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Transcript of Li Xiaoling Office: M1623 QQ: 313320773 E-MAIL: 313320773 @qq.com.
23/4/19
Li Xiaoling
Office: M1623
QQ: 313320773
E-MAIL: 313320773 @qq.com
ContentChapter 1 Introduction
Chapter 2 The Structures of DNA and RNA
Chapter 10 Regulation in Eukaryotes
Chapter 4 DNA Mutation and Repair
Chapter 5 RNA Transcription
Chapter 6 RNA Splicing
Chapter 7 Translation
Chapter 8 The Genetic code
23/4/19
Chapter 9 Regulation in prokaryotes
Chapter 3 DNA Replication
HOW TO LEARN THIS COURSE WELL? To learn effectively
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
To call for reading, thingking and discussing of investigative learning
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EVALUATION (GRADING) SYSTEM
Question in-class and attendance : 10 points Group study and attendance: 20 points Final exam: 70 points Bonus
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Ch 5 : TranscriptionCh 6 : RNA SplicingCh 7 : TranslationCh 8 : The Genetic code
4/3/05
Expression of the GenomeExpression of the Genome
This part concerned with one of the greatest challenges in understanding the gene -how the gene is expressed.
23/4/19 6
The revised central dogma
RNA processing
Maintenance of the Genome Expression of the Genome
CHAPTER 7CHAPTER 7TranslationTranslation
•Molecular Biology Course
What is translation? What is translation?
23/4/19 9
--it is the story about decoding the genetic information contained in messenger RNA (mRNA) into proteins.
Questions addressed in this Questions addressed in this chapterchapterWhat 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?
23/4/19 10
Translation extremely Translation extremely costscosts
In rapid growing bacterial cells, protein synthesis consumes
80% of the cell’s energy50% of the cell’s dry weight
23/4/19 11
Why?
The main challenges of The main challenges of translationtranslation 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.
23/4/19 12
Translation machineryTranslation machinery
1. mRNAs (~5% of total cellular RNA)2. tRNAs (~15%)3. aminoacyl-tRNA synthetases ( 氨
酰 tRNA合成酶 )4. ribosomes (~100 proteins and 3-4
rRNAs--~80%)
23/4/19 13
Outline 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 23/4/19 14
Topic 1: mRNATopic 1: mRNAOnly 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.
23/4/19 15
1-1 polypeptide chains 1-1 polypeptide chains are specified by ORFare specified by ORF
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.
23/4/19 16
Messa
ge R
NA
The start codon —the first codon of an ORFIn 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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23/4/19 18
Prokaryotic mRNA (polycistrionic)
Eukaryotic mRNA (monocistrionic)
23/4/19 19
Fig 7-1 Three possible reading frames of the E. coli trp leader sequence
1-2 Prokaryotic mRNAs 1-2 Prokaryotic mRNAs have a have a ribosome binding ribosome binding sitesite that recruits the that recruits the translational machinerytranslational machinery
23/4/19 20
Messa
ge R
NA 1-3 Eukaryotic mRNA
are modified at their 5’ and 3’ ends to facilitate translation.
Ribosome binding site (RBS) or SD-sequence in prokaryotic mRNA, complementary with the sequence at the 3’ end of 16S rRNA.
23/4/19 21Fig 7-2-a structure of prokaryotic mRNA
Once Once
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.23/4/19 22
Kozak sequence
Fig 7-2-b
23/4/19 23
Fig 7-2-c
Topic 2: tRNATopic 2: tRNA
23/4/19 24
At the heart of protein synthesis is the translation of nucleotide sequence information into amino acids. This work is accomplished by tRNA.
1. The are many types of tRNA molecules in cell (~40).
2. Each tRNA molecule is attached to a specific amino acids (20) and each recognizes a particular codon, or codons (61), in the mRNA.
