6. Translation

144

145

Composition of Ribosomes

Large and small subunits are named according to the velocity with which they

sediment in cenrifugation. The physical unit for sedimentation velocity is Svedberg

(S), which is the larger the faster a molecule/particle sediments.

146

The Ribosome Cylcle Each time a protein molecule is synthesized the large and small subunits of the

ribosome associate with each other, the mRNA and the initiator tRNA. After

completion of synthesizing the protein molecule, the ribosome subunits dissociate.

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tRNA binding Sites on the Ribosome The riboosome has 3 binding sites for tRNAs:

- a tRNA that contains an attached amino acid (Aminoacylated tRNA) binds to

the A site

- a tRNA that contains the growing polypeptide chain (Peptidyl-tRNA) binds to

the P site

- a tRNA that contains neiter an amino acid nor a peptide chain and is about to

Exit from the ribosome, binds to the E site

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Tranfer RNA (tRNA) tRNAs produced from pre-tRNA by post-transcriptional processing (see p. 123/4).

Theoretically, each cell needs 62 different tRNAs = 1 tRNA for each codon that

specifies an amino acid + 1 initiator tRNA: However, the maximum number of

tRNAs observed per cell is 41 (reason: see Wobble base pairing)

All tRNAs share a common 2D (cloverleaf) and 3D (L shape) structure.

contains

dihydrouridines

contains

pseudouridine

contains

dihydrouridines

varies between

3-21 nt

contains the 3 nt anticodon

that binds to the mRNA

contains the CCA 3‘-

end to which the

amino acid gets

attached

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Wobble Base Pairing The 5‘-base in the anticodon has relatively high conformational flexibility as there

are no bases blockin its movement in the 5‘ direction

→ bases in the 5‘ anticodon position have relaxed base pairing rules, i.e. they

can wobble into position to achieve H-bonding with various organic bases

→ a total of 31 different tRNAs are sufficient for translation

Wobble Base Pairing The modified purine base Inosine occurs in tRNA, and has a wider spectrum of

base pairing partners: A, G, U

In all wobble pase pairs the ribose-ribose distances are close to standard A-U and

G-C pairs

150

151

Aminoacyl-tRNA Synthetases (AAtRS) Each cell produces 20 AAtRS, each of which are specific for a single amino acid

→ all AAtRRs, except for those specific for M and W, have at least 2 different tRNA

substrates

Class I AAtRS (generally monomeric) attach the amino acid to the 2‘-OH group of

the 3‘ A, whereas class II AAtRS (dimeric or tetrameric) attach the amino acid to

the 3‘-OH group.

AAtRS recognice their specific tRNA(s) based on multiple structural features in the

acceptor stem and the anticodon loop (also outside the anticodon!).

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Charging tRNAs with Amino Acids

Step 1: activation of the amino acid

anhydride

Step 2: tRNA carging

ester

Nomenclature:

• tRNAGly : tRNA specific for amino acid glycine = glycyl-tRNA

• Gly-tRNAGly : glycyl-tRNA charged with glycine

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Charging tRNAs with Amino Acids

Step 1: activation of the amino acid

anhydride

Step 2: tRNA carging

ester

Nomenclature:

• tRNAGly : tRNA specific for amino acid glycine = glycyl-tRNA

• Gly-tRNAGly : glycyl-tRNA charged with glycine

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Initiation of Translation: Overview Three events are required for successful tranlation initiation:

- recruitment of the ribosome to the mRNA

- positioning of the ribosome precisely at the START codon

- binding of a charged tRNA at the P site

fMet mimics a peptide

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Initiation of Translation in Prokaryotes:

Mechanism

The translation Initiation Factors IF1, IF2 and IF3 catalyze the assembly of the 70S

initiation complex:

1. IF3 binds to the 30S subunit and prevents association with the 50S subunit

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Initiation of Translation in Prokaryotes:

Mechanism2. IF1 binds to the region of the 30S subunit that will become part of the A site.

3. IF2 is a GTPase that binds to IF1 provided that GTP is bound to IF2.

4. The IF1-IF2-GTP complex promotes binding of mRNA and fMet-tRNAfMet

(initiator tRNA) to the 30S subunit → 30S Initiation Complex.

The 16S rRNA interacts with the ribosomal binding

site (RBS) to precisely position the start AUG at the

future P site of the 30S subunit:

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Initiation of Translation in Prokaryotes:

Mechanism

5. Base pairing between the start AUG and the anticodion of the initiator tRNA

induces a conformational change of the 30S subunit, which results in a

release of IF3.

6. In the absence of IF3, the 50S subunit binds to the 30S subunit.

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Initiation of Translation in Prokaryotes:

Mechanism

7. Binding of the 50S subunit stimulates the GTPase activity of IF2 leading to

the formation of IF2-GDP.

8. IF3-GDP has a decreased affinity for the 30S subunit, initiator tRNA and IF1,

which results in dissociation of these componemts from the ribosome

→ 70S initiation complex.

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Initiation of Translation in Eukaryotes:

Mechanism Eukaryotes use a fundamentally different mechanism to identify the start AUG in

mRNA

1. Four eukaryotic Initiation Factors eIF1, eIF1A, eIF3 and eIF5 bind to the 40S

thus preventing its associatin with the 50S subunit.

2. A complex (termed ternary complex) between eIF2-GTP an the eukaryotic

initiator tRNA (Met-tRNAiMet) binds to eIF5 at the 40S subunit

→ 43S preinitiation complex.

3. In parallel to steps 1 and 2, eIF4E binds to the 5‘-cap of the mRNA.

4. Subsequently eIFG and eIFA bind to the 5‘-untranslated region (UTR) near the

5‘-cap.

5. The helicase eIF4B binds to eIFA, which stimulates the helicase activity

removing any potential hairpin structures in mRNA.

6. Once all hairpin structures are removed the mRNA-eIFA, -B, -E, -G complex

associates with the 43S preinitiation complex.

160

Initiation of Translation in Eukaryotes:

Mechanism

161

Initiation of Translation in Eukaryotes:

Mechanism

7. The complex of initiator tRNA and eIFs moves in 5‘ → 3‘ direction along the

mRNA while hydrolysing ATP (scanning).

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Initiation of Translation in Eukaryotes:

Mechanism8. During scanning, the start AUG is recognized by base pairing with the

initiator tRNA. This triggers a conformational change in eIF5 which stimulates

the GTPase activity of eIFG2 leading to the formation of eIF2-GDP.

9. eIF2-GDP has no affinity for the initiator tRNA and eIF5, and thus eIF2-GDP

and eIF5 along with IF1, -3, and -4B are released.

10. Loss of eIF2 allows binding of eIF5B-GTP to the complex small ribosomal

subunit via interaction with the inititator tRNA and eIF1A.

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Initiation of Translation in Eukaryotes:

Mechanism11. eIF5B-GTP stimulates the association of the 60S subunit with the 40S

subunit, thus establishing a 80S ribosome that contains the inititator tRNA

base paired with the start codonm of the mRNA.

12. Binding of the large subunit stimulates the GTPase activity of eIF5B leading to

the formation of eIF5B-GDP, which is released from the ribosome along with

eIF1A → 80S initiation complex.