Download attachment of amino acids to tRNA

Li Xiaoling
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2017/5/23
Content
Chapter 1 Introduction
Chapter 2 The Structures of DNA and RNA
Chapter 3 DNA Replication
Chapter 4 DNA Mutation and Repair
Chapter 5 RNA Transcription
Chapter 6 RNA Splicing
Chapter 7 Translation
Chapter 8 The Genetic code
Chapter 9 Regulation in prokaryotes
Chapter 10 Regulation in Eukaryotes
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HOW TO LEARN THIS COURSE
WELL？



To preview and review

Problem-base learning

Making use of class time effectively
Active participation

Bi-directional question in class

Group discussion

Concept map
Tutorship
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To call for reading, thingking and discussing of investigative
learning
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 To learn effectively
EVALUATION (GRADING) SYSTEM
in-class and attendance : 10 points
 Group study and attendance: 20 points
 Final exam: 70 points
 Bonus
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 Question
Ch 5 : Transcription
Ch 6 : RNA Splicing
Ch 7 : Translation
Ch 8 : The Genetic code
4/3/05
Expression of the Genome
 This
part concerned with one of the
greatest challenges in understanding
the gene－how the gene is
expressed.
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The revised central dogma
Maintenance of the Genome
Expression of the Genome
RNA processing
•Molecular Biology Course
CHAPTER 7
Translation
What is translation?
--it is the story about decoding
the genetic information contained
in messenger RNA (mRNA) into
proteins.
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Questions addressed in this chapter




What are the main challenges of translation and how do
organisms overcome them?
What is the organization of nucleotide sequence information
in mRNA?
What is the structure of tRNAs, and how do aminoacyl
tRNA synthetases recognize and attach the correct amino
acids to each tRNA?
How does the ribosome orchestrate the translation process?
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Translation extremely costs
In rapid growing bacterial cells, protein
synthesis consumes
 80% of the cell’s energy
 50% of the cell’s dry weight
Why?
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The main challenges of
translation
The genetic information in mRNA cannot be
recognized by amino acids.
 The genetic code has to be recognized by an
adaptor molecule (translator), and this
adaptor has to accurately recruit the
corresponding amino acid.

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Translation machinery
1.
2.
3.
4.
mRNAs (~5% of total cellular RNA)
tRNAs (~15%)
aminoacyl-tRNA synthetases (氨酰tRNA
合成酶)
ribosomes (~100 proteins and 3-4 rRNAs-~80%)
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Outline
Topics 1-4: Four components of translation
machinery.
T1-mRNA;T2-tRNA;T3-Attachment of
amino acids to tRNA (aminoacyl-tRNA
synthetases);T4-The ribosome
Topic 5-6:Translation process.
T5-initiation;T6-elongation;T7-termination.
Topic 8:Translation-dependent regulation of
mRNA and protein stability
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Topic 1: mRNA
Only a portion of each mRNA can be
translated.
 The protein-coding region of the mRNA
consists of an ordered series of 3-nt-long
units called codons that specify the
order of amino acids.

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1-1 polypeptide chains are
specified by ORF
Message RNA
The protein coding region of each mRNA is
composed of a contiguous, non-overlapping string of
codons called an opening reading frame (ORF) .
 Each ORF begins with a start codon and ends with a
stop codon.

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The start codon
—the first codon of an ORF
In bacteria : AUG, GUG, or UUG (5’-3’)
In eukaryotic cells: 5’-AUG-3’
Functions:
1.Specifies the first amino acid to be incorporated
into the growing polypeptide chain.
2.Defines the reading frame for all subsequent
codons.
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Prokaryotic mRNA (polycistrionic)
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Eukaryotic mRNA (monocistrionic)
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Fig 7-1 Three possible reading frames
of the E. coli trp leader sequence
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Message RNA
1-2 Prokaryotic mRNAs have a
ribosome binding site that
recruits the translational
machinery
1-3 Eukaryotic mRNA
are modified at their 5’
and 3’ ends to facilitate
translation.
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
Ribosome binding site (RBS) or SD-sequence
in prokaryotic mRNA, complementary with the
sequence at the 3’ end of 16S rRNA.
Fig 7-2-a structure of prokaryotic
mRNA
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Once
Kozak sequence
Fig 7-2-b
Eukaryotic mRNA uses a methylated cap to
recruit the ribosome. Once bound, the ribosome
scans the mRNA in a 5’-3’ direction to find the
AUG start codon.
 Kozak sequence increases the translation
efficiency.
 Poly-A in the 3’ end promotes the efficient
recycling of ribosomes.