3. All tRNAs end with the sequence 5’-CCA-3’ at the 3’ end, where the aminoacyl tRNA synthetase adds the amino acid.
23/4/19 25
2-1: tRNA are adaptors between codons and amino acids
TR
AN
SFE
R R
NA
23/4/19 26
1. tRNAs are 75-95 nt in length. 2. 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.
3.3. There are many modified bases, which There are many modified bases, which sometimes accounting for 20% of the total sometimes accounting for 20% of the total bases in one tRNA molecule. Over 50 bases in one tRNA molecule. Over 50 different types of them have been different types of them have been observed.observed.
1. tRNAs are 75-95 nt in length. 2. 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.
3.3. There are many modified bases, which There are many modified bases, which sometimes accounting for 20% of the total sometimes accounting for 20% of the total bases in one tRNA molecule. Over 50 bases in one tRNA molecule. Over 50 different types of them have been different types of them have been observed.observed.
Primary structure
23/4/19 27
Fig 7-3 unusual bases
4. Pseudouridine (U) is a modified base. These modified bases in tRNA lead to improved tRNA function
2-2:2-2: tRNAs share a common tRNAs share a common secondary structure that secondary structure that resembles a resembles a cloverleaf
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.
23/4/19 28
TR
AN
SFE
R R
NA
23/4/19 29
Fig 7-4 the secondary structure
2-3:2-3: tRNAs have an L- tRNAs have an L-shaped 3-D structureshaped 3-D structure
23/4/19 30Fig 7-5 the 3-D structure of tRNA
D loop U loop
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.
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.
23/4/19 31
23/4/19 32
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 =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.
http://www.ncbi.nlm.nih.gov/books/
Base pairing between residues in the D-and T-arms 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.
23/4/19 33
Topic 3: attachment of Topic 3: attachment of amino acids to tRNAamino 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.
23/4/19 34
3-1 3-1 tRNAs are charged by tRNAs are charged by attachment of an amino acid attachment of an amino acid to the 3’ terminal A of the to the 3’ terminal A of the tRNA via a high energy acyl tRNA via a high energy acyl linkagelinkage
Energy: The energy released when the high-energy bond is broken helps drive the peptide bond formation during protein synthesis.
Enzyme: Aminoacyl tRNA synthetase Aminoacyl tRNA synthetase catalyzing the reaction has three binding sites for ATP, amino acid and tRNA.
23/4/19 35
ATTA
CH
MEN
T O
F AM
INO
AC
IDS
TO
tRN
A
3-23-2 Aminoacyl tRNA Aminoacyl tRNA synthetases charge tRNA in synthetases charge tRNA in two steps (reactions)two steps (reactions)
1. Adenylylation (腺苷酰化 ) of amino amino acidsacids: transfer of AMP to the COO- end of the amino acids.
2. tRNAtRNA charging: transfer of the adenylylated amino acids to the 3’ end of tRNA, generating aminoacyl-tRNAs (charged tRNA).
23/4/19 36
ATTA
CH
MEN
T O
F AM
INO
AC
IDS
TO
tRN
A
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.
23/4/19 37
There are two classes of tRNA synthetases.
23/4/19 38
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 As a result, the adenylylated aa binds to the synthetase a result, the adenylylated aa binds to the synthetase tightlytightly. [This step is also called activation of amino acids p65]
23/4/19 39
A class II tRNA synthetaseA class II tRNA synthetase
23/4/19 40
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.
23/4/19 41
23/4/19 42
Nature structural and Molecular Biology, 2005, 12:915-922
3-33-3 :: each aminoacyl tRNA each aminoacyl tRNA synthetase attaches a single synthetase attaches a single amino acids to one or more amino acids to one or more cognate/appropriate tRNAscognate/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.
23/4/19 43
ATTA
CH
MEN
T O
F AM
INO
AC
IDS
TO
tRN
A
The same synthetase is responsible for charging all tRNAs for a particular amino acid (one synthetaseone amino acid).
Consequently, most organisms have 20 synthetases for 20 different amino acids.
