Kinetic studies of the rates and mechanism of assembly of the protein synthesis initiation complex

Goss, Dixie J.; Parkhurst, Lawrence J.; Wahba, Amy

doi:10.1016/s0006-3495(80)84957-5

Cited by 14 publications

(10 citation statements)

References 30 publications

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“…Many other proteins identified for spermidine immobilization are involved in translation such as translation elongation faction ef1 (PF1375), LSU ribosomal protein L7AE (PF1367), translation elongation factor eIF-5a (PF1264). Spermidine is a well characterized polyamine and is known to play a critical role for eukaryotic organisms in translation, ribosomal assembly and protein elongation and their interaction may reflect the specific affinity of spermidine for these proteins 32–34. While some of proteins identified may represent secondary interactions with the newly discovered metabolite, the immobilization/proteomics screen presented here can be a powerful tool for getting a glimpse of where a previously unknown metabolite discovered through untargeted metabolomics, fits into the biochemical landscape.…”

Section: Resultsmentioning

confidence: 99%

Identification of a New Endogenous Metabolite and the Characterization of Its Protein Interactions through an Immobilization Approach

et al. 2008

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The emerging field of global mass-based metabolomics provides a platform for discovering unknown metabolites and their specific biochemical pathways. We report the identification of a new endogenous metabolite, N 4 -(N-acetylaminopropyl)spermidine and the use of a novel proteomics based method for the investigation of its protein interaction using metabolite immobilization on agarose beads. The metabolite was isolated from the organism Pyrococcus furiosus, and structurally characterized through an iterative process of synthesizing candidate molecules and comparative analysis using accurate mass LC-MS/MS. An approach developed for the selective preparation of N 1 -acetylthermospermine, one of the possible structures of the unknown metabolite, provides a convenient route to new polyamine derivatives through methylation on the N 8 and N 4 of the thermospermine scaffold. The biochemical role of the novel metabolite as well as that of two other polyamines: spermidine and agmatine is investigated through metabolite immobilization and incubation with native proteins. The identification of eleven proteins that uniquely bind with N 4 -(N-acetylaminopropyl)spermidine, provides information on the role of this novel metabolite in the native organism. Identified proteins included hypothetical ones such as PF0607 and PF1199, and those involved in translation, DNA synthesis and the urea cycle like translation initiation factor IF-2, 50S ribosomal protein L14e, DNA-directed RNA polymerase, and ornithine carbamoyltransferase. The immobilization approach demonstrated here has the potential for application to other newly discovered endogenous metabolites found through untargeted metabolomics, as a preliminary screen for generating a list of proteins that could be further investigated for specific activity.

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Section: Resultsmentioning

confidence: 99%

Identification of a New Endogenous Metabolite and the Characterization of Its Protein Interactions through an Immobilization Approach

et al. 2008

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“…Goss et al (1980) have shown that spermidine alone can associate E. coli ribosomal subunits. Weiss & Morris (1973), however, showed that while either spermidine or putrescine could replace Mg2+ in 30s ribosomal subunits, the subunits lost their structural and functional integrity when incubated in low Mg2+ and high putrescine concentrations.…”

Section: Discussionmentioning

confidence: 99%

Influence of various cations on the equilibriums between wheat germ ribosomes and their subunits

