Characterization and crystallization of ribosomal particles from <i>Halobacterium marismortui</i>

Shevack, A.; Gewitz, H.S.; Hennemann, B.; Yonath, Ada; Wittmann, H. G.

doi:10.1016/0014-5793(85)80655-4

Cited by 85 publications

(39 citation statements)

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“…70 Purification of H. volcanii ribosomes H. volcanii ribosomes were prepared essentially as described. 35 H. volcanii cells were grown at 40 8C to midexponential phase, to a concentration of 3 g of cells per litre. The cells were harvested, frozen in liquid nitrogen and stored at 2 80 8C.…”

Section: Methodsmentioning

confidence: 99%

“…As a first step towards describing the putative interaction between ribosomes and the plasma membrane in Archaea, ribosomes were purified from the cytosol of H. volcanii cells relying on protocols previously used for purification of ribosomes from other haloarchaea such as H. salinarum, 34 Haloarcula marismortui, 35 Halobacterium cutiruburm 36 and Haloferax mediterranei. 37 In our hands, 20 A 260 (500 pmol) units of 70 S ribosomes were isolated from 1 g of wet H. volcanii cells.…”

Section: Introductionmentioning

confidence: 99%

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Membrane Binding of Ribosomes Occurs at SecYE-based Sites in the Archaea Haloferax volcanii

Ring

Eichler

2004

Journal of Molecular Biology

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Membrane Binding of Ribosomes Occurs at SecYE-based Sites in the Archaea Haloferax volcanii

Ring

Eichler

2004

Journal of Molecular Biology

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“…Ribosomes from H. marismortui and H. volcanii were isolated as described (Mandal et al 2010) using the basic procedure of Wittman and coworkers (Shevack et al 1985) with minor modifications. Cells from a 2 L culture grown to midlog phase (OD 600 ∼3.5) were suspended in ice-cold 10 mM Hepes pH 7.5, 100 mM Mg(OAc) 2 , 3.4 M KCl and 6 mM 2-mercaptoethanol ("high-salt ribosome buffer"), and lysed by French Press (Constant Cell Disruption System; 10,000 Psi, 2 passages).…”

Section: Isolation Of H Marismortui and H Volcanii Ribosomesmentioning

confidence: 99%

Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Mandal

Köhrer

et al. 2013

RNA

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Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA 2 Ile ) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA 2 Ile binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.

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“…Microbiol. 2004.58:233-251 (36,61,74), and the small subunit from Thermus thermophilus, T30S (82). However, because H. marismortui is an archaeon sharing properties with prokaryotes as well as eukaryotes, the structural information obtained from its crystals is only partially relevant to the vast amount of functional knowledge accumulated over the years from the Escherichia coli ribosome.…”

mentioning

confidence: 99%

Ribosomal Crystallography: Initiation, Peptide Bond Formation, and Amino Acid Polymerization are Hampered by Antibiotics

Yonath

Bashan

2004

Annu. Rev. Microbiol.

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■ Abstract High-resolution structures of ribosomal complexes revealed that minute amounts of clinically relevant antibiotics hamper protein biosynthesis by limiting ribosomal mobility or perturbing its elaborate architecture, designed for navigating and controlling peptide bond formation and continuous amino acid polymerization. To accomplish this, the ribosome contributes positional rather than chemical catalysis, provides remote interactions governing accurate substrate alignment within the flexible peptidyl-transferase center (PTC) pocket, and ensures nascent-protein chirality through spatial limitations. Peptide bond formation is concurrent with aminoacylatedtRNA 3 end translocation and is performed by a rotatory motion around the axis of a sizable ribosomal symmetry-related region, which is located around the PTC in all known crystal structures. Guided by ribosomal-RNA scaffold along an exact pattern, the rotatory motion results in stereochemistry that is optimal for peptide bond formation and for nascent protein entrance into the exit tunnel, the main target of antibiotics targeting ribosomes. By connecting the PTC, the decoding center, and the tRNA entrance and exit regions, the symmetry-related region can transfer intraribosomal signals, guaranteeing smooth processivity of amino acid polymerization. CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . INTRODUCTIONRibosomes are the universal cellular riboprotein assemblies that catalyze amino acid polymerization into proteins. In this process, called protein biosynthesis, the ribosomes interact with the cellular environment, select the genetic frame to be translated, form the peptide bonds, and act as polymerases to ensure smooth and efficient elongation of the newly born protein chains. The ribosomes are composed of two riboprotein subunits of unequal size that associate upon the initiation of the process and dissociate at its termination. Protein biosynthesis is performed by a cooperative effort of both subunits, and the ribosome possesses features that facilitate transmission between its various functional sites, leading to efficient processivity of protein formation essential for cell vitality. The small ribosomal subunit facilitates the initiation of the translation process and is involved in selecting the frame to be translated, decoding the genetic message, and controlling the fidelity of codon-anticodon interactions. The large ribosomal subunit forms the peptide bond, ensures smooth amino acid polymerization, and channels the nascent proteins through their exit tunnel. Messenger RNA (mRNA) carries the genetic instructions to the ribosome, and aminoacylated transfer RNA (tRNA) molecules deliver the amino acids within a ternary complex containing GTP and the elongation factor Tu. The ribosome possesses three tRNA binding sites. The A-site hosts the aminoacylated-tRNA, the P-site hosts the peptidyl tRNA, and the E-site designates the location of the exiting free tRNA once a p...

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Characterization and crystallization of ribosomal particles from Halobacterium marismortui

Cited by 85 publications

References 13 publications

Membrane Binding of Ribosomes Occurs at SecYE-based Sites in the Archaea Haloferax volcanii

Membrane Binding of Ribosomes Occurs at SecYE-based Sites in the Archaea Haloferax volcanii

Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

Ribosomal Crystallography: Initiation, Peptide Bond Formation, and Amino Acid Polymerization are Hampered by Antibiotics

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