23/4/19 44
3-4 tRNA synthetases 3-4 tRNA synthetases recognize recognize unique structure unique structure featuresfeatures of cognate tRNAs of cognate tRNAs The recognition has to ensure two
levels of accuracy: (1) each tRNA synthetase must recognizerecognize the correct set of tRNAs for a particular amino acids; (2) each synthetase must chargecharge all of these isoaccepting tRNAs isoaccepting tRNAs ( 即由一种synthetase所识别的不同 tRNAs)
23/4/19 45
ATTA
CH
MEN
T O
F AM
INO
AC
IDS
TO
tRN
A
23/4/19 46
The specificity determinants for accurate recognition are clusters at two distinct sites: the acceptor stem and the anti-codon loop.
23/4/19 47
Fig 7-8
Fig 7-7
23/4/19 48
Identity elements (specificity determinants ) in various tRNA molecules
3-5 3-5 Aminoacyl-tRNA Aminoacyl-tRNA formation is very accurate: formation is very accurate: selection of the correct selection of the correct amino acidamino acid
The aminoacyl tRNA synthetases discriminate different amino acids according to different natures of their side-chain groups.
23/4/19 49
ATTA
CH
MEN
T O
F AM
INO
AC
IDS
TO
tRN
A
23/4/19 50
Fig. 7-9
3-6 3-6 Some aminoacyl tRNA Some aminoacyl tRNA synthetase use an editing synthetase use an editing pocket to charge tRNAs with pocket to charge tRNAs with high accuracy. high accuracy.
23/4/19 51
ATTA
CH
MEN
T O
F AM
INO
AC
IDS
TO
tRN
A
23/4/19 52
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.
Val
Ile
23/4/19 53
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.
1. Ribosome recognize tRNAs but not 1. Ribosome recognize tRNAs but not amino acids (how to prove?). amino acids (how to prove?). 2. 2. ItIt is responsible to place the is responsible to place the charged tRNAs onto mRNA through charged tRNAs onto mRNA through base pairing of the codon in mRNA base pairing of the codon in mRNA and anticodon in tRNA. and anticodon in tRNA.
3-7 Ribosomes is unable to discriminate between correctly or incorrectly charged tRNAs ( 是否携带正确的氨基酸 )
23/4/19 54
ATTA
CH
MEN
T O
F AM
INO
AC
IDS
TO
tRN
A
Topic 4: the ribosomeTopic 4: the ribosome
23/4/19 55
1. Ribosome composition2. Ribosome cycle3. Peptide bond formation4. Ribosome structure
4-14-1 the ribosome is composed the ribosome is composed of a large and a small subunitof a large and a small subunit
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.
23/4/19 56
RIB
OSO
MES
23/4/19 57
Fig 7-13** Ribosome
4-2:4-2: the large and the small the large and the small subunits undergone subunits undergone association and dissociation association and dissociation during each cycle of during each cycle of translation.translation.
23/4/19 58
RIB
OSO
MES
23/4/19 59
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.
23/4/19 60
Fig 7-14 Overview of the events of translation/ribosome cycle
23/4/19 61
Polysome/polyribosome: an mRNA bearing multiple ribosomes
• Each mRNA can be translated simultaneously by multiple ribosomes
Fig 7-15 A polyribosome
23/4/19 62
23/4/19 63
RIB
OSO
MES
4-3 New amino acids are attached to the C-terminus of the growing polypeptide chain.
Protein is synthesized in a N- to C- terminal direction
4-4 Peptide bonds are formed by transfer of the growing peptide chain from peptidyl- tRNA to aminoacyl-tRNA.
23/4/19 64
Fig 7-16
The structure of the The structure of the ribosomeribosome
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.
