The reaction of Escherichia coli ribosomes with kethoxal

Delihas, Nicholas; Zorn, Glenn A.; Strobel, Eric J.

doi:10.1016/s0300-9084(74)80327-5

Cited by 9 publications

(2 citation statements)

References 30 publications

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“…Reconstitution experiments with the rRNA and proteins of normal and kethoxalinactivated ribosomes suggested that the inactivation was primarily the result of a modified rRNA molecule. However, this evidence needs further investigation since more recent findings (Delihas et al, 1973) have indicated that kethoxal also forms stable complexes with several 30S and 50S proteins. Avadhani & Buetow (1973) have shown that although RNAase hydrolyses all species of rRNA no loss in E. coli ribosomal activity was observed until the 5S RNA was cleaved.…”

Section: Discussionmentioning

confidence: 97%

The role of ribosomal ribonucleic acid in the structure and function of mammalian brain ribosomes

Grove

Johnson

1974

Biochemical Journal

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In order to resolve the functional role of intact rRNA in polypeptide chain elongation mouse brain ribosomes were treated with dilute pancreatic or T(1) RNAase (ribonuclease). After RNAase treatment, several physical-chemical properties as well as the functional activity of the ribosomes were measured. RNAase treatment resulted in the extensive hydrolysis of both 18S and 28S rRNA; however, the sedimentation properties of mono-ribosomes were unaltered and more than 90% of the relatively low-molecular-weight RNA fragments remained associated with ribosome particles. Analysis of the ability of RNAase-treated ribosomes to participate in cell-free protein synthesis showed that ribosomes with less than 2% intact rRNA retained more than 85% of their activity in polyphenylalanine incorporation. Proof that the incorporation of phenylalanine by ribosomes with hydrolysed rRNA actually represented active translocation was obtained by the effective inhibition of incorporation by diphtheria toxin. In addition, the oligopeptide products of protein synthesis could be identified by BD (benzoylated diethylaminoethyl)-cellulose column chromatography. Analysis of the size distribution of oligopeptides synthesized by normal and RNAase-treated ribosomes showed no significant differences which indicated that there was no change in the proportion of ribosomes engaged in protein synthesis. Thus strong RNA-protein and protein-protein interactions must serve to maintain the functional integrity of ribosomes even when the rRNA is extensively degraded. The ability of the enzyme-treated ribosomes to efficiently incorporate amino acids clearly demonstrated that ;intact' rRNA is not required for protein-synthetic activity.

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Section: Discussionmentioning

confidence: 97%

The role of ribosomal ribonucleic acid in the structure and function of mammalian brain ribosomes

Grove

Johnson

1974

Biochemical Journal

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“…An Arg labeling reagent that is not a vicinal dicarbonyl compound is represented by 3-ethoxy-1,1-dihydroxy-2-butanone (kethoxal), which is a popular RNA footprinting reagent that reacts with the guanine group. , Kethoxal was later demonstrated to react with Lys residues in proteins. − MS-based Arg footprinting by kethoxal was first demonstrated by Fabris and co-workers in 2005, where they used Arg-containing derivatized amino acids, di- and tripeptides, and model proteins to investigate the reaction product between kethoxal (structures depicted in Scheme ) and an Arg residue and to demonstrate its capability as a Arg footprinting reagent. As the initial addition of kethoxal forms a diol product, the reaction can also include borate for higher yield.…”

Section: Targeted-labeling Reagentsmentioning

confidence: 99%

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

2020

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Proteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS determination of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS analysis, through which different covalent labeling approaches “mark” the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen–deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resolution, we present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biological questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a reference for investigators seeking a MS-based tool to address structural questions in protein science.

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