Introduction

Bielka, H

doi:10.1007/978-3-642-68272-8_1

Cited by 3 publications

(3 citation statements)

References 26 publications

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“…To examine the binding of RTA to the yeast ribosomes by SPR, the His-tagged RTA was immobilized on the NTA chip as the ligand and ribosomes were used in a mobile solution as the analyte. Since the molecular mass of the ribosome (3600 kDa) is ∼120 times larger than the molecular mass of the N-terminally (31.6 kDa) or C-terminally His-tagged RTA (31.2 kDa), if every immobilized RTA molecule interacted with one ribosome when 1 RU of RTA was immobilized on the chip, 120 RU should be obtained as the ribosome binding signal. However, at ligand concentrations below 500 RU, we could not detect any interaction, suggesting that not every RTA molecule immobilized on the chip may be available to interact with the ribosomes.…”

Section: Resultsmentioning

confidence: 99%

A Two-Step Binding Model Proposed for the Electrostatic Interactions of Ricin A Chain with Ribosomes

et al. 2009

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Ricin is a ribosome inactivating protein that catalytically removes a universally conserved adenine from the α-sarcin/ricin loop (SRL) of the 28S rRNA. We recently showed that ricin A chain (RTA) interacts with the P1 and P2 proteins of the ribosomal stalk to depurinate the SRL in yeast. Here we examined the interaction of RTA with wild type and mutant yeast ribosomes deleted in the stalk proteins by surface plasmon resonance. The interaction between RTA and wild type ribosomes did not follow a single step binding model, but was best characterized by two distinct types of interactions. The AB1 interaction had very fast association and dissociation rates, was saturable and required an intact stalk, while the AB2 interaction had slower association and dissociation rates, was not saturable and did not require the stalk. RTA interacted with the mutant ribosomes by a single type of interaction, which was similar to the AB2 interaction with the wild type ribosomes. Both interactions were dominated by electrostatic interactions and the AB1 interaction was stronger than the AB2 interaction. Based on these results we propose a two-step interaction model. The slow and ribosomal stalk nonspecific AB2 interactions concentrate the RTA molecules on the surface of the ribosome. The AB2 interactions facilitate the diffusion of RTA towards the stalk and promote the faster, more specific AB1 interactions with the ribosomal stalk. The electrostatic AB1 and AB2 interactions work together allowing RTA to depurinate the SRL at a much higher rate on the intact ribosomes than on the naked 28S rRNA.Ricin is a ribosome inactivating protein (RIP) isolated from the castor bean plant, Ricinus communis that consists of a Ricin Toxin A chain (RTA) and a galactose-binding B chain (RTB). RTA and RTB are linked by a disulfide bond between the Cys 259 near the C-terminus of RTA and the Cys 4 of RTB (1). The holotoxin is not active on ribosomes (2-6). The enzymatic activity of ricin is due to RTA, which is an N-glycosidase that specifically cleaves the Nglycosidic bond at A4324 of the 28S rRNA of rat ribosomes (7) and inhibits the elongation factor dependent ribosomal functions (8-10). RTA can also cleave the A2660 in naked 23S rRNA from E. coli but not the ribosomes from E. coli (11)(12)(13)(14). Both A4324 of rat 28S rRNA and A2660 of E. coli 23S rRNA are located at a universally conserved rRNA stem loop called the α-sarcin/ricin loop (SRL) named after the toxins (9). The SRL is the site where elongation factors interact with the ribosome and is involved in peptide translocation during protein synthesis (15). RTA depurinates the 28S rRNA on rat ribosomes about 5000 times faster than the naked rat 28S rRNA (7), indicating that the conformation of the rRNA on intact ribosomes and the ribosomal proteins play an important role in ribosome depurination by ricin.The α-sarcin/ricin loop (SRL) is located on the 60S subunit in close proximity to the ribosomal stalk, which is a lateral flexible structure on the large subunit. The stalk region was ...

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Section: Resultsmentioning

confidence: 99%

A Two-Step Binding Model Proposed for the Electrostatic Interactions of Ricin A Chain with Ribosomes

et al. 2009

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“…The same phosphoprotein forms were identified in the in vivo 3zP-labelling experiments.The large ribosomal subunits of pro-and eukaryotic organisms studied so far, contain a set of unusually acidic proteins of Mr about 13 kDa. These proteins are involved in the interaction of translation factors with the ribosomes during protein synthesis (for the review see BIELKA 1982). In contrast to most of the ribosomal proteins they are present in multiple copies in the ribosomal particles, however, their amount in eukaryotic ribosomes is variable and seems to be related to the metabolic activity of the cells.…”

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confidence: 99%

The acidic ribosomal proteins of different yeast species. Phosphorylation by ribosome‐associated protein kinases

Cytryńska

Wojda

Jakubowicz

1995

J. Basic Microbiol.

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Two major ribosomal proteins of Mr 13 kDa and 38 kDa were identified as phosphorylation substrates for ribosome-bound protein kinases from different yeast species. The phosphorylation level was much higher in ribosomes from the cells collected at the exponential growth phase, than in those from the stationary phase. Isoelectrofocusing of the protein band of 13 kDa and subsequent silver staining allowed to identify from four in the case of Pichia stipitis up to ten in Saccharomyces cerevisiae. Saccharomycodes Ludwigii, Torulopsis utilis and Kloeckera apiculata individual peptides. In the most of the yeast species studies five phosphorylated peptides were observed. However, only one or two such phosphopeptides were detected in Pichia stipitis and Trichosporon cutaneum ribosomes, respectively. The same phosphoprotein forms were identified in the in vivo 32P-labelling experiments.

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“…Particularly relevant in this regard is phosphorylation of acidic proteins (P-proteins) of Mr 13 kDa, the components of the large ribosomal subunit. These proteins are involved in the interaction of translation factors with ribosomes during protein synthesis (for the review see BIELKA 1982). It was reported that P-proteins are the only multicopy components of the ribosomal particles.…”

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Phosphorylation of ribosomal proteins by ribosome‐associated protein kinases of Trichosporon cutaneum

Wojda

Cytryńska

Jakubowicz

1996

J. Basic Microbiol.

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Four ribosomal proteins of Mr 13 kDa, 15 kDa, 19 kDa and 38 kDa were identified as phosphorylation substrates for protein kinases tightly associated with Trichosporon cutaneum ribosomes. It was found that proteins of 13 kDa, 19 kDa and 38 kDa were phosphorylated by multifunctional casein kinase II while the protein of 15 kDa by casein kinase I. Proteins of 13 kDa and 38 kDa were detected in the large subunits while 15 kDa and 19 kDa in the small ribosomal subunits. By using isoelectrofocusing the protein of 13 kDa was resolved into two individual phosphorylated forms. The phosphorylation level of both forms was much higher in ribosomes from the cells collected at the exponential growth phase than in those from the stationary phase. The same phosphoprotein forms were identified in ribosomes from in vitro and in vivo [32P]-labelling experiments.

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Introduction

Cited by 3 publications

References 26 publications

A Two-Step Binding Model Proposed for the Electrostatic Interactions of Ricin A Chain with Ribosomes

A Two-Step Binding Model Proposed for the Electrostatic Interactions of Ricin A Chain with Ribosomes

The acidic ribosomal proteins of different yeast species. Phosphorylation by ribosome‐associated protein kinases

Phosphorylation of ribosomal proteins by ribosome‐associated protein kinases of Trichosporon cutaneum

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