Volume 1

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    It seems likely that the stepwise reconstitution of the 30S subunit which can be achieved in vitro mimics the process of assembly in vivo. This idea is supported by the close relationship between RI particles and 21S precursors (see below). But an important difference between the two processes is that assembly in vivo takes place on precursor RNA, which is both longer and also less modified than the mature rRNA which has been used in vitro. The success of reconstitution experiments in vitro makes it difficult to see what function the precursor RNA serves; it seems unlikely that it contains sequences which must be recognised by ribosomal proteins. Although the maturation of the rRNA takes place as the precursor particles associate with further ribosomal proteins, we do not know whether the maturation of particles depends upon that of the rRNA or whether the two processes, although simultaneous, are independent. One possible speculation is that the precursor rRNA can inhibit the maturation process unless its extra sequences are removed and proper modifications made; this might provide some control over the assembly of ribosome subunits. Reconstitution of 50S Subunits In Vitro The reconstitution of 50S subunits in vitro has proved more difficult than that of the smaller subunits. One possible reason is that a higher temperature of incubation is needed but that this would inactivate the the subunits made in vitro. Nomura and Erdmann (1970) and Fahnestock, Erdmann and Nomura (1973) therefore reconstituted 50S particles from Bacillus stearother- mophilus, which lives at higher temperatures than E.coli and whose protein synthetic apparatus, including the ribosomes, is stable in these conditions. They found that although this reaction is successful, it is less efficient than the reconstitution of the 30S subunits of E.coli. The small 5S rRNA must be present in the reconstitution mixture, for Erdmann et al. (1971) found that its omission severely restricts all the catalytic activities of the reconstituted subunits. Particles reconstituted without 5S rRNA appear also to be more readily unfolded when their magnesium ions are removed, which suggests that 5S rRNA may play some role in maintaining the compact structure of the large subunit. By using a gentle procedure to isolate the proteins of E.coli 50S subunits, in which the particles are unfolded by dialysis against 1M NH 4 C1 and treated with ribonuclease II to degrade their RNA, Maruta et al. (1971) were able to reconstitute the subunits in vitro. This may open the way to an analysis of 50S subunit assembly. However, the behaviour of the sad mutants which inhibit both 50S and 30S subunit maturation in vivo implies that proper assembly of the 50S subunit may depend upon simultaneous assembly of the 30S subunit; in this case reconstitution of the 50S subunit alone may not be typical of its assembly within the cell. But one possible mechanism, which is supported by an analysis of the interaction of ribosomal proteins with rRNA (see below) is