When the gene dosage for the primary rRNA-binding ribosomal protein L25 in yeast cells was raised about 50-fold, the level of mature L25 transcripts was found to increase almost proportionally. The plasmid-derived L25 transcripts were structurally indistinguishable from their genomic counterparts, freely entered polysomes in vivo and were fully translatable in a heterologous in vitro system. Nevertheless, pulse-labelling for periods varying from 3-20 min did not reveal a significant elevation of the intracellular level of L25-protein. When pulse-times were decreased to 10-45 s, however, we did detect a substantial overproduction of L25. We conclude that, despite the strong RNA-binding capacity of the protein, accumulation of L25 is not controlled by an autogenous (pre-)mRNA-targeted mechanism similar to that operating in bacteria, but rather by extremely rapid degradation of excess protein produced.

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Treatment of yeast 60s ribosomal subunits with 0.5 M LiCl was found to remove all but six of the ribosomal proteins. The proteins remaining associated with the (26s + 5.8s) rRNA complex were identified as L4, L8, LIO, L12, L16 and L25. These core proteins were split off sequentially in the order (L16 + L12), L10, (L4 + L8), L25by further increasing the LiCl concentration. At 1 .O M LiCl only ribosomal protein L25 remains bound to the rRNA. Upon lowering the LiCl concentration the core proteins reassociate with the rRNA in the reverse order of their removal. The susceptibility of the ribosomal proteins to removal by LiCl corresponds quite well with their order of assembly into the 60s subunit in viva as determined earlier [Kruiswijk et al. (1978) Biochim. Biophys. Acta 517,378 -3891. Binding studies in vitro using partially purified L25 showed that this protein binds specifically to 26s rRNA. Therefore our experiments for the first time directly identify a eukaryotic ribosomal protein capable of binding to high-molecular-mass rRNA. Binding studies in vitro using a blot technique demonstrated that core proteins L8 and L16 as well as protein L21, though not present in any of the core particles, are also capable of binding to 26s rRNA to approximately the same extent as L25. About nine additional 60s proteins appeared to interact with the 26s rRNA, though to a lesser extent.Structural and functional studies on prokaryotic ribosomes have benefited enormously from our ability to take ribosomal subunits apart and reassemble the components again into biologically active particles. A widely used technique for controlled dissociation of ribosomal constituents is treatment of ribosomal subunits with high concentrations of monovalent cations, notably LiC1. Such treatment results in removal of a portion of the ribosomal proteins, leaving discrete core particles on which a well-defined subset of the ribosomal proteins remains [I -31. Such cores have played an important role in establishing structure-function relationships for various prokaryotic ribosomal components (see for instance [4]) and continue to be used for this purpose [5, 61. Moreover, the split protein fractions obtained by LiCl treatment are an excellent source of partially purified ribosomal proteins, the conformation of which appears not to be unduly disturbed by this extraction method [7].

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