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Chapter: Genetics and Molecular Biology: Biological Assembly, Ribosomes and Lambda Phage

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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.

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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