Identification of yeast 60 S ribosomal proteins crosslinked to rRNA by 2-iminothiolane (original) (raw)
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Journal of Biological Chemistry, 1989
We have investigated the protein-protein cross-links formed within the 60 S subunit of the Escherichia coli ribosome using 2-iminothiolane as the cross-linking reagent. The members of the cross-links have been identified by immunoblotting from one-dimensional and two-dimensional diagonal sodium dodecyl sulfatepolyacrylamide gels using antisera specific for the individual ribosomal proteins. This method also allowed a quantitation of the yield of cross-linking for each cross-link. A total of 14 cross-links have been identified Ll
Tetrahymena ribozyme disrupts rRNA processing in yeast
Journal of Biological Chemistry
The intervening sequence (IVS) of lletruhymenu thermophila nucleolar DNA interrupts a highly conserved sequence in the RNA core structure of the large ribosomal subunit. This location in nuclear DNA is unusual as most group I introns are in mitochondrial and chloroplast DNA. To examine the effect of a ribozyme insertion in another nuclear genome, the !&truhymenu IVS was introduced into the analogous position in a cloned Schizosaccharomyces pombe ribosomal gene, and the mutant rDNA was expressed in vivo. RNA analyses indicated that mature 5.8 S rRNA was not formed from the mutant gene transcript and the amount of 27 S nRNA was significantly reduced. In contrast, hybridization analyses indicated that RNA splicing continued, and normal forms of free ribozyme were present. The results show that the IVS sequence can interfere with rRNA processing and suggest that the unusual amplification of a single rDNArepeat may have forced lletrahymena to accommodate its ribozyme.
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...
Probing the conformation of 26S rRNA in yeast 60S ribosomal subunits with kethoxal
Biochemistry, 1984
The conformation and accessibility of 26s rRNA in yeast 6 0 s ribosomal subunits were probed with kethoxal. Oligonucleotides originating from reactive sites were isolated by diagonal electrophoresis and sequenced. From over 70 oligonucleotide sequences, 26 kethoxal-reactive sites could be placed in the 26s rRNA sequence. These are in close
Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli
Biochemistry, 1989
We have carried out an extensive protein-protein cross-linking study on the 50s ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-Ll9, L10-L11, L13-L21, and L14-LI9. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (1 2 A),
Probing the conformation of 18S rRNA in yeast 40S ribosomal subunits with kethoxal
Biochemistry, 1984
Yeast 40s ribosomal subunits have been reacted with kethoxal to probe the conformation of 18s rRNA. Over 130 oligonucleotides were isolated by diagonal electrophoresis and sequenced, allowing identification of 48 kethoxal-reactive sites in the 18s rRNA chain. These results generally support a secondary structure model for 18s rRNA derived from comparative sequence analysis. Significant reactivity at positions 1436 and 1439, in a region shown to be base paired by comparative analysis, lends support to the earlier suggestion R i b o s o m e s from the cytoplasm of eucaryotes are distinguishable from those of procaryotic cells or organelles by their size, shape, and chemical composition (Wool, 1979). They are usually referred to as 80s ribosomes, dissociable into 40s and 6 0 s subunits, in contrast with the 70S, 30S, and 50s subunits of procaryotes. They possess morphological characteristics that distinguish them from eubacterial or archaebacterial ribosomes (Lake et al., 1982) and have a higher
Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly
The Journal of Cell Biology, 1990
Two strains of Saccharomyces cerevisiae were constructed that are conditional for synthesis of the 60S ribosomal subunit protein, L16, or the 40S ribosomal subunit protein, rp59. These strains were used to determine the effects of depriving cells of either of these ribosomal proteins on ribosome assembly and on the synthesis and stability of other ribosomal proteins and ribosomal RNAs. Termination of synthesis of either protein leads to diminished accumulation of the subunit into which it normally assembles. Depletion of L16 or rp59 has no effect on synthesis of most other ribosomal proteins or ribo-bly of the Saccharomyces cerevisiae 40S ribosomal subunit protein, rp59, and the 60S ribosomal subunit protein, L16, we constructed strains that are conditional for synthesis of either protein. We describe the effect of terminating synthesis of these r-proteins on ribosome assembly. The work presented in this paper demonstrates that synthesis rates of most 40 or 60S subunit proteins and RNAs are not affected by terminating synthesis of an individual r-protein. However, most proteins and RNAs that are members of the same subunit as the protein whose synthesis is terminated are degraded rapidly in the absence of assembly into ribosomes. The stability of protein and RNA components of the opposite subunit is not affected.