Differences between the protein moieties of active subunits and EDTA-treated subunits of rat liver ribosomes with specific references to A 5 S rRNA · protein complex

Terao, Kazuo; Takahashi, Yoshiaki; Ogata, Kazuhiro

doi:10.1016/0005-2787(75)90042-8

Cited by 62 publications

(21 citation statements)

References 15 publications

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“…Rat ribosomal protein L5 was isolated as the 5s RNP complex by the method of Terao et al [6]. The 5s RNP obained was used for the amino acid sequence determination by automatic Edman degradation in an Applied Biosystems gas-phase microsequencer.…”

Section: Preparation Of Synthetic Probe Dnamentioning

confidence: 99%

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

Tamura¹,

Kuwano²,

Nakayama³

et al. 1987

Eur J Biochem

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cDNA clones specific for rat ribosomal protein L5, which is the protein moiety of 5s RNA protein particles, were isolated by using a mixture of tetradecamer deoxyribonucleotide probes reflecting the amino acid sequence of the N-terminal portion of L5. A cDNA clone (pRL5-1) lacking the 3'-terminal region of L5 was isolated by hybrid-selected translation and shown to be L5-specific by the identity of the deduced amino acid sequence and that of the N-terminus of protein L5. pRL5-1 was then used to select another clone @RL5-2) by colony hybridization which contained the entire coding region, 3'-non-coding region and poly(A) addition signal. The nucleotide sequence of L5-specific cDNA was determined by using these two clones. It contained a total of 1069 base pairs: 123 base pairs in the 5'-non-coding region, 894 base pairs in the coding region and 52 base pairs in the 3'-non-coding region. The poly(A) addition signal appeared at the 33rd nucleotide after the termination codon. The amino acid sequence deduced from this nucleotide sequence consists of 296 amino acids. The homology of amino acid sequence between L5 and other 5s RNA-binding proteins is discussed.The large ribosomal subunits of both procaryotes and eucaryotes contain 5s RNA, which can be released in the form of a complex with one or more ribosomal proteins by various treatments (for review, see [l]). A 5s RNA-protein complex is, for example, released from rat liver 60s subunits by EDTA treatment [2] or other methods 13 -51, and we have identified its protein moiety as protein L5 [6] according to the proposed uniform nomenclature [7]. The existence of the 5S-RNA-L5-protein complex in the 60s ribosomal subunits is shown by cross-linking of 5 s RNA to L5 by ultraviolet irradiation of this particle [8] or by treating it with sodium periodate followed by reduction by sodium borohydride [9]. We have also shown [lo] that ribosomal protein L5 is crosslinked to mRNA besides proteins S3a, S6 and L6 by ultraviolet irradiation of the total polysomal fraction of rat liver and to proteins S4 and S25 by treatment of rat liver 80s ribosomes with 2-iminothiolane [ll]. These results suggest that the 5S-RNA-L5 complex is located at the boundary between the large and the small subunits and interacts with mRNA.Others have reported that L5 is located at the peptidyl (P) site in the 80s initiation complex [12].The interaction between 5s RNA and protein L5 has been extensively studied and the L5 binding site in 5s RNA has been identified by Aoyama et al. [13] and Huber and Wool [14]. 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.To elucidate further the function, the mode of interaction with 5s RNA and...

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Section: Preparation Of Synthetic Probe Dnamentioning

confidence: 99%

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

Tamura¹,

Kuwano²,

Nakayama³

et al. 1987

Eur J Biochem

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“…1 B and D). This shadow has been reported below rat liver L5 [7] and probably represents a proteolytic derivative of this protein.…”

Section: Resultsmentioning

confidence: 60%

“…The protein moiety exists in a 1 : 1 ratio with 5-S RNA and has been identified by two-dimensional gel electrophoresis as protein L5 [7] using the standard nomenclature for mammalian ribosomal proteins [S]. A protein complexed with 5-S RNA is also released from yeast 60-S subunits by EDTA [9,10].…”

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confidence: 99%

Identification of Homologous Ribosomal Proteins in HeLa Ceils and in Tetrahymena pyriformis. A Study of Proteins Binding 5-S RNA and Acidic Proteins Released from 40-S Subunits by EDTA

