Translation, cont.

Ribosomes

Ribosomes are the machinery for protein translation. As noted in the table (Table 27-6; Fig 27-14) the ribosome is a very large and complex suprmolecular structure, approximately 2/3 RNA and 1/3 protein. Note that though quite similar overall, the eukaryotic particles are significantly larger, with larger main RNAs and an additional 5.8 s RNA and over 50% more proteins than the bacterial ribosome (Fig 27-13).

Note also that the ribosomes are self-assembling particles.

Ribosome Structure

Ribosome Structure (E. coli): [overhead, Figure 26-16, p 863 of text]

1. The 3' end of the 16s RNA (small rRNA) participates in binding mRNA (via base-pairing) and is located on the "platform" of the small ribosome. The mRNA molecule bends across the middle of the small (30s) ribosome.
2. The anticodon-binding sites occur in the small ribosome's cleft region.
3. The four subunits forming the large (50s) ribosome's "stalk" participate in various GTPase reactions.
4. The peptidyl transferase function, (P) is in the "valley" on the large ribosome.
5. The peptide is directed into a tunnel through the large ribosome as it is synthesized
6. The ribosome-membrane binding site is below the "ridge" on the outside of the 50s ribosome, while the polypeptide exit site is below this, directing newly synthesized peptide into the membrane when the ribosome is bound to a membrane.

Eukaryote ribosomes are larger and more complex than than the bacterial ribosomes, but probably have similar structures, since the RNA secondary structures are conserved.

Polypeptide Synthesis

The synthesis of polypeptides takes place on the ribosomes. Frequently see polysomes, that is groups of ribosomes arranged on a single mRNA with gaps of 50-150Å (maximum ribosome density occurs with about one ribosome every 80Å).

Note that the peptide is synthesized starting at the amino-terminus (N-terminus) and proceeds to the C-terminus as the messenger is read 5' 3'.

The synthesis of a polypeptide can be envisioned as taking place in three phases:

1. Initiation
2. Elongation
3. Termination.

We will first look at the process in the bacteria, E. coli.

Initiation

A number of characteristics of initiation may be noted (Fig 27-25):

• AUG is the first codon, but it is read by a special tRNA:
  • N-formylmethionine-formylmethionyl-tRNA (fMet-tRNA_f^Met) in eubacteria,
    • the peptides are then deformylated, and often the methionine is also removed, usually on the nascent polypeptide, so that mature proteins all lack fMet in E. coli.
  • N-methionine-initiator methionyl-tRNA (Met-tRNA_i^Met) in eukaryotes.
    • The terminal Met is often removed form the peptide, so all amino acid residues may exist as N-terminii.
• Base-pairing between mRNA and a specific sequence of the mRNA helps select each initiation site in eubacterial (generally polycistronic) mRNA. [overhead, Figure 26-27 on p 873 of your text]
  • The rRNA has partial complementarity to a purine rich tract of 3-10 bases (the Shine-Dalgarno sequence) centered about 10 bases upstream of the start codon.
  • In eukaryotes just the first AUG needs to be found since mRNA is monocistronic.

Initiation can be viewed as a three part process [overhead, Figure 26-27 on p 873 of your text]

1. The 30s and 50s particles exist as an inactive 70s ribosome. initiation factor 3 (IF-3) binds to the 30s particle, and with the help of IF-1, dissociates the ribosome.
2. IF-2-GTP-fMet-tRNA_f^Met and the mRNA bind to the 30s particle. The IF-3 aids in the mRNA-16s RNA binding. (Note that in this instance the tRNA binding is not codon directed.)
3. Finally, the 70s initiation complex is formed by:
   1. the release of IF-3,
   2. the binding of the 50s particle to the complex with the hydrolysis of GTP to GDP and P_i,
   3. and the release of IF-1 and IF-2

Elongation

Elongation occurs as a three stage cycle (Figure 27-28)

1. Aminoacyl tRNA binding occurs via a four step subcycle:
   1. GTP binds to EF-Tu°EF-Ts (Elongation Factor Tu and Elongation Factor Ts), releasing EF-Ts to form EF-Tu°GTP.
   2. EF-Tu°GTP binds to aminoacyl-tRNA to form a aminoacyl-tRNA°EF-Tu°GTP complex.
   3. The aminoacyl-tRNA°EF-Tu°GTP binds to the mRNA-Ribosomal complex releasing EF-Tu°GDP + P_i and the ribosomal-mRNA charged with aminoacyl-tRNA in the A-site.
   4. EF-Ts binds to EF-Tu°GDP, displacing GDP and forming the EF-Tu°EF-Ts complex, returning to 1.1 above.
2. Transpeptidation: The peptide bond is formed via a nucleophilic displacement of the P-site peptidyl-tRNA by the NH₂ group of the aminoacyl-tRNA in the A-site. (Figure 27-29)
3. Translocation: The uncharged tRNA in the P-site is expelled or (transferred to the E-site and subsequently expelled), and the new peptidyl-tRNA is moved to the P-site as the mRNA is translocated by one codon. EF-G aids this process with the hydrolysis of GTP to GDP + P_i. (Figure 27-30)

Termination

Three codons, UAA, UGA and UAG are used to signal termination:

• Normally there is no tRNA to bind to these codons.
• These codons are recognized by releasing factors:
  • RF-1 recognizes UAA and UAG
  • RF-2 recognizes UAA and UGA
• Releasing factors can't bind simultaneously with EF-G. RF-3°GTP helps the binding of RF-1 or RF-2 in the A-site, binding as well.
• RF-1 or RF-2 catalyzes the transfer of the peptide to water instead of an aminoacyl-tRNA, releasing the peptide.
• The releasing factors and the uncharged tRNA are then released with the hydrolysis of GTP to GDP + P_i.
• The mRNA is then released to give the inactive 70s ribosome for reuse.

Eukaryotic peptide synthesis is very similar, however;

• Initiation:
  • Eukaryotes have many more initiation factors, designated, for example as eIF-2.
  • Met-tRNA_i^Met is used instead of fMet-tRNA_f^Met.
  • Eukaryotic mRNAs don't have a complementary sequence to bind to the 18s rRNA (no analog to the Shine-Delgarno sequence) since they are always monocistronic and it is not needed.
• Elongation:
  • The functions of EF-Tu and EF-Ts are assumed by different subunits of the single releasing factor, eEF-1.
  • The function of EF-G is accomplished by eEF-2, which is not interchangeable.
• Termination:
  • One releasing factor is required, eRF, which binds the ribosome and GTP

Pathway Diagrams

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Last modified 9 April 2009