Transcript CHAPTER 6 Gene Expression: Translation

Peter J. Russell
A molecular Approach 2nd Edition
CHAPTER 6
Gene Expression: Translation
edited by Yue-Wen Wang Ph. D.
Dept. of Agronomy,台大農藝系
NTU
遺傳學 601 20000
Chapter 5 slide 1
Proteins
Chemical Structure of Proteins
1. Proteins are built from amino acids held together by peptide bonds. The
amino acids confer shape and properties to the protein.
2. Two or more polypeptide chains may associate to form a protein complex.
Each cell type has characteristic proteins that are associated with its
function.
3. All amino acids (except proline) have a common structure (Figure 6.1).
a. The α-carbon is bonded to:
i. An amino group (NH2), which is usually charged at cellular pH
(NH3+).
ii. A carboxyl group (COOH), which is also usually charged at cellular
pH (COO-).
iii. A hydrogen atom (H).
iv. An R group, which is different for each amino acid, and confers
distinctive properties. The R groups in an amino acid chain give
polypeptides their structural and functional properties.
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Chapter 5 slide 2
Fig. 6.1 General structural formula for an amino acid
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Chapter 5 slide 3
4. There are 20 amino acids used in biological
proteins. They are divided into subgroups
according to the properties of their R groups
(acidic, basic, neutral and polar, or neutral and
nonpolar) (Figure 6.2).
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Chapter 5 slide 4
Fig. 6.2 Structures of the 20 naturally occurring amino acids organized according to
chemical type
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Chapter 5 slide 5
Fig. 6.2 Structures of the 20 naturally occurring amino acids organized according to
chemical type (continued)
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Chapter 5 slide 6
Fig. 6.2 Structures of the 20 naturally occurring amino acids organized according to
chemical type (continued)
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Chapter 5 slide 7
5. Polypeptides are chains of amino acids joined by
covalent peptide bonds. A peptide bond forms
between the carboxyl group of one amino acid,
and the amino group of another (Figure 6.3).
6. Polypeptides are unbranched, and have a free
amino group at one end (the N terminus) and a
carboxyl group at the other (the C terminus). The
N-terminal end defines the beginning of the
polypeptide.
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Chapter 5 slide 8
Fig. 6.3 Mechanism for peptide bond formation between the carboxyl group of one
amino acid and the amino group of another amino acid
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Chapter 5 slide 9
Proteins
Molecular Structure of Proteins
1. Proteins have up to four levels of organization (Figure 6.4):
a. Primary structure is the amino acid sequence of the polypeptide. This is
determined by the nucleotide sequence of the corresponding gene.
b. Secondary structure is folding and twisting of regions within a
polypeptide, resulting from electrostatic attractions and/or hydrogen
bonding. Common examples are α-helix and β-pleated sheet.
c. Tertiary structure is the three-dimensional shape of a single polypeptide
chain, often called its conformation. Tertiary structure arises from
interactions between R groups on the amino acids of the polypeptide,
and thus relates to primary structure.
d. Quaternary structure occurs in multi-subunit proteins, as a result of the
association of polypeptide chains. Hemoglobin is an example, with two
141-amino-acid a polypeptides, and two 146-amino-acid β polypeptides
(each associated with a heme group).
2. More than amino acid sequence alone determines the folding of a
polypeptide into a functional protein. Cell biology experiments show
that proteins in the molecular chaperone family assist other proteins in
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Chapter 5 slide 10
folding.
Fig. 6.4 Four levels of protein structure
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Chapter 5 slide 11
The Nature of the Genetic Code
1. How many nucleotides are needed to specify one
amino acid? A one-letter code could specify four
amino acids; two-letters specify 16 (4 X 4). To
accommodate 20, at least three letters are needed.
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Chapter 5 slide 12
The Genetic Code Is a Triplet Code
1. Evidence for a triplet code came from experiments in bacteriophage T4. A
virulent phage, T4 produces 100–200 progeny phage per infected E. coli cell,
and produces plaques on a “lawn” of E. coli.
