Gene Regulation Translation Russell.ppt

Gene Expression: Translation
Peter J. Russell

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.
台大農藝系遺傳學 601 20000
Chap
ter 5
slide
2

Fig. 6.1 General structural formula for an amino acid
Chap
ter 5
slide
3
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

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).
Chap
ter 5
slide
4

Fig. 6.2 Structures of the 20 naturally occurring amino acids organized according to
chemical type
Chap
ter 5
slide
5

chemical type (continued)
Chap
ter 5
slide
6

chemical type (continued)
Chap
ter 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.
Chap
ter 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
Chap
ter 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 folding.
Chap
ter 5
slide
10

Fig. 6.4 Four levels of protein structure
Chap
ter 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.
Chap
ter 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.
Chap
ter 5
slide
13

Chap
ter 5
slide
14

Fig. 6.5 Reversion of a deletion frameshift mutation by a nearby addition mutation
Chap
ter 5
slide
15

Fig. 6.6 Hypothetical example showing how three nearby + (addition) mutations
restore the reading frame, giving normal or near-normal function
Chap
ter 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-Ser-Leu-Ser), indicating that UCU is one and CUC
is the other, but not which is which.
Chap
ter 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.
Chap
ter 5
slide
18

Fig. 6.7 The genetic code
Chap
ter 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 chain-terminating 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).
Chap
ter 5
slide
20

Fig. 6.8 Example of base-pairing wobble
Chap
ter 5
slide
21

iActivity: Determining Causes of Cystic Fibrosis
Chap
ter 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.
Chap
ter 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 Ala-tRNA.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.
Chap
ter 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).
Chap
ter 5
slide
25

Fig. 6.9 Charging of a tRNA molecule by aminoacyl-tRNA synthetase to produce an
aminoacyl-tRNA (charged tRNA)
Chap
ter 5
slide
26

Fig. 6.10 Molecular details of the attachment of an amino acid to a tRNA molecule
Chap
ter 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).
Chap
ter 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 purine-rich 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
Chap
ter 5
slide
29

Fig. 6.11 Initiation of protein synthesis in prokaryotes
Chap
ter 5
slide
30

Fig. 6.12 Sequences involved in the binding of ribosomes to the mRNA in the
initiation of protein synthesis in prokaryotes
Chap
ter 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.
Chap
ter 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.
Chap
ter 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.
Chap
ter 5
slide
34

Fig. 6.13 Elongation stage of translation in prokaryotes
Chap
ter 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.
Chap
ter 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.
Chap
ter 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
Chap
ter 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).
Chap
ter 5
slide
39

Fig. 6.15 Diagram of a polysome, a number of ribosomes each translating the same
mRNA sequentially
Chap
ter 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.
Chap
ter 5
slide
41

Fig. 6.16 Termination of translation
Chap
ter 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.
Chap
ter 5
slide
43

Fig. 6.17x Movement of secretory proteins through the cell membrane system
Chap
ter 5
slide
44

Fig. 6.17 Model for the translocation of proteins into the endoplasmic reticulum in
eukaryotes
Chap
ter 5
slide
45

Gene Regulation Translation Russell.ppt

Recommended

Recommended

More Related Content

Similar to Gene Regulation Translation Russell.ppt

Similar to Gene Regulation Translation Russell.ppt (20)

More from Pudhuvai Baveesh

More from Pudhuvai Baveesh (20)

Recently uploaded

Recently uploaded (20)

Gene Regulation Translation Russell.ppt