Analysis and reconstruction of an RNP particle which stores 5S RNA and tRNA in amphibianoocytes

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Abstract
Previtellogenic oocytes of Triturus cristatus accumulate a free cytoplasmic RNP which sediments at 40S and contain 5S RNA and tRNA in association with two proteins of MW 45,000 and 39,000 daltons (P45 and P39). The 40S particle has a buoyant density of 1.53 g . cm-3 in CsCl and consists of four identical RNP subunits. Each monomeric subunit contains one molecule of 5S RNA, three molecules of tRNA, two molecules of P45 and one molecule of P39. The 40S particle can be completely dissociated by SDS treatment into its individual components, and the subunits, and even the complete 40S particle, can be reformed by removal of SDS in the presence of 0.2 M NaCl. RNA/protein binding experiments with isolated components, and analysis of reformed RNP complexes in CsCl gradients, demonstrate that the stable interactions are: 5S RNA/P45, 3(tRNA)/P45, 5S RNA/P39 and 5S RNA/P45/P39. Immunological studies show that P45 has also a nuclear location and may bind to the 5S RNA transcript in the chromatin, whereas P39 is predominantly cytoplasmic and is possibly related to proteins associated with 5S RNA in the ribosomal 60S subunit. It is suggested that the 40S RNP particle not only stores 5S RNA and tRNA but also provides a means for the exchange of the 5S RNA transcript binding protein (P45) for the 5S RNA ribosome associated protein (P39).