Transcript Translation I

Translation I
Gerhard Wagner
BCMP200
12/2/2002
[email protected]
Overview
• Lecture 1
– Events of translation:
• initiation, elongation, termination, folding
– Machinery involved in translation
• tRNAs, synthetases, ribosomes, translation factors
– General regulatory mechanisms
• Lecture 2
– Initiation
• Lecture 3
– Elongation, termination, protein folding
– regulation
– Methods for studying translation
Translation - big picture
Initiation: Recruit
fMet-tRNAMet, mRNA, large particle
Elongation:
Synthesize protein
Termination:
Stop synthesis, release protein
Events of Translation
• Initiation (very different between prokaryotes and eukaryotes)
–
–
–
–
–
–
Dissociation of ribosome
Binding of initiation factor (IF1/eIF1A) to A-site of small subunit
Binding of other factors
Recruitment of f-Met-tRNAMet to P-site of small particle
Recruitment of mRNA to small particle
Binding of large particle - dissociation of initiation factors
• Elongation (similar between pro- and eukaryotes)
–
–
–
–
Entry of aa-tRNA to A-site
Peptide-bond formation
Translocation of mRNA and tRNAs to P and E sites
Entry of next aa-tRNA to A-site etc.
• Termination (similar between pro- and eukaryotes)
– Entry of release factor to recognize termination codon
– Exit of polypeptide and release factors
• Protein folding
Players of translation
•
•
•
•
•
Ribosome (RNA, proteins)
mRNA
tRNA
Aminoacyl-tRNA synthetases
Translation factors (initiation,
elongation,termination)
Differences between eubacteria and eukaryotes
Bacteria
Eukaryotes
• Ribosome: 30S+50S -> 70S
• Few initiation factors:
• Ribosome: 40S+60S-> 80S
• Many initiation factors
– IF-1(eIF1A), IF-2(eIF5B), IF-3 (?)
• Elongation factors
– EF1A (EF-Tu), EF1B (EF-Ts), EF2
(EF-G)
• Release factors
– RF-1, RF2, RF3
• Ribosome recycling factor
– RRF
• mRNA is not capped
• Direct binding of 30S particle next to
initiation codon (AUG) at ShineDalgarno sequence, 5’-AGGAGGU3’
• Translation coupled to transcription
– eIF1, eIF1A, eIF2, eIF2B, eIF3,
eIF4A, eIF4B, eIF4E, eIF4F,
eIF4G, eIF4H, eIF5, eIF5B, eIF6
• Elongation factors
– eEF1, eEF2
• Release factors
– eRF1, eRF3
• Most mRNA is capped at 5’ end
and polyadenylated at 3’ end
• 40S particle is recruited to 5’ cap
structure or poly(A) tail or an
internal ribosome entry site (IRES)
• Translation in always (?) in
cytoplasm apart from transcription
tRNA
• Up to 50 (eukaryotes), or 30-35
(bacteria) different tRNAs
• Cloverleaf structure
• Unusual bases - covalent
modification after transcription but
before tRNAs leave nucleus
• Acceptor arm:
•
7 base pairs followed by xCCA-3’
aa attached to 2’ or 3’-OH of terminal
A by ClassI and ClassII aa-tRNA
synthetases, respectively
TYC arm forms one continuous helix
with acceptor arm
• D arm (dihydro-uridine) interacts
with TYC loop via unusual Hbonds
• V loop short in Class I tRNAs, long
in Class II.
• Anticodon arm contains base triplet
that pairs with mRNA codon
3’
TYC
D
5’
V
Anticodon arm
TYC loop
D loop
acceptor arm
C C
5’
V loop
anticodon loop
3’
A
Aminoacyl-tRNA synthetases
•
Synthetase attaches aa to tRNA in a two-step
process:
–
•
•
•
•
adenylation of aa
20 aa-tRNA synthetases, one for each aa.
