code for an amino acid, but not necessarily
the right amino acid
–
Nonsense mutations change an amino acid codon into a stop codon,
nearly always leading to a nonfunctional protein
Fig. 17-23a
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)
Fig. 17-23b
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
Fig. 17-23c
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
Insertions and Deletions
•
Insertions and deletions are additions or losses of nucleotide pairs in a
gene
–
These mutations have a disastrous effect on the resulting protein more
often than substitutions do
–
Insertion or deletion of nucleotides may alter the reading frame,
producing a frameshift mutation
• All the nucleotides that are downstream of the deletion or insertion
will be improperly grouped into codons
• Unless the frameshift is very near the end of a gene, the protein is
almost certain to be nonfunctional
Fig. 17-23d
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)
Fig. 17-23e
Wild type
DNA template 3
strand 5
5
3
mRNA 5
3
Protein
Stop
Amino end
Carboxyl end
missing
5
3
3
5
missing
5
3
Frameshift causing extensive missense (1 base-pair deletion)
Fig. 17-23f
Wild type
DNA template 3
strand 5
5
3
mRNA 5
3
Protein
Stop
Amino end
Carboxyl end
missing
5
3
3
5
missing
5
3
Stop
No frameshift, but one amino acid missing (3 base-pair deletion)
Mutagens
•
Mutations can arise in a number of ways
–
Spontaneous mutations can occur during DNA replication,
recombination, or repair
• In both prokaryotes and eukaryotes, the rate of spontaneous
mutation is about one in every 1010 nucleotide
–
A number of physical and chemical agents (mutagens) can also
interact with DNA in ways that cause mutations
• Ex) X-rays and other forms of high-energy radiation are physical
mutagens
Concept Check 17.5
• 1) What happens when one nucleotide pair is lost from the
middle of the coding sequence of a gene?
• 2) A gene whose template strand contains the sequence 3’-
TACTTGTCCGATATC-5’ is mutated to 3’TACTTGTCCAATATC-5’. For both normal and mutant genes,
draw the double-stranded DNA, the resulting mRNA, and the
amino acid sequence each encodes. What is the effect of the
mutation on the amino acid sequence?
Concept 17.6:
While gene expression differs among
the domains of life, the concept of a
gene is universal
Gene Expression in Prokaryotes vs. Eukaryotes
•
Prokaryotes in domain Archaea share many features of gene expression with
eukaryotes
–
Bacteria and eukarya differ in their RNA polymerases, termination of
transcription and ribosomes, while archaea tend to resemble eukarya in these
respects
–
Fig. 17-24
RNA polymerase
Bacteria can simultaneously transcribe
DNA
mRNA
and translate the same gene
•
In eukarya, transcription and
translation are separated by the
Polyribosome
RNA
polymerase
Direction of
transcription
DNA
nuclear envelope
Polyribosome
•
In archaea, transcription and
translation are likely coupled
0.25 µm
Polypeptide
(amino end)
Ribosome
mRNA (5 end)
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
Concept Check 17.6
• 1) Would the coupling of processes shown in Figure
17.24 (pp. 347) be found in a eukaryotic cell? Explain.
• 2) In eukaryotic cells, mRNAs have been found to
have a circular arrangement in which the poly-A tail is
held by proteins near the 5’ end cap. How might this
increase translation efficiency?
You should now be able to:
1.
Describe the contributions made by Garrod, Beadle, and Tatum to our
understanding of the relationship between genes and enzymes
2.
Briefly explain how information flows from gene to protein
3.
Compare transcription and translation in bacteria and eukaryotes
4.
Explain what it means to say that the genetic code is redundant and
unambiguous
5.
Include the following terms in a description of transcription: mRNA,
RNA polymerase, the promoter, the terminator, the transcription unit,
initiation, elongation, termination, and introns
6.
Include the following terms in a description of translation: tRNA,
wobble, ribosomes, initiation, elongation, and termination