2. A mutant T4 phage strain call rII can be identified in two ways:
a. T4 phage rII mutants produce clear plaque when grown on E. coli strain B, while the
wild-type r+ phage make turbid plaques on E. coli B.
b. T4 phage rII mutants do not grow in E. coli strain K12(λ), while r+ T4 phage do.
3. The rII mutant strain used in the experiments was produced by treating r+
phage with proflavin. Proflavin causes frameshift mutants by inserting or
deleting base pairs of DNA.
4. Crick and colleagues(1961) reasoned that reversion of a deletion (a – mutation)
could be caused by a nearby insertion (a + mutation) , and vice versa.
Revertants of rII to r+ can be detected by plaques on E. coli K12(λ)
5. Combine genetically distinct rII mutants of the same type (either all + or all -),
and only when it was a combination of three (or multiple of three) were there
high levels of reversion. This indicates that the genetic code is a triplet code.
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Chapter 5 slide 13
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Chapter 5 slide 14
Fig. 6.5 Reversion of a deletion frameshift mutation by a nearby addition mutation
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Chapter 5 slide 15
Fig. 6.6 Hypothetical example showing how three nearby + (addition) mutations
restore the reading frame, giving normal or near-normal function
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Chapter 5 slide 16
Deciphering the Genetic Code
1. The relationship between codons and amino acids was determined by
Nirenberg and Khorana (1968) using cell-free, protein-synthesizing
systems from E. coli that included ribosomes and required protein
factors, along with tRNAs carrying radiolabeled amino acids.
2. To begin determining the genetic code, synthetic mRNAs were used in
the cell-free translation system, and the resulting polypeptides
analyzed:
a. When the mRNA contained one type of base, the results were clear
(e.g., poly(U) was responsible for a chain of phenylalanines).
b. Synthetic random copolymers of mRNA (a mix of two different
nucleotides, A and C for example) can contain eight possible codons,
including two with only one nucleotide (e.g., AAA and CCC) whose
amino acid is already known. By altering the concentrations of the two
nucleotides and analyzing the polypeptides produced, the codons can
be deduced.
c. Copolymers with a known repeating sequence (e.g., UCUCUCUCU)
will produce polypeptides with alternating amino acids (e.g., Leu-SerLeu-Ser), indicating that UCU is one and CUC is the other, but not
which is which.
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Chapter 5 slide 17
3. Ribosome-binding assay is another approach:
a. An in vitro translation system is made that includes:
i. ribosomes.
ii. tRNAs charged with their respective amino acids.
iii. an RNA trinucleotide (e.g., UUU).
b. Protein synthesis does not occur, because the mRNA template contains
only one codon. When the ribosome binds the trinucleotide, only one
type of charged tRNA will bind.
c. The amino acid carried by that tRNA corresponds with the codon.
About 50 codons were clearly identified using this approach.
4. Both of these techniques were important in understanding the genetic
code, and all 61 codons have now been assigned to amino acids; the
other three codons do not specify amino acids (Figure 6.7).
5. By convention, a codon is written as it appears in mRNA, reading in the
5’3’ direction.
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Chapter 5 slide 18
Fig. 6.7 The genetic code
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Chapter 5 slide 19
Characteristics of the Genetic Code
1. Characteristics of the genetic code:
a. It is a triplet code. Each three-nucleotide codon in the mRNA specifies 1 amino in the
polypeptide.
b. It is comma free. The mRNA is read continuously, three bases at a time, without
skipping any bases.
c. It is non-overlapping. Each nucleotide is part of only one codon, and is read only once
during translation.
d. It is almost universal. In nearly all organisms studied, most codons have the same amino
acid meaning. Examples of minor code differences include the protozoan Tetrahymena
and mitochondria of some organisms.
e. It is degenerate. Of 20 amino acids, 18 are encoded by more than one codon. Met
(AUG) and Trp (UGG) are the exceptions; all other amino acids correspond to a set of
two or more codons. Codon sets often show a pattern in their sequences; variation at
the third position is most common (Figure 6.8).
f. The code has start and stop signals. AUG is the usual start signal for protein synthesis.
