Studies on the RNA and protein binding sites of the E.coli ribosomal protein L10

Pettersson, Ingvar

doi:10.1093/nar/6.7.2637

Cited by 43 publications

(23 citation statements)

References 23 publications

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“…The N Terminus of P0 Is Crucial for rRNA Binding-It has been reported that E. coli L10 and L10⅐L7/L12 complex bind directly to the 1070 region of 23 S rRNA (3,38,39), and the L10 fragment lacking the N-terminal 1-69 amino acids fails to assemble into the ribosome, together with L7/L12 (35). In eukaryotes, the yeast P0 variant lacking the C-terminal 87 residues can assemble into the ribosome in vivo with weakly bound P1/P2 (25).…”

Section: Discussionmentioning

confidence: 99%

A Mode of Assembly of P0, P1, and P2 Proteins at the GTPase-associated Center in Animal Ribosome

Hagiya

Naganuma

Maki

et al. 2005

Journal of Biological Chemistry

108

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Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants C⌬65 and C⌬81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0⅐P1-P2 binding site. The mutant C⌬107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants N⌬21-N⌬92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants N⌬6, N⌬14, and C⌬18-C⌬81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10⅐L7/L12 complex and L11. Analysis of incorporation of 32 P-labeled P1/P2 into the 50 S subunits with WT-P0 and C⌬81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but C⌬81 bound only one copy each. The hybrid ribosome with C⌬81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on eukaryotic elongation factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.The ribosomal large subunits from all organisms contain an active site termed the "GTPase-associated center" that is responsible for the GTPase-related events in protein biosynthesis. This active site is composed of the two highly conserved domains around 1070 and 2660 (Escherichia coli numbering is used throughout) of 23 S/28 S rRNA and the ribosomal proteins bound to the 1070 region (1-3). The protein components of this site in prokaryotic ribosomes constitute a characteristic pentameric complex, L10(L7/L12) 2 (L7/L12) 2 (4, 5), in which two L7/L12 homodimers bind to the C-terminal regions of L10 (6) and constitute a highly flexible and functionally important lateral protuberance, the so-called "stalk" (7). Although the ribosomal stalk is observed by cryo-electron microscopy (8), the detailed structure of this pentameric complex has not been resolved by x-ray crystallography of ribosomes (9 -11). The chemical features of protein-protein and proteinrRNA interactions in the GTPase-associated center remain to be clarified.The animal ribosomal phosphoproteins P0 and P1/P2 (P proteins) are counterparts of prokaryotic L10 and L7/L12, respectively, although P1 and P2 are related but different proteins, unlike prokaryotic L7/L12 (12-14). In yeast cells, there are two P1-type proteins, P1␣ and P1␤, and two P2-type proteins, P2␣ and P2␤ (15). It is believed that P proteins constitute a pentameric complex, designated here as...

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Section: Discussionmentioning

confidence: 99%

A Mode of Assembly of P0, P1, and P2 Proteins at the GTPase-associated Center in Animal Ribosome

Hagiya

Naganuma

Maki

et al. 2005

Journal of Biological Chemistry

108

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“…This is comparable with the fact that rat L12 and anti-28 S recognize a similar tertiary structure of the eukaryotic GTPase domain. The E. coli-L8 complex (a counterpart of the rat P complex) binds to the GTPase domain of 23 S rRNA through L10 moiety (Pettersson et al, 1976;Pettersson, 1979;Beauclerk et al, 1984) and protects mainly the internal loop region comprising residues 1044 -1050 and 1109 -1111 against chemical reagents and nuclease (Egebjerg et al, 1990;Rosendahl and Douthwaite, 1993). This loop region is highly conserved; 7 of 10 bases are preserved between E. coli and rat.…”

Section: Conserved Protein Binding Features Of the Gtpase Domain-mentioning

confidence: 99%

Binding of Mammalian Ribosomal Protein Complex P0·P1·P2 and Protein L12 to the GTPase-associated Domain of 28 S Ribosomal RNA and Effect on the Accessibility to Anti-28 S RNA Autoantibody