23/4/19 65
RIB
OSO
MES
4-54-5: Ribosome structure: Ribosome structure
23/4/19 66
Fig 7-17 two views of the ribosome
4-64-6 Three binding site for tRNAs Three binding site for tRNAs
23/4/19 67
Fig 7-18
A site: to bind the aminoacylated-tRNAP-site: to bind the peptidyl-tRNAE-site: to bind the uncharged tRNA
23/4/19 68Fig
7-1
9 3
-D s
truct
ure
of
the
riboso
me incl
udin
g 3
boun
d t
RN
A
23/4/19 69
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 7-20
23/4/19 70
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
Translation processTranslation process
T5: T5: Initiation of translationInitiation of translationT6:T6: Elongation of translation Elongation of translationT7: T7: termination of termination of translationtranslation
Watch the animation Watch the animation on your study CDon your study CD
23/4/19 71
QuestionsQuestions
1. Compare the mechanism of translation initiation in prokaryotes and eukaryotes (similarity and difference)
2. How do aminoacyl-tRNA synthetases and the ribosomes contribute to the fidelity of translation, respectively?
23/4/19 72
Overview of the events of translationOverview of the events of translation
23/4/19 73Termination Elongation
Initiation
Fig 7-14
T5: T5: Initiation of translation Initiation of translation
Initiation in prokaryotic cells (1-3)Initiation in prokaryotic cells (1-3)
Initiation in eukaryotic cells (4-6)Initiation in eukaryotic cells (4-6)
23/4/19 74
5-15-1 Prokaryotic mRNAs are initially Prokaryotic mRNAs are initially recruited to the small subunit by recruited to the small subunit by base pairing to rRNA.base pairing to rRNA.
23/4/19 75
INIT
IATIO
N O
F TR
AN
SLA
TIO
N
Fig 7-23
RBS is also called Shine–Dalgarno sequence
5-25-2 A specialized tRNA (initiator A specialized tRNA (initiator tRNA) charged with a modified tRNA) charged with a modified methionine (f-Met) binds directly to methionine (f-Met) binds directly to the prokaryotic small subunit.the prokaryotic small subunit.
23/4/19 76
INIT
IATIO
N O
F TR
AN
SLA
TIO
N Fig 7-24
5-35-3 Three initiator factors direct Three initiator factors direct the assembly of an initiation the assembly of an initiation complex that contains mRNA and complex that contains mRNA and the initiator tRNA.the initiator tRNA.
23/4/19 77
INIT
IATIO
N O
F TR
AN
SLA
TIO
N
1. Formation of the 30S initiation complex: IFs1-3 + 30S + mRNA + fmet-tRNA.
2. Formation of the 70S initiation complex: 50S + 30S + mRNA + fmet-tRNA
23/4/19 78
Eukaryotic initiationEukaryotic initiation5-45-4 Eukaryotic ribosomes are Eukaryotic ribosomes are recruited to the 5’ cap.recruited to the 5’ cap.
5-55-5 The start codon is found The start codon is found by scanning downstream from by scanning downstream from the 5’ end of the mRNA.the 5’ end of the mRNA.
23/4/19 79
INIT
IATIO
N O
F TR
AN
SLA
TIO
N
Figs 7-26 and -27
5-65-6 Translation initiation factors Translation initiation factors hold eukaryotic mRNAs in circleshold eukaryotic mRNAs in circles
23/4/19 80
Try to explain how the mRNA poly-A tail contributes to the translation efficiency?