Stop signals are codons with no corresponding tRNA, the nonsense or chainterminating codons. There are generally three stop codons: UAG (amber), UAA (ochre)
and UGA (opal).
g. Wobble occurs in the anticodon. The 3rd base in the codon is able to base-pair less
specifically, because it is less constrained three-dimensionally. It wobbles, allowing a
tRNA with base modification of its anticodon (e.g., the purine inosine) to recognize up
to three different codons (Figure 6.8).
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Chapter 5 slide 20
Fig. 6.8 Example of base-pairing wobble
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Chapter 5 slide 21
iActivity: Determining Causes of Cystic Fibrosis
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Chapter 5 slide 22
Translation: The Process of Protein Synthesis
1. Ribosomes translate the genetic message of mRNA into
proteins.
2. The mRNA is translated 5’3’, producing a
corresponding N-terminal  C-terminal polypeptide.
3. Amino acids bound to tRNAs are inserted in the proper
sequence due to:
a. Specific binding of each amino acid to its tRNA.
b. Specific base pairing between the mRNA codon and tRNA
anticodon.
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Chapter 5 slide 23
The mRNA Codon Recognizes the tRNA
Anticodon
1. tRNA.Cys normally carries the amino acid cysteine.
Ehrenstein, Weisblum and Benzer attached cysteine to
tRNA.Cys (making Cys-tRNA.Cys), and then
chemically altered it to alanine (making AlatRNA.Cys).
2. When used for in vitro synthesis of hemoglobin, the
tRNA inserted alanine at sites where cysteine was
expected.
3. The concluded that the specificity of codon recognition
lies in the tRNA molecule, and not in the amino acid it
carries.
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Chapter 5 slide 24
Charging tRNA (Adding amino acid to tRNA)
1. Aminoacyl-tRNA synthetase attaches amino acids to their specific
tRNA molecules. The charging process (aminoacylation) produces a
charged tRNA (aminoacyl-tRNA), using energy from ATP hydrolysis.
2. There are 20 different aminoacyl-tRNA synthetase enzymes, one for
each amino acid. Some of these enzymes recognize tRNAs by their
anticodon regions, and others by sequences elsewhere in the tRNA.
3. The amino acid and ATP bind to the specific aminoacyl-tRNA
synthetase enzyme. ATP loses two phosphates and the resulting AMP is
bound to the amino acid, forming aminoacyl-AMP (Figure 6.9).
4. The tRNA binds to the enzyme, and the amino acid is transferred onto
it, displacing the AMP. The aminoacyl-tRNA is released from the
enzyme.
5. The amino acid is now covalently attached by its carboxyl group to the
3’r end of the tRNA. Every tRNA has a 3’r adenine, and the amino acid
is attached to the 3’r–OH or 2’r–OH of this nucleotide.(Figure 6.10).
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Chapter 5 slide 25
Fig. 6.9 Charging of a tRNA molecule by aminoacyl-tRNA synthetase to produce an
aminoacyl-tRNA (charged tRNA)
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Chapter 5 slide 26
Fig. 6.10 Molecular details of the attachment of an amino acid to a tRNA molecule
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Chapter 5 slide 27
Initiation of Translation
Animation: Initiation of Translation
1. Protein synthesis is similar in prokaryotes and eukaryotes. Some
significant differences do occur, and are noted below.
2. In both it is divided into three stages:
a. Initiation.
b. Elongation.
c. Termination.
3. Initiation of translation requires:
a. An mRNA.
b. A ribosome.
c. A specific initiator tRNA.
d. Initiation factors.
e. Mg2+ (magnesium ions).
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Chapter 5 slide 28
4. Prokaryotic translation begins with binding of the 30S ribosomal
subunit to mRNA near the AUG codon (Figure 6.11). The 30S comes to
the mRNA bound to:
a. All three initiation factors, IF1, IF2 and IF3.
b. GTP.
c. Mg2+.
