Reconstitution of a GTPase Activity a 50S Ribosomal Protein from E. coli

Kischa, K.; Möller, W.; Stöffler, Georg

doi:10.1038/newbio233062a0

Cited by 100 publications

(68 citation statements)

References 12 publications

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“…In contrast, removal of L7/L12 from the ribosome resulted in a substantial reduction in the GTPase activity of wild type EF-G, in accord with previous studies (7,8,10,12). From these results, we anticipate that residues Glu-224 and Glu-228 in helix A GЈ are involved in ion pairs with positively charged residues of L7/L12, which have been identified previously (10).…”

Section: Discussionsupporting

confidence: 79%

“…Early research identified L7/L12, 4 a protein component of one of the peripheral stalks of the ribosome, as an important contributor to GTPase activation of both factors (7,8). More recent studies identified residues of the C-terminal domain (CTD) of L7/L12, important for GTPase activation and rapid association of both factors with the ribosome (9,10).…”

Section: Together These Results Provide Evidence For Functionally Immentioning

confidence: 99%

“…Studies during the 1970s first demonstrated the requirement of L7/L12 for the functions of a wide variety of essential translation factors, including G and non-G proteins, in protein synthesis in vitro (7,8,27,28). Subsequent studies isolated mutants of L7/L12, which altered the fidelity and speed of protein synthesis in vivo (29).…”

Section: Discussionmentioning

confidence: 99%

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Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor

Murataliev

Wilson

2007

Journal of Biological Chemistry

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Protein L7/L12 of the bacterial ribosome plays an important role in activating the GTP hydrolytic activity of elongation factor G (EF-G), which promotes ribosomal translocation during protein synthesis. Previously, we cross-linked L7/L12 from two residues (209 and 231) flanking ␣-helix A G in the G subdomain of Escherichia coli EF-G. Here we report kinetic studies on the functional effects of mutating three neighboring glutamic acid residues (224, 228, and 231) to lysine, either singly or in combination. Two single mutations (E224K and E228K), both within helix A G , caused large defects in GTP hydrolysis and smaller defects in ribosomal translocation. Removal of L7/L12 from the ribosome strongly reduced the activities of wild type EF-G but had no effect on the activities of the E224K and E228K mutants. Together, these results provide evidence for functionally important interactions between helix A G of EF-G and L7/L12 of the ribosome.

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Section: Discussionsupporting

confidence: 79%

Section: Together These Results Provide Evidence For Functionally Immentioning

confidence: 99%

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Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor

Murataliev

Wilson

2007

Journal of Biological Chemistry

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“…The ribosomal proteins L7 and L12 of Escherichia coli have been shown to be involved in the elongation factor Tu (EF-Tu)-dependent binding of aminoacyl-tRNA to ribosomes (1,2) and in the elongation factor G (EF-G)-dependent hydrolysis of GTP (3)(4)(5). These same reactions are also inhibited by the antibiotic thiostrepton (6)(7)(8)(9)(10)(11)(12).…”

mentioning

confidence: 86%

“…It has been shown that these two proteins are identical except for the presence of an acetyl group on the NH2-terminal serine of L7 (19). The reactions affected by L7 and L12 include: (a) initiation factor-dependent binding of fMet-tRNA to the ribosomet (13); (b) EF-Tu-catalyzed binding of aminoacyl-tRNA to the ribosome (1, 2, 4, 5); and (c) EF-G-mediated reactions, including EF-G-dependent GTP hydrolysis, binding of EF-G to the ribosome, and translocation (1)(2)(3)(4)(5).…”

