Lecture 9 Translation - Rutgers Universitychem.rutgers.edu/~kyc/Teaching/Files/543-05/09 544-09...
Transcript of Lecture 9 Translation - Rutgers Universitychem.rutgers.edu/~kyc/Teaching/Files/543-05/09 544-09...
Lecture 9 Translation
1. Aminoacyl-tRNA synthetase makes aminoacyl-tRNA in an anti-codondependent manner.
2. Aminoacyl-tRNA reads the mRNA codon messages.
3. Peptide formation and chain elongation are carried out in ribosome.
4. A ribosome has three t-RNA binding sites that bridges the 30S and 50S subunits.
5. Translation initiation, elongation, and termination.
Reactions involved in peptide bond formation
Three types of RNA in protein synthesis
Genetic code: non-overlapping, commaless
binding of lysyl-tRNA to ribosome in response to various codons
Gobind Khorana
Lysyl-tRNA + ribosome + trinucleotide filter paper assay
Two-step decoding
1st: Aminoacyl-tRNA synthetase couples aa to its corresponding tRNA2nd: Anticodon in the tRNA base-pairs with correct codon
Aminoacyl tRNA synthetase
Amino acid
tRNA
General structure of tRNA
Cloverleaf73 to 93 nt
about 25kD7 to 15 unusual bases, 1/2 of nt base paired CCA terminus/acceptor stemTΨC loopextra armDHU loopAnticodon loop 5'-G phosphorylated
Yeast alanine-tRNA
Robert Holley, 1965
A skeletal model of yeast phe-tRNA
4 helices form an L-shaped structure
Helix stacking in tRNA
Amino acid is coupled to tRNA through an esterLinkage to either the 2'-or 3'-OH of the 3'-A of the CCA terminus of the tRNA.
Aminoacyl-tRNA
Aminoacyl sththetase reaction
xx
Aminoacylation of tRNA
Activation of aa with ATP toaminoacyl adenylate.
Transfer it to the A of CCA.
Class I to 2'-OHClass II to 3'-OH
Two steps reaction
class I monomeric, class II dimeric,
bind ATP in different conformations
class I to 2'-OHclass II to 3'-OH)
Two classes of synthetases in E. coli
Synthetase binding to tRNA
I and II synthetase recognize different faces of tRNA, I monomer, 2-OH; II dimer, 3-OH
Asp-tRNA SynthetaseGln-tRNA Synthetase
Microhelix with acceptor stem and a loop
Microhelix with acceptor stem and a loop (24 nt out of 76 nt) can be recognized by alanyl-tRNA synthetase
The important question is how does synthetase recognize its cognate tRNA?(i) Anticodon loop on tRNA(ii) Acceptor stem(iii) Other parts of tRNA
Threonyl-tRNAsynthetase and tRNAThr
Recognizes both the acceptor stem and the anticodon loop.
CCA arm extends into the zinc-containing activation site.
CGU anticodonH-bonding to enzyme.
Glutaminyl-tRNAsynthetase and tRNAGln
in addition to acceptor stem and anticodonloop, contact at G10:C25 bp
x
Proofreading
fidelity <1/104,
ser-tRNAThr + Thr-tRNA synthetaseimmediate hydrolysis of ser-tRNAThr,
Editing site identified at 20Å from active center by X-ray and mutagenesis
Flexible CCA arm for editing
if aa fits into editing site, it is removed
Synthetase active site
Large fragment of one subunit of Thr-tRNA synthetase
Zn coordinates with incoming Thr
Low resolution EM
Ribosomes
X-ray structure of Ribosome
23S RNA(2904 nt) =yellow, 5S=orange, 16S (1541 nt) =green
L proteins=red, S proteins=blue
Composition of prokaryotic and mammalian ribosome
30S = S1 S21 + 16S RNA, 50S = L1 L34 + 23S and 5S RNA, 70S = 30S + 50S, S20=L26, 2 copies L7, L12
EM of prokaryotic ribosomes
cryoEM
CryoEM at 25 A (4300 projections analysed)
Stereoview of A, P, E sites
anticodon loops and mRNA codons on 30S
Tertiary structure of 16S1542 nt,
5'-part=red,center=green, 3'-part=blue
2nd structure showing regions involved in three sites
16S rRNA of 30S small subunit
Magenta: A site; red: P site; yellow: E site
X-ray of 50SAt 2.4 Å
Proteins that appear on the surface of the large ribosomal subunit shown in gold, rRNA in gray; (a) front or crown view, (b) back view, the 180o rotated crown view orientation; (c) A view from the bottom of the subunit down the polypeptide tunnel exit which lies in the center; The proteins visible in each image are identified in the small images at the lower left of the figure.