5. Ribosome binding to mRNA requires more than the AUG:
a. RNase protection experiments have shown that the ribosome binds at a
ribosome-binding site, where it is oriented to the correct reading frame
for protein synthesis (Figure 6.13)
b. The AUG is clearly identified in these studies.
c. An additional sequence 8–12 nucleotides upstream from the AUG is
commonly involved. Discovered by Shine and Dalgarno, these purinerich sequences (e.g., AGGAGG) are complementary to the 3’r end of
the 16S rRNA (Figure 6.12)
d. Complementarity between the Shine-Dalgarno sequence and the 3’r
end of 16S rRNA appears to be important in ribosome binding to the
mRNA
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Chapter 5 slide 29
Fig. 6.11 Initiation of protein synthesis in prokaryotes
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Chapter 5 slide 30
Fig. 6.12 Sequences involved in the binding of ribosomes to the mRNA in the
initiation of protein synthesis in prokaryotes
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Chapter 5 slide 31
6. Next, the initiator tRNA binds the AUG to which the 30S subunit is
bound. AUG universally encodes methionine. Newly made proteins
begin with Met, which is often subsequently removed.
a. Initiator methionine in prokaryotes is formylmethionine (fMet). It is
carried by a specific tRNA (with the anticodon 5’r-CAU-3’r).
b. The tRNA first binds a methionine, and then transformylase attaches a
formyl group to the methionine, making fMet-tRNA.fMET (a charged
initiator tRNA).
c. Methionines at sites other than the beginning of a polypeptide are
inserted by tRNA.Met (a different tRNA), which is charged by the
same aminoacyl-tRNA synthetase as tRNA.fMet.
7. When Met-tRNA.fMet binds the 30S-mRNA complex, IF3 is released
and the 50S ribosomal subunit binds the complex. GTP is hydrolysed,
and IF1 and IF2 are relased. The result is a 70S initiation complex
consisting of (Figure 6.14):
a. mRNA.
b. 70S ribosome (30S and 50S subunits) with a vacant A site.
c. fMet-tRNA in the ribosome’s P site.
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Chapter 5 slide 32
8. The main differences in eukaryotic translation are:
a. Initiator methionine is not modified. As in prokaryotes, it is attached to
a special tRNA.
b. Ribosome binding involves the 5’r cap, rather than a Shine-Dalgarno
sequence.
i. Eukaryotic initiator factor (eIF-4F) is a multimer of proteins,
including the cap binding protein (CBP), binds the 5’r mRNA cap.
ii. Then the 40S subunit, complexed with initiator Met-tRNA, several
eIFs and GTP, binds the cap complex, along with other eIFs.
iii. The initiator complex scans the mRNA for a Kozak sequence that
includes the AUG start codon. This is usually the 1st AUG in the
transcript.
iv. When the start codon is located, 40S binds, and then 60S binds,
displacing the eIFs and creating the 80S initiation complex with
initiator Met-tRNA in the ribosome’s P site.
c. The eukaryotic mRNA’s 3’r poly(A) tail also interacts with the 5’r cap.
Poly(A) binding protein (PABP) binds the poly(A), and also binds a
protein in eIF-4F on the cap, circularizing the mRNA and stimulating
translation.
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Chapter 5 slide 33
Elongation of the Polypeptide Chain
Animation: Elongation of the Polypeptide Chain
1. Elongation of the amino acid chain has three steps
(Figure 6.13):
a. Binding of aminoacyl-tRNA to the ribosome.
b. Formation of a peptide bond.
c. Translocation of the ribosome to the next codon.
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Chapter 5 slide 34
Fig. 6.13
Elongation stage of translation in prokaryotes
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Chapter 5 slide 35
Binding of Aminoacyl-tRNA
1. Protein synthesis begins with fMet-tRNA in the P
site of the ribosome. The next charged tRNA
approaches the ribosome bound to EF-Tu-GTP.
When the charged tRNA hydrogen bonds with the
codon in the ribosome’s A site, hydrolysis of GTP
releases EF-Tu-GDP.
2. EF-Tu is recycled with assistance from EF-Ts,
which removes the GDP and replaces it with GTP,
preparing EF-Tu-GTP to escort another aminoacyl
tRNA to the ribosome.
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Chapter 5 slide 36
Peptide Bond Formation
1. The two aminoacyl-tRNAs are positioned by the
ribosome for peptide bond formation, which occurs in
two steps:(Fig. 6.14)
a. In the P site, the bond between the amino acid and its tRNA is
cleaved.
b. Peptidyl transferase forms a peptide bond between the now-free
amino acid in the P site and the amino acid attached to the tRNA
in the A site. Experiments indicate that the 23S rRNA is most
likely the catalyst for peptide bond formation.
c. The tRNA in the A site now has the growing polypeptide chain
attached to it.