Bacteria have often fewer synthetases, and
one synthetase attaches different amino acids
to tRNA. Another enzyme then chemically
modifies the incorrectly attached aa so that it
corresponds to the anticodon of the tRNA
Two classes of aa-tRNA synthetases
Class I binds minor groove of acceptor arm,
Class II binds major groove of acceptor arm
(there are newly found exceptions)
aa-tRNA synthetases have been engineered to
incorporate unusual amino acids (P. Schultz,
S. Yokoyama)
Asp-tRNA-synthetase
O
H2N
CH
O
+ ATP
C
CH
O
CH3
ribose
P
O
H2N
O
adenine
CH3
AMP + 2Pi
C
O
ribose
A of tRNA
aa-tRNA synthase
Complex of ClassI Tyr-tRNA synthetase with tRNAtyr
Fig. 3. Interactions between tyrosyl-tRNA synthetase and tRNAtyr. (A) The C-terminal domain (orange) binds in the
elbow between the long variable arm and the anti-codon stem of the tRNA (red backbone, green bases). The
anti-codon stem loop interacts with both the C-terminal domain and the -helical domain (pink). The tRNA makes no
contact with the catalytic domain of the same subunit (cyan). (B) The unusual conformation of the anti-codon triplet in
which Ade-36 is stacked on Gua-34, while Psu-35 bulges out. (C) Base-specific interactions of Asp-259 from the
-helical domain with Gua-34 and Asp-423 from the C-terminal domain with Psu-35.
Tyr-tRNA synthase complex with tRNATyr
mRNA
• Linear in bacteria - can circularize in eukaryotes (via Pabp, eIF4G and
eIF4E)
• In bacteria, ribosome is recruited to AUG codon via a Shine-Dalgarno
sequence
– 5’
AGGAGGU-(X)3-10-AUG
3’
• In eukaryotes, mRNA is usually capped and poly-adenylated - a
consensus sequence is found around the initiation codon -ACCAUGG(Kozak sequence)
O
N
HN
2HN
Me
N
5’
N
HO
O
O
O
O
O P O P O P O
O
O
O
OH
Base
5’
O
HO
OH
• 5’end-5’UTR-AUG-coding region-stop codon-3’UTR-poly(A)tail
Capped mRNA
•
•
Capping happens right after transcription, after about 25 nucleotides have been
synthesized
Capping by three enzymes:
–
–
–
–
–
•
•
•
Phosphatase removes one phosphate from 5’ end
Guanyl transferase adds a GMP in reverse linkage (5’ to 5’ instead of 5’ to 3’)
Methyl transferase adds a methyl to the guanosine
Some RNAs are also methylated at the second nucleotide
All three enzymes bind to the phosphorylated RNA polymerase tail
Cap addition distinguishes mRNA from other RNAs and helps to direct the ribosome to
mRNA
Cap is recognized by the cap-binding complex (CBC), consisting of two proteins,
CBP80 and CBP20. Cap is stacked between two tyrosines Y20 and Y43 of CBP20.
Bindin is achieved via the p-stacking effect. CBC stabilizes the mRNA and interacts
with nuclear pore complex during export of mRNA.
In cytoplasm, CBC is replaced with eIF4E that helps to recruit the ribosome to mRNA.
Here the m7G is stacked between two tryptophanes (p-stacking )
Polyadenylation of mRNA - binding of Pabp and other factors
• 3’-end is polyadenylated by CstF (cleavage stimulating factor), CPSF
(cleavage and polyadenylation specificity factor) and PAP (poly(A)
polymerase).
• Poly(A) tail binds multiple copies of Pabp (poly(A)-binding protein)
• Other factors bind mRNA, SR proteins, hnRNPs etc. bind to mRNA
and make it ready for export
• Some but not all of the attached proteins (CBC, Pabp) are exported
with the mRNA
• In cytosole, CBC is replaced with eIF4E for translation.
mRNA structure
•
Eukaryotic mRNA can circularize by coupling the cap-binding protien eIF4E
and Pabp to the scaffold protein eIF4G
eIF4E
m7GpppN--
AUG
eIF4G
Pabp
AAAAAAAA
•
•
5’UTR sometimes contains long GC-rich regions that tend to form secondary
structure and inhibit ribosome scanning. This is found particularly in mRNAs
for growth-promoting proteins (growthfactors, oncogene products) and is
thought to be a regulatory element to prevent uncontrolled cell growth.