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Fig 7-2-c
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Topic 2: tRNA
At the heart of protein synthesis
is the translation of nucleotide
sequence information into amino
acids. This work is accomplished
by tRNA.
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2-1: tRNA are adaptors between
codons and amino acids
TRANSFER RNA
1.
2.
3.
The are many types of tRNA
molecules in cell (~40).
Each tRNA molecule is attached to a
specific amino acids (20) and each
recognizes a particular codon, or
codons (61), in the mRNA.
All tRNAs end with the sequence 5’CCA-3’ at the 3’ end, where the
aminoacyl tRNA synthetase adds the
amino acid.
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Primary structure
1.
2.
3.
tRNAs are 75-95 nt in length.
There are 15 invariant and 8 semi-invariant
residues. The position of invariant and
semi-variant nucleosides play a role in
either the secondary and tertiary structure.
There are many modified bases, which
sometimes accounting for 20% of the total
bases in one tRNA molecule. Over 50
different types of them have been
observed.
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4.
Pseudouridine (U) is a modified base.
These modified bases in tRNA lead to
improved tRNA function
Fig 7-3 unusual bases
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2-2: tRNAs share a common secondary
structure that resembles a cloverleaf
TRANSFER RNA

The cloverleaf structure is a common
secondary structural representation of
tRNA molecules which shows the base
paring of various regions to form four
stems (arms) and three loops.
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Fig 7-4 the secondary structure
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2-3: tRNAs have an L-shaped
3-D structure
D loop
U loop
Fig 7-5 the 3-D structure of tRNA
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Formation of the 3-D structure :
9 hydrogen bonds (tertiary
hydrogen bonds) mainly involving in
the base paring between the
invariant bases help the formation of
tRNA tertiary structure.
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http://www.ncbi.nlm
.nih.gov/books/
The cloverleaf structure of
a tRNA. The tRNA is drawn
in the conventional
cloverleaf structure, with
the different components
labeled. 15 invariant
nucleotides (A, C, G, T, U,
Y, where Y =pseudouridine)
and 8 semi-invariant
nucleotides (abbreviations:
R, purine; Y, pyrimidine)
are indicated. Optional
nucleotides not present in
all tRNAs are shown as
smaller dots. The standard
numbering system places
position 1 at the 5’ end and
position 76 at the 3’ end; not all of the optional nucleotides are
included. The invariant and semi-invariant nucleotides are at positions
8, 11, 14, 15, 18, 19, 21, 24, 32, 33, 37, 48, 53, 54, 55, 56, 57, 58, 60, 61, 74,
75 and 76. The nucleotides of the anticodon are at positions 34, 35 and 36.
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
Base pairing between residues in the D-and Tarms fold the tRNA molecule into an L-shape,
with the anticodon loop at one end and the
amino acid acceptor site at the other (Fig. 14-5).
The base pairing is strengthened by base
stacking interactions.
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Topic 3: attachment of amino
acids to tRNA
Amino acids should be attached to tRNA
first before adding to polypeptide chain.
 tRNA molecules to which an amino acid is
attached are said to be charged, and
tRNAs lacking an amino acid are said to
uncharged.

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ATTACHMENT OF AMINO ACIDS TO tRNA
3-1 tRNAs are charged by
attachment of an amino acid to the 3’
terminal A of the tRNA via a high
energy acyl linkage
Energy: The energy released when the highenergy bond is broken helps drive the peptide
bond formation during protein synthesis.
 Enzyme: Aminoacyl tRNA synthetase catalyzing
the reaction has three binding sites for ATP, amino
acid and tRNA.

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ATTACHMENT OF AMINO ACIDS TO tRNA
3-2 Aminoacyl tRNA synthetases
charge tRNA in two steps (reactions)
1.Adenylylation (腺苷酰化) of amino acids:
transfer of AMP to the COO- end of the
amino acids.
2. tRNA charging: transfer of the adenylylated
amino acids to the 3’ end of tRNA,
generating aminoacyl-tRNAs (charged tRNA).
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There are two classes of tRNA
synthetases.
Class I: attach the amino acids to the 2’OH of
the tRNA, and is usually monomeric.
Class II: attach the amino acids to the 3’OH of
the tRNA, and is usually dimeric or
tetrameric.
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Step 1-Adenylylation of amino acids: the
aminoacyl-tRNA synthetase attaches AMP to the-COOH
group of the amino acid utilizing ATP to create an
aminoacyl (氨酰的) adenylate (腺苷酸) intermediate. As
a result, the adenylylated aa binds to the synthetase
tightly.
[This step is also called activation of amino acids p65]
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A class II tRNA synthetase
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Step 2- tRNA charging: transfer of the adenylated amino
acid to the 3’ end of the appropriate tRNA via the 2’ or 3’OH group, and the AMP is released as a result.
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Nature structural and Molecular Biology, 2005, 12:915-922
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ATTACHMENT OF AMINO ACIDS TO tRNA
3-3： each aminoacyl tRNA
synthetase attaches a single amino
acids to one or more
cognate/appropriate tRNAs
Each of the 20 amino acids is attached to the
appropriate tRNA (s) by aminoacyl-tRNA
synthetases.
 Most amino acids are specified by more than
one codon, and by more than one tRNA as well.