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Chapter 5 slide 37
Fig. 6.14 The formation of a peptide bond between the first two amino acids of a
polypeptide chain is catalyzed on the ribosome by peptidyl transferase
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Chapter 5 slide 38
Translocation
1. The ribosome now advances one codon along the mRNA. EF-G is used in
translocation in prokaryotes. EF-G-GTP binds the ribosome, GTP is
hydrolyzed and the ribosome moves 1 codon while the uncharged tRNA leaves
the P site. Eukaryotes use a similar process, with a factor called eEF-2.
2. Release of the uncharged tRNA involves the 50S ribosomal E (for Exit) site.
Binding of a charged tRNA in the A site is blocked until the spent tRNA is
released from the E site.
3. During translocation the peptidyl-tRNA remains attached to its codon, but is
transferred from the ribosomal A site to the P site by an unknown mechanism.
4. The vacant A site now contains a new codon, and an aminoacyl-tRNA with the
correct anticodon can enter and bind. The process repeats until a stop codon is
reached.
5. Elongation and translocation are similar in eukaryotes, except for differences in
number and type of elongation factors and the exact sequence of events.
6. In both prokaryotes and eukaryotes, simultaneous translation occurs. New
ribosomes may initiate as soon as the previous ribosome has moved away from
the initiation site, creating a polyribosome (polysome); an average mRNA
might have 8-10 ribosomes (Figure 6.15).
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Chapter 5 slide 39
Fig. 6.15 Diagram of a polysome, a number of ribosomes each translating the same
mRNA sequentially
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Chapter 5 slide 40
Termination of Translation
Animation: Translation Termination
1. Termination is signaled by a stop codon (UAA, UAG, UGA), which has no
corresponding tRNA (Figure 6.16).
2. Release factors (RF) assist the ribosome in recognizing the stop codon and
terminating translation.
a. In E. coli:
i. RF1 recognizes UAA and UAG.
ii. RF2 recognizes UAA and UGA.
iii. RF3 stimulates termination.
b. In eukaryotes, there is only one termination factor, eRF.
3. Termination events triggered by release factors are:
a. Peptidyl transferase releases the polypeptide from the tRNA in the ribosomal P site.
b. The tRNA is released from the ribosome.
c. The two ribosomal subunits and RF dissociate from the mRNA.
d. The initiator amino acid (fMet or Met) is usually cleaved from the polypeptide.
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Chapter 5 slide 41
Fig. 6.16 Termination of translation
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Chapter 5 slide 42
Protein Sorting in the Cell
1. Localization of the new protein results from signal (leader) sequences in the
polypeptide.
2. In eukaryotes, proteins synthesized on the rough ER (endoplasmic reticulum)
are glycosylated and then transported in vesicles to the Golgi apparatus. The
Golgi sorts proteins based on their signals, and sends them to their destinations.
a. The required signal sequence for a protein to enter the ER is 15–30 N-terminal
amino acids.
b. As the signal sequence is produced by translation, it is bound by a signal
recognition particle (SRP) composed of RNA and protein.(Fig. 6.17)
c. The SRP suspends translation until the complex (containing nascent protein,
ribosome, mRNA and SRP) binds a docking protein on the ER membrane.
d. When the complex binds the docking protein, the signal sequence is inserted into
the membrane, SRP is released, and translation resumes. The growing polypeptide is
inserted through the membrane into the ER, an example of cotranslational transport.
e. In the ER cisternal space, the signal sequence is removed by signal peptidase and
the protein is usually glycosylated.
f. Proteins destined for other organelles are translated completely, and then specific
amino acid sequences direct their transport into the appropriate organelle.
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Chapter 5 slide 43
Fig. 6.17x Movement of secretory proteins through the cell membrane system
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Chapter 5 slide 44
Fig. 6.17 Model for the translocation of proteins into the endoplasmic reticulum in
eukaryotes
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Chapter 5 slide 45