Some mRNAs contain secondary structures that allow for direct binding of the
small ribosomal particle, aided by segments of eIF4G. This is called an
internal ribosome entry site, IRES.
Genetic Code and Codon Usage
Escherichia coli [gbbct]: 11985 CDS's (3688954 codons)
•
Genetic Code is degenerate.
–
–
–
–
•
•
•
64 codons
20 amino acids
< 50 tRNAs
20 synthetases
Wobble base pairing: in some
tRNAs, 3rd base of anticodon can
pair with different bases of
codons
Common aa have multiple
codons and multiple tRNAs,
rarest amino acids, trp and met
are each encoded by only one
codon
Codon usage frequency varies
with organism. Can be looked up
at:
http://www.kazusa.or.jp/codon/
•
Important for expression of
mammalian proteins in E.coli.
Optimize codons for expression.
fields: [triplet] [amino acid] [fraction] [frequency: per thousand] ([number])
UUU F 0.58 22.2 ( 81958)
UUC F 0.42 16.0 ( 59150)
UUA L 0.14 14.4 ( 53048)
UUG L 0.13 13.0 ( 47827)
UCU S 0.17 10.4 ( 38427) UAU Y 0.59 17.5 ( 64717) UGU C 0.46 5.2 ( 19357)
UCC S 0.15 9.1 ( 33697) UAC Y 0.41 12.2 ( 44909) UGC C 0.54 6.1 ( 22348)
UCA S 0.14 9.0 ( 33177) UAA * 0.61 2.0 ( 7408) UGA * 0.30 1.0 ( 3684)
UCG S 0.14 8.5 ( 31383) UAG * 0.08 0.3 ( 996) UGG W 1.00 13.9 ( 51416)
CUU L 0.12 11.9 ( 43948) CCU P 0.18 7.5 ( 27601) CAU H 0.58 12.5 ( 46295) CGU R 0.36 19.9 ( 73524)
CUC L 0.10 10.2 ( 37561) CCC P 0.13 5.4 ( 19840) CAC H 0.42 9.3 ( 34207) CGC R 0.36 19.6 ( 72420)
CUA L 0.04 4.2 ( 15655) CCA P 0.20 8.6 ( 31840) CAA Q 0.34 14.6 ( 53879) CGA R 0.07 3.8 ( 13999)
CUG L 0.47 48.2 (177820) CCG P 0.49 20.8 ( 76842) CAG Q 0.66 28.4 (104717) CGG R 0.11 5.9 ( 21773)
AUU I 0.49 29.8 (109873) ACU T 0.19 10.4 ( 38312) AAU N 0.49 20.7 ( 76457) AGU S 0.16 9.9 ( 36590)
AUC I 0.39 23.6 ( 87131) ACC T 0.40 21.9 ( 80904) AAC N 0.51 21.4 ( 78873) AGC S 0.24 15.1 ( 55819)
AUA I 0.12 7.0 ( 25709) ACA T 0.17 9.4 ( 34580) AAA K 0.74 35.3 (130185) AGA R 0.07 3.7 ( 13500)
AUG M 1.00 26.4 ( 97325) ACG T 0.25 13.7 ( 50690) AAG K 0.26 12.5 ( 45938) AGG R 0.04 2.1 ( 7787)
GUU V 0.28 19.8 ( 73179)
GUC V 0.20 14.3 ( 52706)
GUA V 0.17 11.6 ( 42768)
GUG V 0.35 24.3 ( 89623)
GCU A 0.18 17.1 ( 62923) GAU D 0.63 32.8 (120820) GGU G 0.35 25.4 ( 93737)
GCC A 0.26 24.2 ( 89153) GAC D 0.37 19.2 ( 70721) GGC G 0.37 27.0 ( 99602)
GCA A 0.23 21.2 ( 78120) GAA E 0.68 39.0 (144050) GGA G 0.13 9.6 ( 35295)
GCG A 0.32 30.0 (110528) GAG E 0.32 18.7 ( 68998) GGG G 0.15 11.3 ( 41635)
