Page 103 - Academic Press Encyclopedia of Physical Science and Technology 3rd Molecular Biology

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Encyclopedia of Physical Science and Technology EN017F-788 August 3, 2001 16:27

44 Translation of RNA to Protein

direction. Immediately after synthesis of the first pep- In prokaryotes, two release factors have been identified,
tide bond, the ribosomal A site contains dipeptidyl-tRNA one (RF1) recognizing UAA and UAG, the other (RF2)
while uncharged initiator tRNA remains in the P site. functioning with UGA. Ribosomal binding and release of
Thus, both these sites are occupied, and to allow the next RF1 and RF2 are stimulated by a third factor, RF3, which
aminoacyl-tRNA to enter the A site it is necessary to eject interacts with GTP and GDP. In eukaryotic cells such as
the uncharged tRNA and shift the dipeptidyl-tRNA from reticulocytes, one release factor (eRF) has been found to
theAintothePsite.Thistranslocation(Fig.7d)takesplace function with all three termination codons, and the binding
as a concerted process involving movement of both mes- of this factor to ribosomes is stimulated by GTP but not
senger RNA and dipeptidyl RNA together into the P site, GDP. Although the details are not entirely clear, GTP
leaving the A site occupied by the next mRNA codon and hydrolysis appears to be required for the release of the
free to accept the cognate aminoacyl-tRNA (see Fig. 7e). finished polypeptide chain by cleavage of the peptidyl-
At the same time, the deacylated tRNA moves first into tRNA bond and completion of the termination process
an E (exit) site with subsequent ejection when the next leading to dissociation of the release factor from the
aminoacyl-tRNA enters the A site. ribosome.
Translocation requires the participation of another elon- Thus, at the end of the ribosome cycle the coding se-
gation factor (EF-G in prokaryotes and EF-2 in eukary- quence of messenger RNA has been translated to produce
otes) and GTP (Table IV). It seems that when EF-G and a particular polypeptide chain, and all the components in-
GTP bind to the ribosome, translocation occurs but GTP volved become available for re-use in another round of
hydrolysis is required only subsequently to release EF-G the cycle (Fig. 7h). Usually, several ribosomes become
and GDP. The location of the EF-G binding site on the ri- attached to one mRNA molecule, giving rise to polyribo-
bosome overlaps with that for EF-Tu, thus EF-G must be somes (also called polysomes; Fig. 9). In eukaryotic cells
released before the EF-Tu–aminoacyl-tRNA–GTP com- the efficiency of protein synthesis is stimulated by factor
plex can enter the A site. Analogous reactions occur in eIF4-G (Table III), which interacts with both factor eIF-4E
eukaryotic systems. and a poly A-binding protein. The resultant circularized
There is little information about the details of the trans- polysomes show an enhanced ability to re-initiate after re-
location mechanism. A continuous polyribonucleotide lease of the ribosomal subunits from the messenger RNA
chain is not essential, as translocation can occur with in- at the end of a round of translation.
dividual trinucleotides. It seems likely that movement of
the mRNA is dependent on and tightly coupled to that
C. High-Resolution Structural Studies
of the tRNA with the binding sites for the tRNA provid-
of the Ribosome
ing the precision for movement by exactly one codon.
Presumably, binding of EF-G and GTP after release of Electron microscopy of ribosomes has provided sufficient
EF-Tu–GDP following peptide bond synthesis induces a information to allow the construction of models showing
conformational change in the ribosome which leads to the general features of the principal functional domains,
translocation. such as the location of mRNA, ribosomal subunits, and
After translocation the ribosomal P site is occupied factors (Fig. 10).
by dipeptidyl-tRNA and the vacant A site contains the During the last ten years, X-ray diffraction studies have
third mRNA codon. Entry of the next aminoacyl-tRNA, ledtoconsiderableadvancesintheelucidationofthestruc-
selected as before by the codon–anticodon interaction, ture of the ribosome in more detail, culminating in the de-
into the A site (Fig. 7f) enables peptide bond synthe- termination of the E. coli ribosome at 0.78-nm resolution
sis to continue and repeated operation of the elongation- (Fig. 11) and of the small and large subunits at resolu-
translocation cycle gives rise to a stepwise elongation of tions of 0.3 nm and 0.25 nm, respectively. The structures
the nascent polypeptide chain, each complete cycle elon- have revealed the identity of each amino acid and each nu-
gating the chain by one amino acid residue and moving cleotide. The findings provide insights at the atomic level

the mRNA by one codon in the 5 to 3 direction. When the into the reactions leading to the decoding of mRNA and
end of the coding sequence is reached and one of the termi- the formation of the peptide bond. Moreover, the impor-
nation (or stop) codons has entered the A site, translation tance of the role of rRNA in ribosome function has be-
stops and the completed polypeptide chain is released. come evident from the structures; the functional regions
of both small and large subunits are rich in RNA. The
c. Termination. (See Figs. 7g–h.) The presence of three-dimensional structures also highlight the dynamic
one of the three termination codons, UAA, UAG, or UGA, aspects of ribosome function leading to the view that the
in the A site results in the binding of a release factor ribosome is a highly sophisticated motor driven by GTP
(Table IV) instead of an aminoacyl-tRNA to the ribosome. with rRNA playing a leading role.