The primary structure of protein eL12'/eL12'‐P from the large subunit of <i>Artemia salina</i> ribosomes

Amons, Reinout; Pluijms, W.; Kriek, Jan; Möller, W.

doi:10.1016/0014-5793(82)80722-9

Cited by 32 publications

(16 citation statements)

References 23 publications

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“…Determination of the nucleotide sequences of these cDNAs revealed that although the three types of cDNA molecules have different sequences, they could be translated into almost identical carboxy-terminal amino acid sequences (KEESEESD(D/E)DMGFGLFD-COOH). This sequence is quite similar to carboxy-terminal regions of published sequences of P-proteins of rat (P2) (KEESEEKKDEM GFGLFD-COOH) (29), brine shrimp (eL12 and eL12') (KKEEKKEESEEEDEDMGFGLFD-COOH) (1,2,30), and yeast YPA1 (KEEEAKEESDDDMGFGLFD-COOH) (23), all of which are recognized by anti-P-protein antibodies (10,50). Although these cDNAs are quite small, containing as few as the last 23 codons of the coding sequence, all of the clones generated fusion proteins that were recognized by lupus-patient as well as monoclonal anti-P-protein antibodies (data not shown) (50).…”

Section: Resultssupporting

confidence: 55%

“…Like L7/L12, these two proteins interact with eucaryotic elongation factors EFI and EF2 and are required for aminoacyl-tRNA binding and EF2-dependent GTPase activity, as well as polypeptide synthesis (31,32,45). In Artemia sailina (brine shrimp), these two P-proteins, eL12 (P2; molecular weight, 11,472) and eL12' (P1; molecular weight, 11,423), are similar to one another in size and amino acid composition but they are derived from distinct genes (1,2,30). They share an identical 22-amino-acid sequence at their C termini that resembles the C-terminal sequences of the other two eucaryotic P-proteins whose sequences have been published, rat P2 (molecular weight, 11,186) (29) and Saccharomn'c es cerevisiae YPA1 (molecular weight, 11,020) (23).…”

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confidence: 99%

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Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly.

Rich¹,

Steitz²

1987

Mol. Cell. Biol.

273

153

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cDNA clones encoding three antigenically related human ribosomal phosphoproteins (P-proteins) P0, P1, and P2 were isolated and sequenced. P1 and P2 are analogous to Escherichia coli ribosomal protein L7/L12, and P0 is likely to be an analog of LIO. The three proteins have a nearly identical carboxy-terminal 17-amino-acid sequence (KEESEESD(D/E)DMGFGLFD-COOH) that is the basis of their immunological cross-reactivity. The identities of the P1 and P2 cDNAs were confirmed by the strong similarities of their encoded amino acid sequences to published primary structures of the homologous rat, brine shrimp, and Saccharomyces cerevisiae proteins. The P0 cDNA was initially identified by translation of hybrid-selected mRNA and immunoprecipitation of the products. To demonstrate that the coding sequences are full length, the PO, P1, and P2 cDNAs were transcribed in vitro by bacteriophage T7 RNA polymerase and the resulting mRNAs were translated in vitro. The synthetic P0, P1, and P2 proteins were serologically and electrophoretically identical to P-proteins extracted from HeLa cells. These synthetic P-proteins were incorporated into 60S but not 40S ribosomes and also assembled into a complex similar to that described for E. coli L7/L12 and L10.Acidic ribosomal proteins from diverse species have been studied extensively by a number of different techniques. These proteins, called A-proteins (acidic) or P-proteins (the phosphorylated eucaryotic A-proteins), are generally present in multiple copies on the ribosome and have isoelectric points in the range of pH 3 to 5, in contrast to most ribosomal proteins, which are single copy and basic. Aproteins have hydrophobic amino acid compositions (notably about 20% alanine) (34). Because of their distinct chemical properties, A-proteins can be easily dissociated from the ribosome by treatment with high salt and 50% ethanol; the A-proteins remain soluble whereas the remainder of the ribosomes or subunits precipitate (17). This procedure provides a uniform, simple protocol for the identification and isolation of A-proteins from many sources.The best characterized A-protein is the L7/L12 protein of Escherichia coli (L7 is the N-acetylated form of L12 [molecular weight, 12,164]), which has been localized to the stalk of the large (50S) ribosomal subunit (5,38). L7/L12 interacts with several translation factors, some of which bind and hydrolyze GTP, including initiation factor IF2, elongation factors EF-Tu and EF-G, and release factors RF1 and RF2 (5, 59). L7/L12 is required for binding of these factors as well as aminoacyl-tRNA to the ribosome (5, 59). Hydrodynamic and circular dichroism studies have suggested that L7/L12 and other A-proteins are prolate and have high alpha-helical contents (16). However, the crystal structure of the Cterminal domain of L7/L12 has been determined (28), and it is remarkably globular in light of the elongated shape of the whole molecule. The results of intraribosomal proteinprotein cross-linking studies and reconstitution experiments have depicted a complex...

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Section: Resultssupporting

confidence: 55%

mentioning

confidence: 99%

Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly.

Rich¹,

Steitz²

1987

Mol. Cell. Biol.

