Yeast ribosomal protein L25 binds to an evolutionary conserved site on yeast 26S and E. coli 23S rRNA (original) (raw)
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Biochimie, 1987
The interaction of ribosomal protein EL23 from E. coli and L25 from yeast with yeast 26S rRNA was analysed by nitrocellulose filter binding and RNase protection experiments using both intact rRNA and various fragments prepared by in vitro transcription of cloned yeast rDNA regions in the SP6 system. The results show that EL23 efficiently and specifically interacts with the region of 26S rRNA previously identified as the binding site for the yeast ribosomal protein L25. A comparison of the oligonucleotides resulting from limited RNase T1 digestion of the heterologous EL23/26S rRNA complex with those obtained by the same treatment of the homologous L25/26S rRNA complex showed that the molecular details of the two r-protein/rRNA interactions are highly similar if not identical. Using the synthetic 26S rRNA fragments we could demonstrate that all information for the formation of a biologically active binding site is located within the region of the rRNA delimited by the sequences protec...
Journal of Molecular Biology, 1987
The heterologous interaction of Escherichia coli ribosomal protein EL1 1 with yeast 26 S and mouse 28 S rRNA was studied by analysing the ability of this protein to form a specific complex with various synthetic rRNA fragments that span the structural equivalent of the ELI 1 binding site present in these eukaryotic rRNAs. The fragments were obtained by SP6 polymerase-directed in-vitro runoff transcription of parts of the yeast or mouse large rRNA gene cloned behind the SP6 promoter. EL11 was found to protect an oligonucleotide fragment of 63 nucleotides from both the yeast and mouse transcripts against digestion by RNase T,. In both cases, the position of this fragment in the L-rRNA § sequence coincides almost exactly with that of the fragment previously found to be protected by EL11 in E. coli 23 S rRNA. Moreover, the protected yeast fragment was shown to be able to re-bind to EL11 by a nitrocellulose filter binding assay. A ribosomal protein preparation from Saccharomyces cerevisiae containing L15 (YL23) as well as the acidic proteins L44', L44 and L45 protects exactly the same oligonucleotide fragment as does EL1 1 in both the yeast and mouse transcripts. Evidence is provided that 1~15, which is known to be structurally and functionally equivalent to EL1 1, is the rRNA-binding protein in this preparation. Thus t,he structural equivalent of the EL11 binding site present in yeast 26 S rRNA constitutes the second example of functional conservation of a ribosomal protein-binding site on rRNA between prokaryotes and eukaryotes.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1990
We have developed a combination of in vivo and in vitro methods which allows us to determine the effect of practically every slrucOwai change, deletions as well as point mutations, on various biological functions of a ribosomal protein (r-protein). We have used this approach to delineate the functional domains of r-protein L25 from Saccharomyces cerevisiae. By analysis of the intracellular distribution of fusion proteins carrying various portions of L25 linked to EschedcMa eoli fl-galactosidase we traced the nuclear localization signal(s) of L25 to the region encompassing the N-terminal 61 amino acids of the protein. On the other hand, using in vitro prepared fragments of L25 we located the domain responsible for its specific binding to 26S rRNA to the region between amino acids 61 and 135. In order to be able to analyze the effect of mutations in L25 on ribosome biogenesis and function in vivo we constructed a mutant yeast strain in which the chromosomal L25 gene is placed under control of the inducible yeast GAL promoter. Since this strain is unable to grow on glucose as a carbon source the L25 gene must be essential for cell viability. Growth on glucose can be restored by introduction of a wild-type L25 gene on a plasmid, demonstrating that under these conditions the cells are dependent upon an extrachromosomaily supplied copy of the gene. * E. coil r-proteins are indicated by the prefix E. For yeast r-proteins the nomenclature of Kruiswijk and Planta [16] is used.