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The same synthetase is responsible for charging
all tRNAs for a particular amino acid (one
synthetaseone amino acid).
 Consequently, most organisms have 20
synthetases for 20 different amino acids.

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ATTACHMENT OF AMINO ACIDS TO tRNA
3-4 tRNA synthetases recognize
unique structure features of
cognate tRNAs

The recognition has to ensure two levels of
accuracy: (1) each tRNA synthetase must
recognize the correct set of tRNAs for a
particular amino acids; (2) each synthetase
must charge all of these isoaccepting
tRNAs (即由一种synthetase所识别的不同
tRNAs)
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
The specificity determinants
for accurate recognition are
clusters at two distinct sites: the
acceptor stem and the anti-codon
loop.
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Fig 7-8
Fig 7-7
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Identity elements (specificity determinants )
in various tRNA molecules
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ATTACHMENT OF AMINO ACIDS TO tRNA
3-5 Aminoacyl-tRNA formation is
very accurate: selection of the
correct amino acid
The aminoacyl tRNA synthetases discriminate
different amino acids according to different
natures of their side-chain groups.
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Fig. 7-9
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ATTACHMENT OF AMINO ACIDS TO tRNA
3-6 Some aminoacyl tRNA
synthetase use an editing pocket to
charge tRNAs with high accuracy.
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Ile
Val
Isoleucyl tRNA synthetase as an example:
1. Its editing pocket near the catalytic pocket allows it
to proof read the product of the adenylation reaction
(step #1).
2. AMP-valine and other mis-bound aa can fit into this
editing pocket and get hydrolyzed. But AMP-Ile is too
big to fit in the pocket. Thus, the binding pocket
serves as a molecular sieve to exclude AMP-valine etc.
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Therefore, Ile-tRNA synthetase
discriminates against valine twice: the
initial binding and adenylylation of the amino
acid, and then the editing of the adenylylated
amino acid. Each step discriminates by a
factor of ~100, and the overall selectivity is
about 10,000-fold.
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ATTACHMENT OF AMINO ACIDS TO tRNA
3-7 Ribosomes is unable to
discriminate between correctly or
incorrectly charged tRNAs (是否携带
正确的氨基酸)
1. Ribosome recognize tRNAs but not amino acids
(how to prove?).
2. It is responsible to place the charged tRNAs
onto mRNA through base pairing of the codon in
mRNA and anticodon in tRNA.
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Topic 4: the ribosome
1.
2.
3.
4.
Ribosome composition
Ribosome cycle
Peptide bond formation
Ribosome structure
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4-1 the ribosome is composed of a
large and a small subunit
RIBOSOMES
The large subunit contains the peptidyl
transferase center, which is responsible for the
formation of peptide bonds.
 The small subunit interacting with mRNA
contains the decoding center, in which charged
tRNAs read or “decode” the codon units of
the mRNA.

