[45] Parameters for the preparation of Escherichia coli ribosomes and ribosomal subunits active in tRNA binding

The translational apparatus is a highly complex structure containing three to four RNA molecules and more than 50 different proteins. In recent years considerable evidence has accumulated to indicate that the RNA participates intensively in the catalysis of peptide-bond formation, whereas a direct involvement of the ribosomal proteins has yet to be demonstrated. Here we report the functional and structural conservation of a peptidyltransferase centre protein in all three phylogenetic domains. In i o replacement studies show that the Escherichia coli L2 protein can be replaced by its homologous proteins from human and

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“…Ribosomes were purified as described in [22]. Polyphenylalanine synthesis was performed according to [23] with slight modifications.…”

Section: In Vitro Translationmentioning

confidence: 99%

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

Ühlein

Weglöhner

Urlaub

et al. 1998

Biochemical Journal

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“…Yeast ribosomes were prepared from Saccharomyces cerevisiae strain ABYS 1 by the method of Rothblatt and Meyer [21]. Escherichia coli ribosomes were prepared from strain JM101 as described [22]. Purified ribosomes were stored at Ϫ70°C in 1ϫEndo buffer (25 mM Tris/ HCl, pH 7.6, 25 mM KCl, 5 mM MgCl 2 ).…”

Section: Methodsmentioning

confidence: 99%

The deoxyribonuclease activity attributed to ribosome‐inactivating proteins is due to contamination

Day

Lord

Roberts

1998

European Journal of Biochemistry

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The mode of action of ribosome-inactivating proteins (RIPs) has, for many years, been considered to be depurination of a specific adenyl residue of ribosomal RNA, resulting in inhibition of protein synthesis. Recently, this view has been challenged by the observation that many RIP preparations have significant DNase activity in addition to their N-glycosidase activity. In this study, we have investigated the putative DNase activity of two RIPs, ricin and pokeweed antiviral protein (PAP), and show that, in both cases, the DNase activity is due to the presence of contaminating nucleases. The N-glycosidase and DNase activities of PAP were separately and specifically inactivated by chemical modification and heat. Gel filtration of ricin allowed physical separation of the two activities. Furthermore, neither recombinant PAP nor recombinant ricin A-chain purified from Escherichia coli displayed DNase activity.Keywords : deoxyribonuclease; ribosome-inactivating protein; RNA N-glycosidase.Ribosome-inactivating proteins (RIPs) are a group of structurally related proteins which depurinate a specific adenyl residue of 28-S ribosomal RNA (A4324 in rat, [1]). Depurination of this residue prevents binding of elongation factors, thereby inhibiting protein synthesis and resulting in cell death. RIPs can be divided into two classes: type I, which consist of a single subunit possessing RNA N-glycosidase activity, and type II, which have a type I-like catalytic subunit (A chain) linked to a cell-binding subunit (B chain). The B chains of plant toxins, e.g. ricin, are typically galactose/N-acetylgalactosamine-specific lectins and are linked to the A chain via a disulphide bond. Bacterial toxins that catalyse the same depurination reaction as RIPs, e.g. Shiga toxin, have a non-covalently bound glycolipidbinding B-chain pentamer.Most type-II RIPs are potent cytotoxins due to their ability to bind to and enter mammalian cells. Once inside the cell, the toxins exploit the cellular retrograde transport system to reach the endoplasmic reticulum from where they translocate into the cytosol by an as yet undetermined mechanism [2]. As holotoxins show no catalytic activity against ribosomes, separation of the A chain and B chain following intoxication is essential in order to exhibit a toxic effect.Type-I RIPs are, by comparison, only weakly cytotoxic when added exogenously to cells. A number of roles have been proposed for type-I RIPs, including defence mechanisms and promotion of senescence. Those which are active on con-specific ribosomes are typically stored in non-cytosolic compartments or as inactive precursors. Such RIPs are believed to be released into the cytosol following cellular damage, such as that caused by viral infection, resulting in local cell suicide around infected areas. An antifungal role has been proposed for RIPs that are inactive against con-specific ribosomes. Barley RIP is co-expressed with a chitinase, and [1, 3] β glucanase, which, while having intrinsic antifungal activity themselves, also facilitate RIP entry i...

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“…Ribosomes and ribosomal subunits were isolated from E. coli strains MRE600 and D10-can20-12E by using standard procedures (18). In all cases of tRNA binding to ribosomes, the tRNAs and the anticodon arms were activated in binding buffer (see below) for 1 min at 65°C and then for 5 min at 37°C before addition to the ribosome-mRNA complex.…”

Section: Generation Of Phosphorothioate-containing Trnas Andmentioning

confidence: 99%

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

Schnitzer

Ahsen

1997

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

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ABSTRACTtRNA binding to the ribosomal P site is dependent not only on correct codon-anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitutioninterference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNA Phe from Escherichia coli which are required for P-site binding. Stereospecific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp-or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNA Phe and E. coli tRNA f Met indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs. N-ethyl-N-nitrosourea modification-interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNA Phe , we propose that specific Rp-phosphate oxygens are required for anticodon loop (''U-turn'') stabilization or are involved in interactions with the ribosome on correct tRNAmRNA complex formation.

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[45] Parameters for the preparation of Escherichia coli ribosomes and ribosomal subunits active in tRNA binding

Cited by 132 publications

References 14 publications

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

The deoxyribonuclease activity attributed to ribosome‐inactivating proteins is due to contamination

Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding

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