Homo sapiens [gbpri]: 50031 CDS's (21930294 codons)
fields: [triplet] [amino acid] [fraction] [frequency: per thousand] ([number])
UUU F 0.46 17.1 (374332) UCU S 0.18 14.7 (323470) UAU Y 0.44 12.1 (264652) UGU C 0.45 10.1 (221863)
UUC F 0.54 20.4 (448127) UCC S 0.22 17.5 (384476) UAC Y 0.56 15.5 (339473) UGC C 0.55 12.4 (271056)
UUA L 0.07 7.3 (160731) UCA S 0.15 11.9 (260418) UAA * 0.28 0.8 ( 16884) UGA * 0.50 1.4 ( 30111)
UUG L 0.13 12.7 (277774) UCG S 0.06 4.5 ( 98166) UAG * 0.22 0.6 ( 12911) UGG W 1.00 13.0 (284246)
CUU L 0.13 12.9 (283480) CCU P 0.28 17.3 (380219) CAU H 0.41 10.6 (231860) CGU R 0.08 4.7 (102673)
CUC L 0.20 19.5 (428574) CCC P 0.33 20.0 (439256) CAC H 0.59 15.0 (329569) CGC R 0.19 10.8 (236986)
CUA L 0.07 7.0 (153837) CCA P 0.27 16.7 (367297) CAA Q 0.26 11.9 (261063) CGA R 0.11 6.3 (138297)
CUG L 0.40 40.1 (880072) CCG P 0.11 7.0 (154028) CAG Q 0.74 34.4 (755209) CGG R 0.21 11.8 (257761)
AUU I 0.36 15.8 (346233) ACU T 0.24 12.9 (283671) AAU N 0.46 16.7 (365457) AGU S 0.15 12.0 (263279)
AUC I 0.48 21.3 (466577) ACC T 0.36 19.1 (419213) AAC N 0.54 19.3 (422697) AGC S 0.24 19.4 (424788)
AUA I 0.16 7.2 (157385) ACA T 0.28 14.9 (325763) AAA K 0.42 24.0 (526117) AGA R 0.21 11.7 (255681)
AUG M 1.00 22.3 (489160) ACG T 0.12 6.2 (135294) AAG K 0.58 32.5 (713826) AGG R 0.20 11.6 (254743)
GUU V 0.18 10.9 (239795) GCU A 0.26 18.6 (408931) GAU D 0.46 22.1 (484271) GGU G 0.16 10.8 (237026)
GUC V 0.24 14.6 (320190) GCC A 0.40 28.4 (622538) GAC D 0.54 25.7 (563848) GGC G 0.34 22.6 (495700)
GUA V 0.11 7.0 (154102) GCA A 0.23 16.0 (350382) GAA E 0.42 29.0 (634985) GGA G 0.25 16.4 (358824)
GUG V 0.47 28.7 (630151) GCG A 0.11 7.6 (165700) GAG E 0.58 40.3 (884368) GGG G 0.25 16.4 (360728)
Ribosomes
80S (4.2M)
70S (2.5M)
60S (2.8M)
50S (1.6M)
30S (0.9M)
5S rRNA (120 nt)
23S rRNA (2900 nt)
34 proteins
16S rRNA (1540 nt)
21 proteins
Prokaryotes
5S rRNA (120 nt)
28S rRNA (4700 nt)
5.8S rRNA (160 nt)
~49 proteins
40S (1.4M)
18S rRNA (1900 nt)
33 proteins
Eukaryotes
Distinct roles of ribosomal particles
• Small particle gathers components, f-Met-tRNAMet,
mRNA, initiation factors; is crucial for decoding
• Some antibiotics (streptomycin) interfere with
decoding process
• Large particle joins after components have been
assembled and performs protein synthesis, in the
presence of small particle.
• Large particle is target of macrolide antibiotics
(erythromycin etc.)