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Fig 7-13** Ribosome
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RIBOSOMES
4-2: the large and the small
subunits undergone association and
dissociation during each cycle of
translation.
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 Ribosome
cycles: In cells, the
small and large ribosome subunits
associate with each other and the
mRNA, translate it, and then
dissociate after each round of
translation. This sequence of
association and dissociation is
called the ribosome cycle.
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Fig 7-14 Overview of the events of
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translation/ribosome cycle
60
Polysome/polyribosome: an
mRNA bearing multiple ribosomes
• Each mRNA can be translated
simultaneously by multiple ribosomes
Fig 7-15 A polyribosome
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RIBOSOMES
4-3 New amino acids are
attached to the C-terminus of
the growing polypeptide
chain.
Protein is synthesized in a Nto C- terminal direction
4-4 Peptide bonds are formed
by transfer of the growing
peptide chain from peptidyltRNA to aminoacyl-tRNA.
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Fig 7-16
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The structure of the ribosome
RIBOSOMES
4-5 Ribosomal RNAs are both structural
and catalytic determinants of the
ribosomes
4-6 The ribosome has three binding sites
for tRNA.
4-7 Channels through the ribosome allow
the mRNA and growing polypeptide to
enter and/or exit the ribosome.
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4-5: Ribosome structure
Fig 7-17 two views of the ribosome
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4-6 Three binding site for tRNAs
A site: to bind the
aminoacylated-tRNA
P-site: to bind the
peptidyl-tRNA
E-site: to bind the
uncharged tRNA
Fig 7-18
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Fig 7-19 3-D structure of the
ribosome including 3 bound tRNA
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4-7 Channel for mRNA entering
and exiting are located in the
small subunit (see Fig. 14-18)
There is a
pronounced kink in
the mRNA between
the two codons at P
and A sites. This kink
places the vacant A
site codon for
aminoacyl-tRNA
interaction.
Fig 7-202017/5/23
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4-7 Channel for polypeptide chain
exiting locates in the large
subunit
The size of the
channel only
allow a very
limited folding
of the newly
synthesized
polypeptide
Fig 7-21
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Translation process
T5: Initiation of translation
T6: Elongation of translation
T7: termination of translation
Watch the animation
on your study CD
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Questions
1.
2.
Compare the mechanism of translation
initiation in prokaryotes and eukaryotes
(similarity and difference)
How do aminoacyl-tRNA synthetases and
the ribosomes contribute to the fidelity
of translation, respectively?
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Overview of the events of translation
Fig 7-14
Initiation
Termination
Elongation
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T5: Initiation of translation
Initiation in prokaryotic cells (1-3)
Initiation in eukaryotic cells (4-6)
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5-1 Prokaryotic mRNAs are initially
recruited to the small subunit by base
pairing to rRNA.
INITIATION OF TRANSLATION
Fig 7-23
RBS is also called Shine–Dalgarno sequence
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INITIATION OF TRANSLATION
5-2 A specialized tRNA (initiator tRNA)
charged with a modified methionine (f-Met)
binds directly to the prokaryotic small
subunit.
Fig 7-24
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5-3 Three initiator factors direct the
assembly of an initiation complex that
contains mRNA and the initiator tRNA.
INITIATION OF TRANSLATION
1. Formation of the 30S initiation
complex: IFs1-3 + 30S + mRNA + fmettRNA.
2. Formation of the 70S initiation
complex: 50S + 30S + mRNA + fmet-tRNA
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INITIATION OF TRANSLATION
Eukaryotic initiation
5-4 Eukaryotic ribosomes are
recruited to the 5’ cap.
5-5 The start codon is found by
scanning downstream from the 5’
end of the mRNA.
Figs 7-26 and -27
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5-6 Translation initiation factors hold
eukaryotic mRNAs in circles
INITIATION OF TRANSLATION
Fig 7-29
Try to explain how the mRNA poly-A tail
contributes to the translation
efficiency?80
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T6: Translation elongation
1. Aminoacyl-tRNA
binding to A site
2. Peptide bond
formation
3. Translocation
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6-1 Aminoacyl-tRNAs are delivered to the A site by
elongation factor EF-Tu
ELONGATION OF TRANSLATION
1. EF-Tu-GTP binds to
aminoacyl-tRNAs
2. Deliver a tRNA to A site
on ribosome
3. When correct codonanticodon occurs, EF-Tu
interacts with the
factor-binding center on
ribosome and hydrolyzes
its bound GTP
4. EF-Tu-GDP leaves
ribosome
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Fig
7-30
82
6-2 The ribosome uses multiple mechanisms to
select against incorrect aminoacyl-tRNAs
ELONGATION OF TRANSLATION
Additional hydrogen bonds
are formed between two
adenine residues of the
16S rRNA and the minor
groove of the anticodoncodon pair only when they
are correctly paired.
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Fig
7-31a
83
Correct base pairing allows
EF-Tu interact with the
factor binding center on
ribosome, which is
important for GTP
hydrolysis and EF-Tu
release.
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Fig
7-31b
84
Only correct base paired
aminoacyl-tRNAs remain
associated with the
ribosome as they rotate
into the correct position
for peptide bond
formation.
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Fig
7-31c
85
6-3 Ribosome is a ribozyme (重点，催化如
何发生？)
ELONGATION OF TRANSLATION
Fig 7-32
Fig 7-16
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ELONGATION OF TRANSLATION
1.The role of L27 protein?
2.The role of A2451 nucleotide
residue in the 23 rRNA?
3.The role of the 2’-OH of the
A residue at the 3’ of the
peptidyl-tRNA (a part of a
proton shuttle)? Figure-14-33
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ELONGATION OF TRANSLATION
6-4 & 5 Elongation factor EF-G drive
translocation of the tRNAs and the
mRNA by displacing the tRNA bound to
the A site
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EF-G mimics a tRNA molecule so as to
displace the tRNA bound to the A site
Fig 7-35
EF-Tu-GDPNP-Phe-tRNA
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EF-G-GDP
89
ELONGATION OF TRANSLATION
6-6 EF-Tu-GDP and EF-G-GDP must
exchange GDP for GTP prior to
participating in a new round of elongation.
1. EF-G-GDP: GDP has a lower
affinity, and GDP is released after
GTP hydrolysis. The free EF-G
rapidly binds a new GTP.
2. EF-Tu-GDP requires a GTP
exchange factor EF-Ts to displace
GDP and recruit GTP to EF-Tu.
(Fig. 14-36)
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Topic 7:Translation termination
1.Releasing factors act to
release the synthesized peptide
from the peptidyl tRNA in the
ribosome.
2.Ribosome recycling factor, EFTu and IF3 act to recycle the
ribosome.
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TERMINATION OF TRANSLATION
7-1 & 2 & 3 The action of Class I and II
releasing factors
7-1 Class I and Class II releasing
factors terminate translation in
response to stop codons.
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TERMINATION OF TRANSLATION
7-2 Class I releasing factors
(bacterial RF1 and RF2, eukaryotic
eRF1) recognize stop codons by its
peptide anticodon and trigger the
release of the peptidyl chain by the
conserved GGQ motif.(Figure 14-37).
RF1 has a structure resembles
tRNA (Figure 14-38), explaining why
it can enters A site.
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TERMINATION OF TRANSLATION
7-3 Class II releasing factor
remove Class I releasing factor
from the A site. And this function
is controlled by GDP/GTP exchange
and GTP hydrolysis (Figure 14-39).
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TERMINATION OF TRANSLATION
7-4 Recycling of the ribosome by a
combined effort of RRF (ribosome
recycling factor), EF-Tu-GTP and IF3.
(Figure 14-40)
RRF mimics a tRNA and enters A site,
and EF-Tu-GTP pushes it into P site.
This action pushes the uncharged
tRNAs from P-site and E-site. Then
mRNA is dissociated from ribosome
(on direct interaction), and the small
subunit is sequestered by IF3.
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Topic 8: translation dependent
regulation of mRNA and protein
stability