INIT
IATIO
N O
F TR
AN
SLA
TIO
N
Fig 7-29
T6: T6: Translation elongation Translation elongation
23/4/19 81
1. Aminoacyl-tRNA binding to A site
2. Peptide bond formation
3. Translocation
6-16-1 Aminoacyl-tRNAs are delivered to the Aminoacyl-tRNAs are delivered to the A site by elongation factor EF-TuA site by elongation factor EF-Tu
23/4/19 82
ELO
NG
ATIO
N O
F TR
AN
SLA
TIO
N
1.1. EF-Tu-GTP binds to EF-Tu-GTP binds to aminoacyl-tRNAsaminoacyl-tRNAs
2.2. Deliver a tRNA to A Deliver a tRNA to A site on ribosome site on ribosome
3.3. When correct codon-When correct codon-anticodon occurs, EF-anticodon occurs, EF-Tu interacts with the Tu interacts with the factor-binding center factor-binding center on ribosome and on ribosome and hydrolyzes its bound hydrolyzes its bound GTPGTP
4.4. EF-Tu-GDP leaves EF-Tu-GDP leaves ribosomeribosome
Fig 7-30
6-26-2 The ribosome uses multiple The ribosome uses multiple mechanisms to select against incorrect mechanisms to select against incorrect aminoacyl-tRNAsaminoacyl-tRNAs
23/4/19 83
ELO
NG
ATIO
N O
F TR
AN
SLA
TIO
N
Additional hydrogen Additional hydrogen bonds are formed bonds are formed between two adenine between two adenine residues of the 16S residues of the 16S rRNA and the minor rRNA and the minor groove of the groove of the anticodon-codon pair anticodon-codon pair only when they are only when they are correctly paired. correctly paired.
Fig 7-31a
23/4/19 84
Correct base pairing Correct base pairing allows EF-Tu interact allows EF-Tu interact with the factor with the factor binding center on binding center on ribosome, which is ribosome, which is important for GTP important for GTP hydrolysis and EF-Tu hydrolysis and EF-Tu release. release.
Fig 7-31b
23/4/19 85
Only correct base paired Only correct base paired aminoacyl-tRNAs aminoacyl-tRNAs remain associated remain associated with the ribosome as with the ribosome as they rotate into the they rotate into the correct position for correct position for peptide bond peptide bond formation.formation.
Fig 7-31c
6-36-3 Ribosome is a ribozyme ( Ribosome is a ribozyme ( 重点,重点,催化如何发生?催化如何发生? ))
23/4/19 86
ELO
NG
ATIO
N O
F TR
AN
SLA
TIO
N
Fig 7-32 Fig 7-16
23/4/19 87
ELO
NG
ATIO
N O
F TR
AN
SLA
TIO
N
1.1.The role of L27 protein? The role of L27 protein? 2.2.The role of A2451 The role of A2451
nucleotide residue in the nucleotide residue in the 23 rRNA?23 rRNA?
3.3.The role of the 2’-OH of The role of the 2’-OH of the A residue at the 3’ of the A residue at the 3’ of the peptidyl-tRNA (a part the peptidyl-tRNA (a part of a proton shuttle)? of a proton shuttle)? Figure-14-33Figure-14-33
6-4 & 56-4 & 5 Elongation factor EF-G Elongation factor EF-G drive translocation of the tRNAs drive translocation of the tRNAs and the mRNA by displacing the and the mRNA by displacing the tRNA bound to the A site tRNA bound to the A site
23/4/19 88
ELO
NG
ATIO
N O
F TR
AN
SLA
TIO
N
EF-G mimics a tRNA molecule so as to EF-G mimics a tRNA molecule so as to displace the tRNA bound to the A sitedisplace the tRNA bound to the A site
23/4/19 89 EF-G-GDPEF-Tu-GDPNP-Phe-tRNA
Fig 7-35
6-6 6-6 EF-Tu-GDP and EF-G-GDP must EF-Tu-GDP and EF-G-GDP must exchange GDP for GTP prior to exchange GDP for GTP prior to participating in a new round of participating in a new round of elongation.elongation.
23/4/19 90
ELO
NG
ATIO
N O
F TR
AN
SLA
TIO
N
1.1. EF-G-GDP: GDP has a lower EF-G-GDP: GDP has a lower affinity, and GDP is released affinity, and GDP is released after GTP hydrolysis. The free after GTP hydrolysis. The free EF-G rapidly binds a new GTP.EF-G rapidly binds a new GTP.
