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

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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

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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