Section: Discussionmentioning

confidence: 99%

The Requirement for Ribosomal Proteins L7 and L12 in Peptide-Chain Termination

Brot¹,

Tate

Caskey

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

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Proteins L7 and L12 from 50S ribosomal subunits of Escherichia coli are required for peptidechain termination. This termination process is inhibited by thiostrepton. Since both thiostrepton-treated ribosomes and those depleted of L7 and L12 have a markedly reduced ability to form release factor UA[3H]A ribosome complexes, the binding of release factors to the ribosome appears to be the primary site of inhibition.The ribosomal proteins L7 and L12 of Escherichia coli have been shown to be involved in the elongation factor Tu (EF-Tu)-dependent binding of aminoacyl-tRNA to ribosomes (1, 2) and in the elongation factor G (EF-G)-dependent hydrolysis of GTP (3-5). These same reactions are also inhibited by the antibiotic thiostrepton (6)(7)(8)(9)(10)(11)(12). In addition, as initially suggested by Hamel et al. (1), L7 and L12 are also required for the enzymatic binding of fMet-tRNA to ribosomest (13). Since these reactions all involve hydrolysis of the -y-phosphate of GTP and since L7 and L12 do not affect ribosomal peptidyl transferase activity (1), it appears that L7 and L12 might be required for reactions involving GTP hydrolysis dependent on a soluble factor and the 50S ribosomal subunit. The hydrolysis of GTP is required for peptide-chain termination in vitro, as studied with reticulocyte release factor (RF) and ribosomes (14). Although no such requirement has been demonstrated for peptide-chain termination in E. coli in vitro, a protein factor, RF-3, and guanine nucleotides are known to influence the ribosomal binding of E. coli termination factors RF-1 and RF-2 (15). Recently the binding of EF-G to E. coli ribosomes, which requires L7 and L12, was shown to inhibit the interaction of RF-1 and RF-2 with ribosomes (16). For these reasons the effects of the ribosomal proteins L7 and L12 and the antibiotic thiostrepton were studied with respect to their influence on peptide-chain termination. The present report provides evidence that L7 and L12 are required for peptidechain termination in E. coli and that this process is inhibited by thiostrepton. MATERIALS AND METHODSMaterials. Ammonium chloride-washed ribosomes were isolated from E. coli B as described (17), and ribosomal subunits were prepared from these ribosomes by the procedure of Pestka and Nirenberg (18). The ribosomal proteins L7 and L12 were extracted from the 50S ribosomal subunits with an Abbreviations: L7 and L12, 50S ribosomal proteins according to the nomenclature of Kaltschmidt and Wittmann (38); release factors, RF-1, RF-2, and RF-3; elongation factor Tu, EF-Tu; elongation factor G, EF-G.

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The conformational stability of ribosomal proteins L₇ and L₁₂ from E. coli. I. Circular dichroism study of the changes induced by ionic strength and solvents

1973

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synopsisRibosomal proteins L7 and LIZ are the only acidic proteins found on the 505 ribosomal subunit of Escherichia coli. The effect of ionic strength, helix-promoting solvents and denaturating agents on the conformation of these proteins has been studied. It has been established that the helicity of L7 and LIz proteins (approx. 45.50% a helix) can be increased to 60-70% when they are exposed to helix-promoting solvents such as methanol or ethanol in the presence of 0.1M salt. High ionic strength by itself was without any effect on the conformation of the proteins. However, the solvent, 2,2,2-trifluoroethanol increased the content of a helices up to 80% even in the absence of salt. Denaturating agents like urea ( 6 M ) or guanidine.HC1 ( 6 M ) , decreased the content of the ordered structure below 20%. All conformational changes induced by salt or solvents were completely reversible and characterized by a broad transition showing a low degree of cooperativity. This might indicate the presence of discrete segments with variations in amino acid sequences and ordered structures with different stabilities. 2083

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Reconstitution of a GTPase Activity a 50S Ribosomal Protein from E. coli

Cited by 100 publications

References 12 publications

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

The Requirement for Ribosomal Proteins L7 and L12 in Peptide-Chain Termination

The conformational stability of ribosomal proteins L₇ and L₁₂ from E. coli. I. Circular dichroism study of the changes induced by ionic strength and solvents

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Reconstitution of a GTPase Activity a 50S Ribosomal Protein from E. coli

Cited by 100 publications

References 12 publications

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

The Requirement for Ribosomal Proteins L7 and L12 in Peptide-Chain Termination

The conformational stability of ribosomal proteins L7 and L12 from E. coli. I. Circular dichroism study of the changes induced by ionic strength and solvents

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The conformational stability of ribosomal proteins L₇ and L₁₂ from E. coli. I. Circular dichroism study of the changes induced by ionic strength and solvents