The sequences of the nontranscribed spacers (NTS) of cloned ribosomal DNA (rDNA) units from both Saccharomyces cerevisiae and Saccharomyces carlsbergensis were determined. The NTS sequences of both species were found to be 93% homologous. The major disparities comprise different frequencies of reiteration of short tracts of six to sixteen basepairs. Most of these reiterations are found within the 1100 basepairs long NTS between the 3'-ends of 26S and 5S rRNA (NTS1). The NTS between the starts of 5S rRNA and 37S pre-rRNA (NTS2) comprises about 1250 basepairs. The first 800 basepairs of NTS NTS2 (adjacent to the 5S rRNA gene) are virtually identical in both strains whereas a variable region is present at about 250 basepairs upstream of the RNA polymerase A transcription start. In contrast to the situation in Drosophila and Xenopus no reiterations of the putative RNA polymerase A promoter are present within the yeast NTS. The strands of the yeast NTS reveal a remarkable bias of G and C-residues. Yeast rDNA was previously shown to contain a sequence capable of autonomous replication (ARS) (Szostak, J.W. and Wu, R (1979), Plasmid 2, 536-554). This ARS, which may correspond to a chromosomal origin of replication, was located on a fragment of 570 basepairs within NTS2.

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].

No abstract

The binding site of the yeast 60S ribosomal subunit protein L25 on 26S rRNA was determined by RNase protection experiments. The fragments protected by L25 originate from a distinct substructure within domain IV of the rRNA, encompassing nucleotides 1465‐1632 and 1811‐1861. The protected fragments are able to rebind to L25 showing that they constitute the complete protein binding site. This binding site is remarkably conserved in all 23/26/28S rRNAs sequenced to date including Escherichia coli 23S rRNA. In fact heterologous complexes between L25 and E. coli 23S rRNA could be formed and RNase protection studies on these complexes demonstrated that L25 indeed recognizes the conserved structure. Strikingly the L25 binding site on 23S rRNA is virtually identical to the previously identified binding site of E. coli ribosomal protein EL23. Therefore EL23 is likely to be the prokaryotic counterpart of L25 in spite of the limited homology displayed by the amino acid sequences of the two proteins.

No abstract