STRUCTURE AND STABILITY OF THE RECONSTITUTED INTERMEDIATE PARTICLES INVOLVED IN THE INVITRO SELF-ASSEMBLY OF THE 30S-RIBOSOMAL SUBUNIT

In an attempt to decipher further the molecular mechanism of the self-assembly of the 30S ribosomal subunit, we have studied the hydrodynamic properties, conformation, and stability of the major reconstitution intermediate, RI, and its heat-activated form, RI*. Preliminary hydrodynamic parameters (f/fmin or Reff) of RI are similar to those of the 30S particle, while those for RI* are similar to those of the 16S RNA. This indicates that, during the course of the 30S assembly, the hydrodynamic shape and/or compactness of 16S RNA appear to switch back and forth as the 16S RNA proceeds through the RI, then the RI*, and finally the 30S subunit. The melting of the 16S RNA, RI*, and 30S subunit was studied simultaneously by three different conformational parameters, and all show complex behavior through the whole temperature range studied (25-90 °C). The RI* is similar to the 16S RNA at low temperature (<50 °C) and closer to the 30S subunit at high temperatures (>80 °C), and its behavior is intermediate between that of the 16S RNA and30S subunit in the middle of the transition (50-80 °C). The bound proteins in the RI* and the 30S subunit appear to stabilize the RNA against melting. The conformational changes of the proteins and the RNA during the heat activation of RI to RI* and the cooling of RI* to RI* at 5 °C have been further investigated by studies of the component RNA and the proteins alone. The conformational changes of the free proteins and RNA upon heating are similar to those observed for the heat activation of RI to RI*. However, complete reversibility was observed for the free proteins and RNA alone, contrary to the complete irreversibility of the proteins and ~75% reversibility of the RNA in the RI* for the heat activation of RI to RI*. The results suggest that the conformational changes are initiated by the 16S RNA and the proteins alone, but the protein-RNA interactions in the RI and RI* particles results in irreversible conformational changes of the proteins upon cooling. © 1979, American Chemical Society. All rights reserved.