273

153

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“…Using the cDNA sequence from C328-15, the amino acid sequence of the putative translation product was deduced and this was used to search the NBRF protein sequence database for homologous protein sequences. A region of strong homology was found between the amino acid sequence of the putative C328-15-derived protein and the amino acid sequence of acidic ribosomal phosphoproteins from rat (Lin et al, 1982) and brine shrimp (Amons et al, 1979(Amons et al, , 1982Maassen et al, 1985 (Rich & Steitz, 1987). Thus, the pLM59 cDNA used as the probe initially corresponds to the mRNA for the human P2 protein.…”

Section: Screeningmentioning

confidence: 99%

A sequence previously identified as metastasis-related encodes an acidic ribosomal phosphoprotein, P2

Sharp

Elvin²,

Walker³

et al. 1990

Br J Cancer

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Summary We have used a metastasis-related human cDNA isolated from a liver metastasis from a colonic adenocarcinoma to screen a human breast carcinoma cDNA library for homologous sequences. Nucleotide sequence analysis of positive clones revealed that the cDNA represents a ribosomal phosphoprotein, P2. The expression of P2 mRNA was significantly higher (Student's t test, one tail; P.0.01) in seven fibroadenomas than in seven carcinomas, with an average five-fold difference. This enhanced expression level of P2 mRNA in benign fibroadenomas compared with malignant carcinomas is contrary to that expected, based on earlier work with normal colonic mucosa, colorectal carcinoma and hepatic metastasis. The identification of gene transcripts which differ in abundance and correlate with the metastatic phenotype may be of considerable importance both as diagnostic aids and in defining the changes associated with tumour progression and metastasis at the molecular level. The possible role that ribosomal proteins may play in the progression of carcinoma of the breast is discussed.

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“…Trypsin cleavage. Purified L7/L12 protein was citraconylated [37] followed by cleavage with I-chloro-4-phenyl-3-tosylamidobutan-2-one-treated trypsin at 37°C [38]. The peptides so obtained were separated by reversed-phase, highperformance liquid chromatography (pBondapak C18 and Baker WidePore C18 columns) with a linear gradient of 0 -75% CH,CN in 0.1% trifluoroacetic acid (see Fig.…”

Section: Preparation Of L7/l12 Proteinsmentioning

confidence: 99%

“…After completion of tryptic digestion [38] the solution was brought to pH 4.3 [35]. The TIMet peptide (positions 5-29) was purified from the precipitate [35].…”

Section: Preparation Of L7/l12 Proteinsmentioning

confidence: 99%

Primary structures of mutationally altered ribosomal protein L7/L12 and their effects on cellular growth and translational accuracy

1986

Self Cite

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The amino acid sequences of mutationally altered ribosomal protein L7/L12 from four different rplL mutants of Escherichia coli were determined and correlated with some features of the mutant ribosomes. Two of the rplL mutations are deletions around position 40, which give rise to a shortened hinge region between the two domains of L7/L12. The other two mutants harbor point mutations at position 74 (Gly+Asp) or at position 82 (Glu+Lys), which are in or close to an evolutionarily conserved sequence in the C-terminal domain. The two latter mutations are associated with decreased rates of growth and translational elongation. All four mutants show increased nonsense codon read-through in vivo. Ribosomes from one of the deletion mutants show clearly increased missense error rates in vitro.Some mutationally altered ribosomal proteins affect translational accuracy both in vivo and in vitro. The best characterized examples of far are mutants with changed S12 (rpsL), which show a more accurate phenotype, and with altered S4 (rpsD), which are less accurate during translation [l -81. These mutants [9, 101 are primarily defective in translational proof-reading step(s) [ll -151. Recent observations show that S5 (rpsE) mutations, which are associated with a ribosomal ambiguity (Ram) phenotype in vivo and in vitro, produce ribosomes which have a decreased proof-reading intensity [16,17,441. Furthermore, mutations in rpsQ (S17) and in rplF (L6) might give a more accurate translation but such mutants are not so well characterized as those affecting S4 and S12 [18, 191. Recently ribosomal protein L7/L12 was shown to be involved in the maintenance of translational accuracy. It was found that some mutations in the gene coding for this protein (rplL) increase the translational error rates both in vivo and in vitro. Furthermore, the increased error level, characteristic of the mutant ribosomes containing altered forms of L7/L12, can be correlated with a decreased proof-reading efficiency. The increased translational misreading in vivo is reversed by the introduction of an rpsL allele, which gives a Str-R phenotype. Therefore it appears that some mutants with altered L7/L12 in many respects are similar to S4 and S5 mutants [7, 9, 20, 441 in that they both increase error frequencies and suppress streptomycin-dependent phenotypes.In the present investigation we have purified the L7/L12 protein [21] from four different rplL mutants and localized the corresponding alterations by determining their amino acid sequences. In addition, we describe the effects of these mutational alterations on translation rates both in vivo MATERIALS AND METHODS Construction of strainsThe E. coli K12 strains used in this study are listed in Table 1. The rplL159, rplL265 and rplL564 alleles were introduced by PI -mediated cotransduction with argE+ into UY128 and UD132 followed by screening for rifampicin sensitivity (Rif-S) as described elsewhere [20]. Strain VT564 was used as the donor of rplL564.Construction of the rplL518 derivatives UY258 and UY278 was...

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The primary structure of protein eL12'/eL12'‐P from the large subunit of Artemia salina ribosomes

Cited by 32 publications

References 23 publications

Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly.

Human acidic ribosomal phosphoproteins P0, P1, and P2: analysis of cDNA clones, in vitro synthesis, and assembly.

A sequence previously identified as metastasis-related encodes an acidic ribosomal phosphoprotein, P2

Primary structures of mutationally altered ribosomal protein L7/L12 and their effects on cellular growth and translational accuracy

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