extended structure to fit into cavities within 23S rRNA
L19
30S and 50S ribosomal subunit
70S ribosome
two oritentations
30S+50S the cavern in between
(a) the large cavernbetween subunits can accommodate 3 tRNAs
(b) tRNA anticodonend touches 30S and acceptor end touches 50S
Schematic view of ribosome
S and L proteins
2D-analysis of E coli ribosomes
The peptidyl transfer mechanism catalyzed by RNA. The general base (adenine 2451in Escherichia coli 23S rRNA) is rendered unusually basic by its environment within the folded structure; it could abstract the proton at any of several steps, one of which is shown here
A ribosome, 50S seen from the viewpoint of 30S
Proteins in purple, 23S rRNA in orange and white, 5S rRNA in burgundy and white, A-site tRNA (green), P-site tRNA (red),
The tunnel surface is shown with backbone atoms of the RNA color coded by domain. Domains I (yellow), II (light blue), III (orange), IV (green), V (light red), 5S ( pink), and proteins are blue
The polypeptide exit tunnel
A space-filling representation of the large subunit surface at the tunnel exit showing the arrangement of proteins, some of which might play roles in protein secretion. The RNA is in white (bases) and orange (backbone) and the numbered proteins are blue. A modeled polypeptide is exiting the tunnel in red
50S tunnel exit
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50S + tRNAs A space-filling representations of the 50S with the three tRNA molecules, in the same relative orientation that they are found in the 70S, docked by model building onto the CCA's bound in the A- and P-site. The proteins are in pink and the rRNA in blue. A backbone ribbon representation of the A-, P-, and E-sites are shown in yellow, red, and white, respectively. (A) The whole subunit in a rotated crown view. (B) A closer view shows the numbered proteins close to the tRNAs
Snapshot of protein synthesisAnother view of three sites (anticodons are in 30S, acceptors in 50S)
The site contains only RNA with no protein within 20 Å
A and P site tRNA acceptor arms converge at the active site, a tunnel passes through the 50S subunit for the growing polypeptide chain to exit.
Peptide tunnel
Peptide bond formation starts when peptidyl-tRNA in the P site and aminoacyl-tRNA in the A site. Translocation occurs via the action of EF-G and deacylated tRNA is pushed to E site to be freed
Peptide synthesis mechanism
Peptide formation
a tetrahedral intermediate is formed
1st and 2nd base of codon form Watson-Crick pairing, 3rd base form wobble pairing; i.e. U:G, A:I, C:I, U:I
U
G
U
I
C
I IA
Wobble base pairs
61 codons, but only 40 or so anticodons…
Alanyl-tRNA has anticodon IGC and it recognizes three codons: GCU, GCC, and GCA. Thus, the degeneracy of the genetic code arises from the wobble in the pairing of the third base of the codonwith the first base of the anticodon. Ala-tRNAcys recognizes UGC, a Cys codon, but incorporates Ala
Codon-anticodon is the key
Condon-anticodon determine the binding
Codon-anticodon recognition Wobble base pairing table
Initiation
tRNAiMet used exclusively for starting protein chains, tRNAMet delivers Met to internal sites. In bacteria, a formyl group is added to Met-tRNAiMet
Two types of Met-tRNA
Formation of N-formylmethionyl-tRNA
the same synthetase attaches met to tRNAf and tRNAm, but transformylase only formylatesmet-tRNAf
Initiation
Initiation factors IF1 and IF3 form complex with 30S,
IF2GTP binds fMet-tRNAf and mRNA and displace IF3 to bring fMet-tRNAf and mRNA to the 30S and forms the 30S initiation complex,
fMet-tRNAf now at the P site
Initiation in prokaryotes
eIF2 and eIF3 have similar counterparts in prokaryotes, eIF4 is the cap binding protein, eIF1 and 1A scan for initiation codon, eIF5 stimulates association between 60S and 48S initiation complex, eIF6 binds to 60S to prevent premature associatio, 60S+40S=80S (4200 kD)
AUG is the only initiation codon. 40S binds to the cap and searches for AUG, a scanning process powered by helicase and ATP. Many initiation factors are involved. For example, eIF4E binds to the 7-mG cap. eIF4A is a helicase.
