Assembly of Ribosomes
How are ribosomes formed? We would like to have an
answer to this question concerning in
vivo assembly because we want to know howthings work. We also would like to
be able to carry out in vitro
assembly so that we can perform sophisticated experiments on the structure and
function of ribosomes.
Study of the in
vivo assembly of ribosomes is difficult because the experimenter can do so
little to alter the system. One or two ribosomal precursors can be
distinguished from mature ribosomes on the basis of their sedimentation
velocities. They contain only a subset of the com-plement or ribosomal
proteins. Additionally, since RNA is methylated in a number of positions, the
degree of methylation of RNA extracted from the precursors can be measured. Not
surprisingly, such studies have not greatly illuminated the subject of ribosome
assembly.
Nomura made pivotal contributions to our
understanding of in vitro ribosome
assembly. As a first step in the attack on this difficult problem, he removed a
few proteins from ribosomes and learned how to replace them and restore the
ribosome’s ability to synthesize protein. The proteins of the smaller ribosomal
subunit were separated into two classes: those few that split off the 30S
subunit when it is centrifuged to equilibrium in CsCl and the remaining cores
that contain the 16S rRNA and the majority of the smaller subunit’s proteins.
He then tried to reassemble the ribosomes from these two fractions. The assay
of assem-bly was sedimentation velocity of the products or their ability to
func-tion in an in vitro translation
assay.
The next step in the study of ribosome assembly was
the reconstitu-tion from isolated rRNA and ribosomal proteins. The RNA was
purified by phenol extraction to remove the proteins and then extensively
dia-lyzed to remove the phenol. The ribosomal proteins were obtained by
extracting purified 30S subunits with urea and lithium chloride. This harsh treatment
denatures the proteins but solves one of the harder technical problems in the
study of ribosomal proteins, that of their insolubility. Much time was invested
by many investigators to find ways of solubilizing these proteins. Why the
proteins should be poorly soluble under some conditions is not at all clear;
perhaps they cannot achieve their correct conformation in the absence of the
correct rRNA-binding site and, as a result, the proteins easily aggregate.
To reconstitute ribosomes, a solution containing
ribosomal proteins is slowly added to a stirred buffer containing the rRNA. In
the solution it is a race between reconstitution and aggregation. A substantial
num-ber of intact 30S functional ribosomal subunits are formed, however. Once
functional 30S subunits could be formed, optimal reconstitution conditions
could be found. Not surprisingly, they were similar to in vivo conditions. They are about 0.3 M KCl, pH 6 to 8, and Mg++ greater
than 10-3 M. Soon it became possible to reconstitute ribosomes from purified 16S
rRNA and the individually purified ribosomal proteins. These results
definitively proved that ribosome assembly requires no addi-tional scaffolding
or assembly proteins that are not found in the mature particle. The remarkable
self-assembly process can be likened to assem-bling watches by shaking their
parts together in a paper bag.
One major reason Nomura succeeded where many others failed was that he tried to reconstitute the ribosomes in buffers closely resembling conditions found in vivo and at temperatures at which cells grow well, 37°. Most other workers had attempted reconstitution at temperatures near freezing to minimize the effects of proteases and nucleases on the ribosomes.
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