2.2. EF-Tu-GDP requires a GTP EF-Tu-GDP requires a GTP exchange factor EF-Ts to exchange factor EF-Ts to displace GDP and recruit GTP displace GDP and recruit GTP to EF-Tu. (Fig. 14-36) to EF-Tu. (Fig. 14-36)
Topic 7: Topic 7: Translation termination Translation termination
23/4/19 91
1.1.Releasing factors act to Releasing factors act to release the synthesized release the synthesized peptide from the peptidyl peptide from the peptidyl tRNA in the ribosome.tRNA in the ribosome.
2.2.Ribosome recycling factor, Ribosome recycling factor, EF-Tu and IF3 act to EF-Tu and IF3 act to recycle the ribosome.recycle the ribosome.
7-1 & 2 & 3 7-1 & 2 & 3 The action of Class I The action of Class I and II releasing factorsand II releasing factors
7-17-1 Class I and Class II Class I and Class II releasing factors terminate releasing factors terminate translation in response to stop translation in response to stop codons. codons.
23/4/19 92
TER
MIN
ATIO
N O
F TR
AN
SLA
TIO
N
7-27-2 Class I releasing factors Class I releasing factors (bacterial RF1 and RF2, (bacterial RF1 and RF2, eukaryotic eRF1) recognize eukaryotic eRF1) recognize stop codons by its stop codons by its peptide peptide anticodon anticodon and trigger the and trigger the release of the peptidyl chain by release of the peptidyl chain by the the conserved GGQ motifconserved GGQ motif..(Figure 14-37). RF1 has a (Figure 14-37). RF1 has a structure resembles tRNA structure resembles tRNA (Figure 14-38), explaining why (Figure 14-38), explaining why it can enters A site. it can enters A site.
23/4/19 93
TER
MIN
ATIO
N O
F TR
AN
SLA
TIO
N
7-37-3 Class II releasing factor Class II releasing factor remove Class I releasing factor remove Class I releasing factor from the A site. And this from the A site. And this function is controlled by function is controlled by GDP/GTP exchange and GTP GDP/GTP exchange and GTP hydrolysis (Figure 14-39). hydrolysis (Figure 14-39).
23/4/19 94
TER
MIN
ATIO
N O
F TR
AN
SLA
TIO
N
7-4 7-4 Recycling of the ribosome Recycling of the ribosome by a combined effort of RRF by a combined effort of RRF (ribosome recycling factor), (ribosome recycling factor), EF-EF-Tu-GTP and IF3. Tu-GTP and IF3. (Figure 14-40)(Figure 14-40)
23/4/19 95
TER
MIN
ATIO
N O
F TR
AN
SLA
TIO
N
RRF mimics a tRNA and enters A RRF mimics a tRNA and enters A site, and EF-Tu-GTP pushes it site, and EF-Tu-GTP pushes it into P site. This action pushes into P site. This action pushes the uncharged tRNAs from P-site the uncharged tRNAs from P-site and E-site. Then mRNA is and E-site. Then mRNA is dissociated from ribosome (on dissociated from ribosome (on direct interaction), and the small direct interaction), and the small subunit is sequestered by IF3.subunit is sequestered by IF3.
Topic 8: translation dependent Topic 8: translation dependent regulation of mRNA and regulation of mRNA and
protein stability protein stability
Here regulation refers cellular processes that deal with defective mRNA and their translated product.
23/4/19 96
23/4/19 97
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
23/4/19 98
Fig 7-39 SsrA rescues the stalled ribosomes
23/4/19 99
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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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
Nonsense mediated mRNA decay
1. Translation of a normal 1. Translation of a normal eukaryotic mRNA displace all eukaryotic mRNA displace all the exon junction complexthe exon junction complex
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Fig 7-40a
2. Nonsense mediated mRNA 2. Nonsense mediated mRNA decaydecay
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Fig 7-40b
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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.
Nonstop mediated decay
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Fig 7-40c
(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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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 )
Key points of the chapter