Ribosome structure
• Ribosome was a main focus of structural biologists, and structure was
solved in a 30-year effort
• X-ray crystallography
– Yonath (30S and 50S of eubacterium, work since 1980)
•
•
Schluenzen et al. Cell, 102, 615 (2000) 30 S particle at 3.3Å resolution
Harms et al., Cell 107, 679 (2002) 50 S particle at 3.1 Å resolution
– Steitz & Moore (50S)
•
•
•
•
Ban et al. Cell, 93, 1105–1115, (1998) 9Å resolution
Ban et al. Nature, 400, 841- (1999) 5Å resolution
Ban et al. Science 289, 905-920 (2000) 2.4Å resolution
Nissen et al. Science 289, 920-930 (2000) Ribosome activity
– Ramakrishnan (30S)
•
•
Clemons et al., Nature, 400, 833 (1999) 5.5 Å
Carter et al. Science, 291, 498 (2001) 3.1 Å structure with IF1 bound
– Noller (70S)
•
•
Cate et al., Science 285 2095-2104 (1999) 7.8Å
Yusupov et al. Science 292, 883-896 (2001) 5.5 Å
• Cryo-EM
–
–
Agarwal et al., PNAS 95, 6134 (1988)
Stark et al. Cell 100, 301 (2000)
50S particle of bacterial ribosome (Steitz & Moore)
• 5Å resolution structure
• 23S RNA (2900 nucleotides), 5S RNA (120 nucleotides), 33 proteins
• Location of some of the proteins
• Deep active site cleft
• Acceptor arms of aa-tRNAs must dive into cleft
• Exit tunnel at the bottom of the large active-site cleft (initial evidence from
work of Unwin et al., 1986, and Yonath and Wittman, 1987
• Positioning of elongation factor EF-G
•Central protuberance (CP), L1 protein, crown view
Ban et al., Nature 400, 841 (1999)
Mapping of the tunnel with tungsten clusters
50S particle
Ban et al. Cell, 93, 1105–1115,
(1998) 9 Å resolution
Ban et al. Nature, 400, 841
(1999) 5Å resolution
Ban et al. Science 289, 905
(2000) 2.4Å resolution
• All RNA and proteins defined
• Catalytic cleft in center
• Exit tunnel
30S particle
(Ramakrishnan)
•
Clemons et al., Nature, 400, 833 (1999)
5.5 Å
• Landmarks:
– Head (H), Neck (N), Platform (P),
Body (Bo), Shoulder (Sh)
30 S particle, Clemons et al. 5.5 Å
Location of prominent helices and proteins
30S particle, location of initiation factor IF1
•
•
•
•
Clemons et al., Science, 2001
Carter et al. Fig. 2B
30S particle with IF1 bound
Landmarks:
– Head (H), Neck (N), Platform (P),
Body (Bo), Shoulder (Sh)
• Helix 44 and S12 interact with
initiation factor IF1, which sits
in the A-site
IF1
S12
H44
30S particle with tRNA-binding sites
Head
Location of tRNA-binding sites
S12
P
Helix 44
E
Neck
Platform
A
Shoulder
S12
H44
Body
Carter et al., Science 2001
70S ribosome (Noller group)
Cate et al., Science 1999
Location of tRNAs
Cate et al., Science 1999
Model of Location of tRNAs in 50S particle (Nissen et al.)
Acceptor arms in catalytic cleft
Anticodon arms stick out
Macrolide bound to exit channel
(Hansen et al. Mol. Cell 10, 117 (2002)
Ribosome is a ribozyme
• First proposed by Crick, J. Mol. Biol. 38, 367(1968)
• Affinity labeling experiments by Noller’s group (Barta et
al., 1984) showed that U2584 and U2585 of a highly
conserved internal loop from domain V of 23S rRNA are
close to CCA-end of P-site
• Ribosome can be depleted of many of its proteins and
maintains peptidyl-transferase activity
• Ribosome structures show that there is no protein near the
site of peptide synthesis (closest distance is 18 Å)
• Good evidence an proposals for the enzymatic mechanism
are shown in Nissen et al. (2000).