Initiation in eukaryotes
it binds to many proteins and help recruiting 40S to the mRNA
Adapter eIF4G to circularize mRNA
AUG or GUG preceded by purine-rich bases for 16S pairing, in bacteria called Shine-Dalgarno sequence, for eukaryotes the Kozaksequence -ACCAUGG- defines the initiation site
Initiation sites
What is the role of IF3 and IF1?
EF-Tu GTP brings an aa-tRNA to A site, peptidyl transferase forms a peptide bond, EF-G with GTP translocates the growing peptide and its mRNA codon to the P site
Three steps elongation
EF-G/GTP binds to 50S EF-Tu site, GTP hydrolysis induces conformational change and drives the stem of EF-G to A site and pushes tRNAs and mRNA by one codon
Translocation mechanism
EF-Tu forms complex with tRNA
EF-Tu cannot bind the fMet-tRNAf, but bind Met-tRNAm. Once the complex is at the A site GTP will be hydrolyzed and EF-Ts will join the complex to release GDP. Peptide bond formation is spontaneous. Once formed, mRNA must move by a distance of 3 nt and the new peptidyl-tRNAmust move to the P site. This translocation is mediated by EF-G (aka translocase).
EF-Tu
molecular mimicry, the N-terminal region is similar to that of EF-Tu.
EF-G structure is remarkably similar to that of EF-Tu-tRNA complex. GTP hydrolysis force EF-G to push peptidyl-tRNA and its associated mRNA to move through ribosome by one codon.
EF-G
Eukaryotic Initiation and Elongation
Eukaryotic protein synthesis
(sites of action for initiation and elongation factors)Factors are abbreviated as: 2, eIF2; 2B, eIF2B; A, eIF4A; E, eIF4E; 4F, eIF4F; G, eIF4G, E1, eEF1; E2, eEF2; S6, ribosomal protein S6; PHAS, phosphorylated, heat- and acid-stable protein.
Those factors shown in colorare targets of the signaling pathways.
Termination
Stop codons (UAA, UGA, or UAG), recognized by RFs;
RF1 for UAA or UAG,RF2 for UAA or UGA,
RF3 G protein homologous to EF-Tu, mediates interaction of RF1 or 2 with ribosome
Ribosome release factor from E. coli
Release factor: RF1 binds UAA, UAG; RF2 binds UAA, UGA; RF3 mediates the interaction between RF1 or RF2 with the ribosome.
Termination
RF brings a H2O molecule to hydrolyze the ester bond in peptidyl-tRNA
RF1 and RF2 recognize the stop codon(how the tripeptide binds to the stop codon).
Nirenberg's assay, AUG polymer + [3H]fMet-tRNAf + ribosome with filter
Assay of releasing factors
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The Nobel Prize in Physiology or Medicine 1968
"for their interpretation of the genetic code and its function in protein synthesis"
Robert W Holley b.1922 d.1993Cornell
Har Gobind Khoranab.1922Wisconsin
Marshall W Nirenbergb.1927NIH
Protein factory Reveals Its SecretsResearchers picture and poke the ribosome to learn how it works
www.CEN-online.orgCEN Feb 19, 2007
http://pubs.acs.org/email/cen/html/022307124511.html movie