Sperrazza¹,

Moore²,

Spremulli³

1981

Biochemistry

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The influence of alkaline earth metal ions, alkali metal ions, ammonium ions, and polyamines upon the equilibria between high-salt-washed wheat germ ribosomes and their subunits was investigated by light scattering. The Mgz+-dependent, reversible association of ribosomal subunits was studied with buffers containing 100 mM concentrations of the chloride salts of various monovalent cations. These studies indicate that K+ and Na+ are most effective in dissociating the ribosomes into subunits, followed by Rb+ and then NH4+. The dissociation curves determined with buffers containing K+, Na', Rb', and NH4+ ions exhibit [Mg"] 1/2 values of 1.4, 1.4, 1.2, and 0.7 mM, respectively. The divalent cations, in general, strongly promote subunit association but differ in their abilities to do so. Ca2+ associates the ribosomal subunits more effectively than Sr2+, which is more effective than Mgz+. In the presence of 100 mM KCl and 0.15 mM MgClz, Ca2+-, Sr2+-, and Mgz+-dependent association curvesx e subunits of both eucaryotic and procaryotic ribosomes are capable of reversible association and dissociation. The types and concentrations of various cations present play a major role in determining the position of the equilibrium between the intact ribosome and its subunits. Kinetic, thermodynamic, and ion-specificity studies have been performed with Escherichia coli ribosomes in an effort to explain the roles of MgZ+, K+, and polyamines in the association of the subunits. Conflicting mechanisms have been proposed as a result of these studies. One model suggests that Mgz+ binds to specific sites on the subunits and thereby promotes subunit association (Petermann, 1964;Goldberg, 1966). This model is supported by the data of Zitomer & Flab (1972), who found that MgZ+, Ca2+, and polyamines promote ribosomal subunit association while K+ promotes subunit dissociation. Ca2+, however, was less effective than Mg2+ in promoting subunit association, suggesting that the ions were acting at specific sites. In contrast, the electrostatic model suggests that Mg2+ neutralizes the charged groups on the ribosomal subunits. This reduction in the electrostatic repulsion between the subunits would allow association. The studies of Walters & Van Os (1970, 1971) on yeast ribosomes and Wishnia & Boussert (1977) on E . coli ribosomes support the electrostatic model. Calculations by these groups also showed that the reduction of the electrostatic repulsion by Mg2+ theoretically is sufficient to promote subunit association. Nieuwenhuysen et al. (1980), using Artemia salina ribosomes, have shown that the molar volume changes produced by ribosomal subunit association are similar to those observed with E. coli type A ribosomes. Furthermore, they have suggested that this large volume change is consistent with two From the exhibit [M2+lIl2 values of 0.9, 1.1, and 1.4 mM, respectively. The abilities of the polyamines to associate ribosomal subunits in buffers containing 100 mM KCl and 0.20 mM MgClz were investigated. Both spermine ( [~p m ]~/~ = 0.09 mM) and sp...

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“…Bacterial and eukaryotic initiation factors have been shown to maintain the pool of small ribosomal subunits largely by preventing joining of the large ribosomal subunit. These discoveries were generally made by the analysis of ribosome complexes using sucrose density gradients (Subramanian & Davis, 1970; Benne et al 1979; Staehelin et al 1979; Chaudhuri et al 1999; Majumdar et al 2003; Unbehaun et al 2004), gel electrophoresis (Lorsch & Herschlag, 1999; Algire et al 2002) as well as with light scattering to observe thermodynamic and kinetic transitions in subunit joining (Grunberg-Manago et al 1975; Gorisch et al 1976; Goss et al 1980, 1982, 1988; Goss & Rounds, 1988; Antoun et al 2004). The conformation of the small ribosomal subunit changes upon association with initiation factors and tRNA.…”

Section: Initiation-factor Binding Sites On the Small Ribosomal Subunitmentioning

confidence: 99%

Quantitative studies of ribosome conformational dynamics

Fraser

Doudna

2007

Quart. Rev. Biophys.

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The ribosome is a dynamic machine that undergoes many conformational rearrangements during the initiation of protein synthesis. Significant differences exist between the process of protein synthesis initiation in eubacteria and eukaryotes. In particular, the initiation of eukaryotic protein synthesis requires roughly an order of magnitude more initiation factors to promote efficient mRNA recruitment and ribosomal recognition of the start codon than are needed for eubacterial initiation. The mechanisms by which these initiation factors promote ribosome conformational changes during stages of initiation have been studied using cross-linking, footprinting, site-directed probing, cryo-electron microscopy, X-ray crystallography, fluorescence spectroscopy and single-molecule techniques. Here, we review how the results of these different approaches have begun to converge to yield a detailed molecular understanding of the dynamic motions that the eukaryotic ribosome cycles through during the initiation of protein synthesis.

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Kinetic studies of the rates and mechanism of assembly of the protein synthesis initiation complex

Cited by 14 publications

References 30 publications

Identification of a New Endogenous Metabolite and the Characterization of Its Protein Interactions through an Immobilization Approach

Identification of a New Endogenous Metabolite and the Characterization of Its Protein Interactions through an Immobilization Approach

Influence of various cations on the equilibriums between wheat germ ribosomes and their subunits

Quantitative studies of ribosome conformational dynamics

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