Uchiumi

Kominami

1997

Journal of Biological Chemistry

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We have investigated binding of rat ribosomal proteins to the "GTPase domain" of 28 S rRNA and its effect on accessibility to the anti-28 S autoantibody, which recognizes a unique tertiary structure of this RNA domain. Ribosomal protein L12 and P protein complex (P complex) consisting of P0, P1, and P2 both bound to the GTPase domain of rat 28 S rRNA in a buffer containing Mg 2 . Chemical footprinting analysis of their binding sites revealed that the P complex mainly protected a conserved internal loop region comprising residues 1855-1861 and 1920 -1922, whereas L12 protected an adjacent helix region encompassing residues 1867-1878 and 1887-1899. These sites are close to but distinct from the binding site for anti-28 S antibody determined previously. The bindings of P complex and L12 increased the anti-28 S accessibility, as revealed by gel retardation and quantitative immunoprecipitation analyses. In a Mg 2؉ -eliminated condition, the RNA failed to bind to either anti-28 S or L12 but assembled into a complex under their coexistence. However, the RNA retained a property of binding to the P complex even in the absence of Mg 2؉ , and this binding conferred high anti-28 S accessibility. These results indicated that the bindings of the P complex and L12 to their respective sites influenced the GTPase domain to increase the accessibility to anti-28 S. A possible RNA conformation adjusted by the protein bindings is discussed.Despite extensive evidence for functional importance of rRNA molecules within the ribosome, it has been difficult to demonstrate biological activities of protein-free rRNA under physiological salt condition. This difficulty appears to be due to involvement of ribosomal proteins that induce and stabilize the higher order structure of rRNA (reviewed by Noller, 1991). Detailed knowledge of rRNA-protein binding and its effect on the RNA conformation is, therefore, important to elucidate the molecular basis of rRNA function. The "GTPase domain" within domain II of Escherichia coli 23 S rRNA is one of the best characterized portions (reviewed by Cundliffe, 1986;Egebjerg et al., 1990;Ryan et al., 1991;Rosendahl and Douthwaite, 1993). This RNA region has been identified as a site of interaction with elongation factor G (Sköld, 1983;Moazed et al., 1988) and with the antibiotic thiostrepton, an inhibitor of elongation factor-dependent processes in protein synthesis (Thompson et al., 1982;Egebjerg et al., 1989). Ribosomal protein L11 and the acidic protein complex L10(L12) 4 cooperatively bind to this RNA domain (Dijk et al., 1979;Beauclerk et al., 1984) and construct a functional site of the 50 S subunit. L11 may participate in induction of a functionally important RNA conformation (Xing and Draper, 1995). On the other hand, the L10(L12) 4 complex appears to affect the RNA conformation through cooperative binding with L11 (Rosendahl and Douthwaite, 1993).The GTPase domain of eukaryotic 28 S rRNA has been also shown to interact with elongation factor 2 (Uchiumi and Kominami, 1994). However, its interaction w...

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“…This raised the questions of whether stoichiometries other than 4:1 and 6:1 exist in bacteria, whether a given stoichiometry is related to a particular taxonomic group or a specific living environment, or which evolutionary processes resulted in a given stoichiometry. Biochemical and mass spectrometry methods that were used so far to quantify the L12 copy numbers 9,[17][18][19][20]21,23 are not suitable for a large-scale screening of hundreds of species. Here we used computational tools to predict the ribosomal L12 stalk composition for a wide range of bacteria, mitochondria and chloroplasts.…”

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confidence: 99%

Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes

et al. 2013

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The emergence of ribosomes and translation factors is central for understanding the origin of life. Recruitment of translation factors to bacterial ribosomes is mediated by the L12 stalk composed of protein L10 and several copies of protein L12, the only multi-copy protein of the ribosome. Here we predict stoichiometries of L12 stalk for 41,200 bacteria, mitochondria and chloroplasts by a computational analysis, and validate the predictions by quantitative mass spectrometry. The majority of bacteria have L12 stalks allowing for binding of four or six copies of L12, largely independent of the taxonomic group or living conditions of the bacteria, whereas some cyanobacteria have eight copies. Mitochondrial and chloroplast ribosomes can accommodate six copies of L12. The last universal common ancestor probably had six molecules of L12 molecules bound to L10. Changes of the stalk composition provide a unique possibility to trace the evolution of protein components of the ribosome.

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Studies on the RNA and protein binding sites of the E.coli ribosomal protein L10

Cited by 43 publications

References 23 publications

A Mode of Assembly of P0, P1, and P2 Proteins at the GTPase-associated Center in Animal Ribosome

A Mode of Assembly of P0, P1, and P2 Proteins at the GTPase-associated Center in Animal Ribosome

Binding of Mammalian Ribosomal Protein Complex P0·P1·P2 and Protein L12 to the GTPase-associated Domain of 28 S Ribosomal RNA and Effect on the Accessibility to Anti-28 S RNA Autoantibody

Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes

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