Svoboda¹,

McConkey

1981

Eur J Biochem

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Tetruhymena pyriformis 60-S ribosomal subunits treated with EDTA release a 7-S particle containing 5-S RNA and a 36000-M,. protein that is similar to mamnialian 5-S-RNA-binding protein L5 in molecular weight, in two-dimensional acrylamide gel mobility, and in peptide pattern as generated by a simple, one-dimensional acrylamide gel technique. Human and T. pyriformis 40-S ribosomal subunits, treated with buffers lacking magnesium or containing EDTA, release varying amounts of two large acidic proteins. We have identified these released proteins by two-dimensional gel electrophoresis.Ciliates of the genus Tetrahymena represent one of the most primitive groups of eukaryotes known. Evolutionary relationships, derived from the amino acid sequences of cytochrome c, suggest that Tetruhymenu pyriformis diverged from a common eukaryotic ancestor earlier than plants or yeast [I], and the histone IV sequence of T. thermophilu is substantially different from the other eukaryotic histone IV sequences thus far examined [2]. Recently the efficient isolation of active ribosomes from T. pyriformis and the two-diniensional gel identification of its ribosomal proteins have been accomplished [3,4]. Therefore, in an effort to study the relationships between ribosomal proteins froin two very distantly related eukaryotes, we have studied selected human and T. pyriformis ribosomal proteins that share similar properties. Some fundamental similarities are expected between ciliate and mammalian ribosomal components because T.pyriforormis 40-S subunits added to rat liver 60-S subunits can translate poly(U) in vitro [5].A 7-S particle containing 5-S RNA and protein is released from EDTA-treated 60-S subunits from rat liver [6]. The protein moiety exists in a 1 : 1 ratio with 5-S RNA and has been identified by two-dimensional gel electrophoresis as protein L5 [7] using the standard nomenclature for mammalian ribosomal proteins [S]. A protein complexed with 5-S RNA is also released from yeast 60-S subunits by EDTA [9,10]. We report here that a similar 5-S-RNA . protein complex is released from EDTA-treated 60-S subunits from T. pyrifbrmis. Both a two-dimcnsional gel comparison and a peptide comparison of the T. pyriformis 5-S-RNA-associated protein and HeLa protein L5 are presented.Eukaryotic 40-S subunits unfold into slower sedimenting particles when they are treated with EDTA [ll]. After resuspension in buffer containing 2.5 mM EDTA, 40-S subunits Dt$niiion. A Z~O unit, the amount of material in 1 ml of a aolution that has an absorbance of 1 when 260-nm light is passcd through a l-cmpathlength cell.Enzvmt7s. RNase A ( K 3.1.27.5), RNase TI (EC 3.1.27.3), chymotrypsin (EC 3.4.21.1); Sfuphylococcus uu~cus VX protease (EC 3.4.21.19), papain (EC 3.4.12.2).~~ from rabbit reticulocytes release two high and two lowmolecular-weight ribosomal proteins that are visible on a one-dimensional sodium dodecyl sulfate gel but have not been identified by two-dimensional gel electrophoresis 1121. We have treated 40-S subunits from HeLa cells and T.pyrifbrrnis...

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“…The post-nuclear supernatant was centrifuged at 21000Xg for 12 min and the resultant supernatant was centrifuged at 264 500Xg for 2 h. The supernatant was used as the liver cytosol [l]. The ribosomes, ribosomal 60s and 40s subunits and 5SRNP were prepared from the microsomal fraction as described previously [9]. The 5SRNP was prepared by sucrose-gradient centrifugation of EDTA-treated 60s subunits [7].…”

Section: Cell Fractionationmentioning

confidence: 99%

Metabolic characteristics of rat liver cytosolic 5SRNA

Ogata

Kurahashi

Nishiyama

et al. 1993

European Journal of Biochemistry

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Institute for Gene Expression, Dobashi Kyoritsu Hospital, Matsuyama, Japan SSRNA were purified from the cytosol, ribosomes, nucleoli and nucleoplasm of rat liver. The 5'-terminal and 3'-terminal sequences of these SSRNA species were shown to be the same. The time course of changes in specific activities of these SSRNA in rat liver, 20 min to 16 h after intraperitoneal injection of ['4C]orotic acid, were investigated. The specific activity of cytosolic SSRNA increased almost linearly for 3 h, without a lag, to a high value, then decreased rapidly. The specific activity of cytosolic SSRNA was much higher than the specific activities of SSRNA from the other cellular components over 45 min to 6 h after intraperitoneal injection, although at 20 min the specific activity was similar to that of nucleoplasmic SSRNA. Thus, cytosolic SSRNA of rat liver is characterized by a high rate of turnover, which was shown to be comparable to that of heterogeneous nuclear RNA. The specific activity of SSRNA, 45 min to 6 h after intraperitoneal injection, was as follows ; cytosolic > nucleoplasmic > nucleolar > ribosomal SSRNA. The specific activity of nucleoplasmic RNA that migrated slightly slower than nucleoplasmic SSRNA on PAGE was much higher than that of cytosolic SSRNA or nucleoplasmic SSRNA, and so may be the precursor of nucleoplasmic SSRNA. Together, these results indicate that cytosolic SSRNA is transferred from the nuclei to the cytosol immediately after maturation of the precursor of nucleoplasmic SSRNA without entering the nucleoplasmic pool of SSRNA.Previously, we reported [l] that most SSRNA in rat liver cytosol is present in two high molecular-mass fractions that can be separated by Sephadex G200 column chromatography. One of these fractions, eluted in the void volume, contained nine aminoacyl-tRNA synthetases. This macromolecular aminoacyl-tRNA synthetase complex was purified and found to contain SSRNA and the ribosomal protein L5, probably as a SSRNA-LS complex (SSRNP). Furthermore, SSRNA and SSRNP were found to function in the complex as positive effectors of methionyl-tRNA and isoleucyl-tRNA synthetases [2].Recently, we purified a fraction containing threonyltRNA and histidyl-tRNA synthetases from a second high molecular-mass fraction (fraction 11) obtained by chromatography on Sephadex G200, and found that this fraction also contained SSRNA and protein L5 (unpublished results).There are only a few reports on the metabolism of cytosolic SSRNA in mammalian cells; Zehabi-Willner and Danon [3] reported that the rate of radiolabeling of cytosolic SSRNA was much lower than that of ribosomal SSRNA in rabbit reticulocytes. Leibowitz et al. (1973) [4] presented a kinetic study on radiolabeling of SSRNA from various cellular components in HeLa cells with [3H]uridine; this study is relevant to our investigations. The authors indicated that newly synthesized SSRNA appeared rapidly in the cytosolic fraction. It then associated with the nucleolus via the nucleoplasm, where it was finally incorporated into large ribosomal subu...

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Differences between the protein moieties of active subunits and EDTA-treated subunits of rat liver ribosomes with specific references to A 5 S rRNA · protein complex

Cited by 62 publications

References 15 publications

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

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

Identification of Homologous Ribosomal Proteins in HeLa Ceils and in Tetrahymena pyriformis. A Study of Proteins Binding 5-S RNA and Acidic Proteins Released from 40-S Subunits by EDTA

Metabolic characteristics of rat liver cytosolic 5SRNA

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