Lecture 18 and 19 The Mechanism of Translation

14.1 Overview of translation

• mRNA+charged-tRNA+ribosome

• Ribosome subunits are assembled in the nucleolus.

• tRNAs are “charged” with their appropriate amino acids.

• All the players in protein synthesis join together in the cytoplasm.

Structure of ribosomes

• Ribosomes consist of two subunits, large and small, composed of rRNAs and many ribosomal proteins

Functional motifs on the ribosome

• Three tRNA binding sites on the ribosome that bridge the larger and small subunits

• (A) Aminoacyl

• (P) peptidyl

• (E) exit

• peptidyl transferase center is in the large subunit

• Decoding the mRNA occurs on the small subunit

Svedberg unit

• S increases with particle mass and density (related to shape).

rRNAs and ribosomal proteins

So 23, 5 and 16 is prokaryotic

28, 5.8 and 5, 18 are eukaryotic

5 is both

The nucleolus

• non-membrane-bound subcompartment of the nucleus.

• Eukaryotic large and small ribosomal subunits are assembled within the nucleolus.

• Site of 45S pre-rRNA transcription by RNA polymerase I.

overall fidelity of translation is dependent on the accuracy of two processes

• Codon-anticodon recognition during translation

• Aminoacyl-tRNA synthesis

Aminoacyl-tRNA charging

• Aminoacyl-tRNA synthetases attach an amino acid to a tRNA by two enzymatic steps:

• The amino acid reacts with ATP to become adenylylated and pyrophosphate is released.

• AMP is released and the amino acid is transferred to the 3′ end of the tRNA

Aminoacyl-tRNA charging 1st adn 2nd steps

tRNA is the translator

High fidelity of aminoacyl-tRNA synthetases

• •Initial high fidelity selection

  • Can easily match AA with corresponding anticodon

• Proofreading

The 21st amino acid

This charged Sec-tRNASec recognizes and translate UGA codon

The 22st amino acid

The charged tRNA can recognize and translate UAG stop codon

Overview of translation

• Initiation is the most complex and tightly controlled.

• Focus on eukaryotic protein synthesis.

Initiation is subdivided into four steps

• Ternary complex formation and loading onto the 40S subunit.

• mRNA Loading.

• Scanning and binding start codon.

• Joining of the 40S and 60S subunits to form 80S ribosomes.

Ternary complex formation and loading onto the 40S ribosomal subunit

• The ternary complex is composed of:

  • Eukaryotic initiation factor 2 (eIF2)

  • GTP

  • The amino acid-charged initiator tRNA (Met-tRNA)

• The ternary complex binds the 40S subunit, plus other initiation factors, including eIF4G/E, to form a 43S complex.

mRNA loading onto the 40S subunit

• eIF4G and eIF4E

  • initiation with RNA helicases unwind with secondary and tertiary structures

• poly(A)- binding protein (PABP)

  • bound to 3′-poly(A) tail.

The closed-loop model of translation initiation

• The 5′-cap and 3′-poly(A) tail of the mRNA join to form a closed loop with eIF4G serving as the bridge between them.

• Some cellular RNAs are translated by a 5′-cap-independent mechanism in which ribosomes are directly recruited by an internal ribosome entry site (IRES)

Scanning and binding start codon

• Once the mRNA is loaded, the 43S complex scans along the message from 5′→3′ looking for the AUG start codon.

• ATP-dependent mechanism.

• AUG is embedded in a Kozak consensus sequence (Shine-Dalgarno sequence in E. coli).

Joining of the 40S and 60S ribosomal subunits to form 80S ribosomes

• eIF2-GTP is hydrolyzed into eIF2-GDP and released.

• eIF2-GDP is converted to eIF2-GTP through a nucleotide exchange reaction mediated by eIF2B.

• The 60S subunit joins with the 40S subunit to form the 80S ribosome initiation complex, in a process that requires a second GTP hydrolysis step.

• eIF5-GDP is converted to eIF5-GTP through a nucleotide exchange reaction mediated by eIF5B

14.5 Elongation

needs 2 GTP(total) for initial binding and translacation

Peptide bond formation and translocation

• Peptidyl transferase activity transfers a growing polypeptide chain from peptidyl-tRNA in the P site to an amino acid esterified with another tRNA in the A site.

• After the tRNAs and mRNA are translocated and the next codon is moved to the A site, the process is repeated.

• Mediated by eEF2; requires GTP hydrolysis.

translocation need 1 GTP faci

A = where oi

Biochemical evidence that 23S rRNA is a ribozyme

“Fragment reaction” used by Harry Noller and colleagues to shown that purified bacterial 23S rRNA has “peptidyl transferase activity” in vitro.

Structural evidence that ribosome is a ribozyme

• rRNA forms the catalytic center, decoding site, A, P and E sites, and the intersubunit interface.

• Ribosomal proteins are abundant on the exterior of the ribosome.

• The peptidyl transferase center is located in domain V of the 23S rRNA.

Cotranslation translocation

• Cotranslation translocation pathway from the ribosome to the endoplasmic reticulum (ER) lumen.

• Signal recognition particle (SRP) binds to a ribosome translating a polypeptide that bears a signal sequence for targeting to the ER.

• The SRP and SRP receptor use a cycle of recruitment and hydrolysis of GTP to control delivery of the ribosome-mRNA complex to ER

14.6 Termination of translation

• The stop codons are recognized by release factor eRF1 in association with eRF3.

• The completed polypeptide is cleaved from the peptidyl-tRNA

• GTP hydrolysis may trigger the release of eRF1 and eRF3.

Phosphorylation of eIF2a blocks ternary complex formation (EXAM)

• Hypoxia, viral infection, amino acid starvation, heat shock, etc. trigger the phosphorylation of the a- subunit of eIF2.

• Phosphorylation of eIF2a inhibits GDP-GTP exchange. stop hydrolysis so no energy is evailable to continue translation

• eIF2a phosphorylation leads to inhibition of translation by blocking ternary complex formation.

• Selective translation of a subset of mRNAs continues, which allows cells to adapt to stress conditions.

eIF2 phosphorylation is mediated by four distinct protein kinases

Protein Kinase RNA (PKR)

• senser to innitiate phosphorylation of elf2a

dsRNA recognise PKR(senser and receptor) and bind to GDP of elf2-GDP to creating an isolated state

Model of the protein kinase RNA (PKR) activation pathway

Viral double-stranded RNA binds to the RNA binding domains of PKR.

PKR catalytic-domain dimerization.

Autophosphorylation of PKR.

Specific recognition of eIF2a.

Phosphorylation of eIF2a. to stop translation from continueing when a viral infectant is present

pathway on exam