The 5‐S RNA Binding Protein from Yeast (Saccharomyces cerevisiae) Ribosomes

Abstract

The ribonucleoprotein complex between 5-S RNA and its binding protein (5-S RNA . protein complex) of yeast ribosomes was released from 60-S subunits with 25 mM EDTA and the protein component was purified by chromatography on DEAE-cellulose. This protein, designated YL3 (MI = 36000 on dodecylsulfate gels), was relatively insoluble in neutral solutions (pH 4-9) and migrated as one of four acidic 60-S subunit proteins when analyzed by the Kaltschmidt and Wittman two-dimensional gel system. Amino acid analyses indicated lower amounts of lysine and arginine than most ribosomal proteins. Sequence homology was observed in the N terminus of YL3, and two prokaryotic 5-S RNA binding proteins, EL 18 from Escherichia coliand HL 13 from Halobacterium cutirubrum : Ala'-Phe2-Gln3-Lys4-AspS-Ala6-Lys7-Ser8-Ser9-Ala'o-Tyr''-Ser'2-Ser'3-Arg'4-Phe'sGln'6-Tyr'7-Pro's-Phe'9-Arg20-Arg21-Arg22-Arg23-G1~24-Gly2s-Ly~26-Thr27-A~p28-Tyr2 ;of particular interest was homology in the cluster of basic residues (18-23). Since the protein contained one methionine residue it could be split into two fragments, CN1 (MI = 24700) and CN2 ( M , = 11 300) by CNBr treatment; the larger fragment originated from the N terminus. The N-terminal amino acid sequence of CN2 shared a limited sequence homology with an internal portion of a second 5-S RNA binding protein from E. coli, EL5, and, based also on the molecular weights of the proteins and studies on the protein binding sites in 5-S RNAs, a model for the evolution of the eukaryotic 5-S RNA binding protein is suggested in which a fusion of the prokaryotic sequences may have occurred. Unlike the native 5-S RNA . protein complex, a variety of RNAs interacted with the smaller CN2 fragment to form homogeneous ribonucleoprotein complexes; the results suggest that the CN1 fragment may confer specificity on the natural 5-S RNA-protein interaction.

Molecular cloning and nucleotide sequence of cDNA specific for rat ribosomal protein L5

“…4, the deduced amino acid sequence of protein L5 is compared with the known sequences of other ribosomal proteins which bind to 5s RNA. As can be seen, the Nterminal amino acid sequence of yeast YL3 [15] and that of rat L5 show high homology: 18 out of the 30 amino acids with a cluster of 4 arginine residues are identical. It is of interest that a reported sequence of 36 amino acids from the N-terminal region of Halobacterium cutirubrum ribosomal protein HL13 [24] may correspond to residues 10-45 of rat L5, since 12 out of the 36 amino acids with the arginine cluster are identical.…”

“…From the limited homology of the partial amino acid sequences of fragments CN1 and CN2, cleaved products of yeast 5s RNA binding protein (YL13), with those of Escherichia coli 5s RNA binding proteins (EL18, EL5 and EL25), Nazar et al [15] and Yaguchi et al [16] proposed that the single large eucaryotic 5s RNA binding protein may have evolved through a fusion of genes for the multiple 5s RNA binding proteins in procaryotes.…”

Accessibility of phosphodiester bonds in the yeast ribosomal 5 S RNA protein complex

“…In a typical experiment 32P-labeled 5 S RNA was dissolved in 25 pl of 25 mM EDTA, pH 7.0, containing 5-6 Az60nm units/ml of unlabeled yeast complex and incubated on ice for 30-45 min. The labeled complex was characterized by electrophoresis on an 8% polyacrylamide gel [IO,22]; 50-90% of the labeled RNA was associated with the ribonucleoprotein complex.…”

“…Yeast (S. cerevisiae, strain S288C) was grown aerobically and both the 5 S RNA and 5 S RNA-protein complexes were isolated from 60 S ribosomal subunits as described [22]. The purified RNA was labeled with cytidine 3',5-[5'-"Plbisphosphate at the 3'-end using RNA ligase (Pharmacia) and repurified on a 12% polyacrylamide sequencing gel [23].…”

Mechanism of resistance to the antibiotic trichothecin in the producing fungi

“…In the cases so far studied in detail, the resistance genetic locus has been shown to be located in the gene encoding ribosomal protein L3 (Fernandez-Lobato et al, 1990;Fried and Warner, 1981). Interestingly, this protein forms a complex with the 5s rRNA (Nazar et al, 1979) located at the central protuberance in the large ribosomal subunit, close to the peptidyl transferase center where the antibiotic is proposed to bind.…”