Here regulation refers cellular
processes that deal with defective
mRNA and their translated product.
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8-1: The SsrA RNA rescues (拯救)
ribosomes that translate broken
mRNAs lacking a stop codon
(prokaryotes)
1.The ribosomes are trapped or
stalled on the broken mRNA
lacking a stop codon
2.The stalled ribosomes are
rescued by the action of a
chimeric RNA molecule that is
part tRNA and part mRNA, called
tmRNA.
3.SsrA is a 457-nt
tmRNA
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Fig 7-39 SsrA rescues the stalled ribosomes
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8-2: Eukaryotic cells degrade
mRNAs that are incomplete or
have premature stop codons.
Translation is tightly linked to the
process of mRNA decay in
eukaryotic cells
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Nonsense mediated mRNA decay
When an mRNA contains a premature
stop codon (nonsense codon), the
mRNA is rapidly degraded by
nonsense mediated mRNA decay.
Pre-releasing the ribosome at the
nonsense codon prior to reaching the
exon-junction complex initiates a talk
between the complex and ribosome
to remove the 5’ cap from the mRNA
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0
1.Translation of a normal eukaryotic
mRNA displace all the exon junction
complex
Fig 7-40a
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1
2. Nonsense mediated mRNA
decay
Fig 7-40b
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2
Nonstop mediated decay
Non-stop mediated mRNA decay
rescues ribosomes that translate
mRNAs lacking a stop codon.
(1)The lack of a stop codon results in
ribosome translation into the poly-A tail
to produce poly-Lys at the C-terminus
of the polypeptide; the poly-Lys marks
the newly synthesized for rapid
degradation.
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3
(2)The ribosome eventually stalls at the 3’ end of
the mRNA, which is bound by the Ski7 protein
that triggers the ribosome dissociation and
recruits a 3’-5’ exonuclease activity to degrade
the “nonstop” mRNA.
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Fig 7-40c 10
4
Key points of the chapter
1. The main challenge of translation
2. The structure and function of four
components of the translation machinery.
(重点)
3. Translation initiation, elongation and
termination (具体过程和翻译因子的作用-注意
起始阶段原核与真核的不同，重点)
4. Translation-dependent regulation of the
stability of defective mRNAs and the
resulted protein (生物学问题是什么，在原核
和真核分别怎么解决的)2017/5/23
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5