The primary and secondary structure of yeast 26S rRNA
Nucleic Acids Research, 1981
We present the sequence of the 26S rRNA of the yeast Saccharomyces carlsbergensis as inferred from the gene sequence. The molecule is 3393 nucleotides long and consists of 48% G+C; 30 of the 43 methyl groups can be located in the sequence. Starting from the recently proposed structure of E_. coli 23S rRNA (see ref. 25) we constructed a secondary structure model for yeast 26S rRNA. This structure is composed of 7 domains closed by long-range base pairings as in the bacterial counterpart. Most domains show considerable conservation of the overall structure; unpaired regions show extended sequence homology and the base-paired regions contain many compensating base pair changes. The extra length of the yeast molecule is due to a number of insertions in most of the domains, particularly in domain II. Domain VI, which is extremely conserved, is probably part of the ribosomal A site. a-Sarcin, which apparently inhibits the EF-1 dependent binding of aminoacyl-tRNA, causes a cleavage between position 3025 and 3026 in a conserved loop structure, just outside domain VI. Nearly all of the located methyl groups, like in E_. coli, are present in domain II, V and VI and clustered to a certain extent mainly in regions with a strongly conserved primary structure. The only three methyl groups of 26S rRNA which are introduced relatively late during the processing are found in single stranded loops in domain VI very close to positions which have been shown in £. coli 23S rRNA to be at the interface of the ribosome.
Nucleic Acids Research, 2010
The majority of constitutive proteins in the bacterial 30S ribosomal subunit have orthologues in Eukarya and Archaea. The eukaryotic counterparts for the remainder (S6, S16, S18 and S20) have not been identified. We assumed that amino acid residues in the ribosomal proteins that contact rRNA are to be constrained in evolution and that the most highly conserved of them are those residues that are involved in forming the secondary protein structure. We aligned the sequences of the bacterial ribosomal proteins from the S20p, S18p and S16p families, which make multiple contacts with rRNA in the Thermus thermophilus 30S ribosomal subunit (in contrast to the S6p family), with the sequences of the unassigned eukaryotic small ribosomal subunit protein families. This made it possible to reveal that the conserved structural motifs of S20p, S18p and S16p that contact rRNA in the bacterial ribosome are present in the ribosomal proteins S25e, S26e and S27Ae, respectively. We suggest that ribosomal protein families S20p, S18p and S16p are homologous to the families S25e, S26e and S27Ae, respectively.
Proceedings of the National Academy of Sciences, 1993
Previous phylogenetic analysis of rRNA sequences for covariant base changes has identified approximately 20 potential tertiary interactions. One of these is present in domain III of the large subunit rRNA and consists of two adjacent Watson-Crick base pairs that, in Saccharomyces cerevisiae 26S rRNA, connect positions 1523 and 1524 to positions 1611 and 1612. This interaction would strongly affect the structure of an evolutionarily highly conserved region that acts as the binding site for the early-assembling ribosomal proteins L25 and EL23 of S. cerevisiae and Escherichia coli, respectively. To assess the functional importance of this tertiary interaction, we determined the ability of synthetically prepared S. cerevisiae ribosomal protein L25 to associate in vitro with synthetic 26S rRNA fragments containing sequence variations at positions 1523 and 1524 and/or positions 1611 and 1612. Mutations that prevent the formation of both base pairs abolished L25 binding completely, whereas...
Nucleotides in 23S rRNA protected by the association of 30S and 50S ribosomal subunits
Journal of Molecular Biology, 1999
We have studied the effect of subunit association on the accessibility of nucleotides in 23 S and 5 S rRNA. Escherichia coli 50 S subunits and 70 S ribosomes were subjected to a combination of chemical probes and the sites of attack identi®ed by primer extension. Since the ribose groups and all of the bases were probed, the present study provides a comprehensive map of the nucleotides that are likely to be involved in subunit-subunit interactions. Upon subunit association, the bases of 22 nucleotides and the ribose groups of more than 60 nucleotides are protected in 23 S rRNA; no changes are seen in 5 S rRNA. Interestingly, the bases of nucleotides A1866, A1891 and A1896, and G2505 become more reactive to chemical probes, indicating localized rearrangement of the structure of the 50 S subunit upon association with the 30 S subunit. Most of the protected nucleotides are located in four stem-loop structures around positions 715, 890, 1700, and 1920. In free 50 S subunits, virtually all of the ribose groups in these four regions are strongly cleaved by hydroxyl radicals, suggesting that these stems protrude from the 50 S subunit. When the 30 S subunit is bound, most of the ribose groups in the 715, 890, 1700 and 1920 stemloops are protected, as are many bases in and around the corresponding apical loops. Intriguingly, three of the protected regions of 23 S rRNA are known to be linked via tertiary interactions to features of the peptidyl transferase center. Together with the juxtaposition of the subunit-protected regions of 16 S rRNA with the small subunit tRNA binding sites, our ®ndings suggest the existence of a communication pathway between the codon-anticodon binding sites of the 30 S subunit with the peptidyl transferase center of the 50 S subunit via rRNA-rRNA interactions.