|Lecture Notes: 8 April
© R. Paselk 2006
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
Ribosome Structure (E. coli): [overhead, Figure
26-16, p 863 of text]
- 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.
- The anticodon-binding sites occur in the small ribosome's
- The four subunits forming the large (50s) ribosome's "stalk"
participate in various GTPase reactions.
- The peptidyl transferase function, (P) is in the "valley"
on the large ribosome.
- The peptide is directed into a tunnel through the large ribosome
as it is synthesized
- 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.
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:
We will first look at the process in the bacteria, E. coli.
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-tRNAfMet)
- 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-tRNAiMet)
- 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
- 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]
- 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.
- IF-2-GTP-fMet-tRNAfMet 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.)
- Finally, the 70s initiation complex is formed by:
- the release of IF-3,
- the binding of the 50s particle to the complex with the hydrolysis
of GTP to GDP and Pi,
- and the release of IF-1 and IF-2
Elongation occurs as a three stage cycle (Figure 27-28)
- Aminoacyl tRNA binding occurs via a four step subcycle:
- GTP binds to EF-Tu°EF-Ts (Elongation Factor Tu and Elongation
Factor Ts), releasing EF-Ts to form EF-Tu°GTP.
- EF-Tu°GTP binds to aminoacyl-tRNA to form a aminoacyl-tRNA°EF-Tu°GTP
- The aminoacyl-tRNA°EF-Tu°GTP binds to the mRNA-Ribosomal
complex releasing EF-Tu°GDP + Pi and the ribosomal-mRNA
charged with aminoacyl-tRNA in the A-site.
- EF-Ts binds to EF-Tu°GDP, displacing GDP and forming
the EF-Tu°EF-Ts complex, returning to 1.1 above.
- 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.
- 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 + Pi.
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 + Pi.
- The mRNA is then released to give the inactive 70s ribosome
Eukaryotic peptide synthesis is very similar, however;
- Eukaryotes have many more initiation factors, designated,
for example as eIF-2.
- Met-tRNAiMet is used instead of fMet-tRNAfMet.
- 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.
- 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.
- One releasing factor is required, eRF, which binds
the ribosome and GTP
Last modified 9 April 2009