Thionucleotides in transfer ribonucleic acid.  Addition of N-ethylmaleimide and formation of mixed disulfides with thiol compounds

Carbon, John; David, H

doi:10.1021/bi00851a010

Cited by 40 publications

(17 citation statements)

References 14 publications

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“…The acp 3 U47 position of tRNA Phe (Sigma) was labeled with Cy5-NHS ester (Amersham Biosciences) following published protocols (27). Labeled tRNA Phe was purified by high-performance liquid chromatography using a TSK-phenyl 5-PW column (Tosohaas) and aminoacylated as described previously (28). Short synthetic DNA oligonucleotides (IDT DNA) were 5′ labeled with 32 P and purified by gel filtration.…”

Section: Methodsmentioning

confidence: 99%

Site-specific labeling of the ribosome for single-molecule spectroscopy

Dorywalska

Blanchard²,

Gonzalez³

et al. 2005

Nucleic Acids Research

110

129

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Single-molecule fluorescence spectroscopy can reveal mechanistic and kinetic details that may not be observed in static structural and bulk biochemical studies of protein synthesis. One approach requires site-specific and stable attachment of fluorophores to the components of translation machinery. Fluorescent tagging of the ribosome is a prerequisite for the observation of dynamic changes in ribosomal conformation during translation using fluorescence methods. Modifications of the ribosomal particle are difficult due to its complexity and high degree of sequence and structural conservation. We have developed a general method to label specifically the prokaryotic ribosome by hybridization of fluorescent oligonucleotides to mutated ribosomal RNA. Functional, modified ribosomes can be purified as a homogenous population, and fluorescence can be monitored from labeled ribosomal complexes immobilized on a derivatized quartz surface.

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Section: Methodsmentioning

confidence: 99%

Site-specific labeling of the ribosome for single-molecule spectroscopy

Dorywalska

Blanchard²,

Gonzalez³

et al. 2005

Nucleic Acids Research

110

129

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“…The formyl donor, 10-formyltetrahydrofolate, was prepared as described (37). Aminoacylation and formylation of tRNA fMet and tRNA fMet (Cy3-s 4 U) was achieved as reported (38), and aminoacylation of tRNA Phe was achieved following standard protocols (39).…”

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confidence: 99%

tRNA dynamics on the ribosome during translation

Blanchard

Kim

Gonzalez

et al. 2004

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

449

575

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Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by Ϸ6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components. P rotein synthesis, catalyzed by the ribosome, is rapid, processive, and highly regulated. In bacteria, two RNA-protein subunits consisting of a small 30S subunit and a large 50S subunit assemble around a mRNA template into a roughly spherical 70S particle that translates the nucleotide sequence into a polypeptide chain through repetitive, codon-dependent binding of aminoacylated tRNA.The landmark structures of the 30S (1, 2), 50S (3, 4), and 70S (5) ribosomal particles provide a molecular basis for understanding ribosome function. tRNA molecules bind to the ribosome in a solvent-accessible channel at the subunit interface. Three binding sites for tRNA, called the aminoacyl site (A site), peptidyl site (P site), and exit site (E site), have been identified on both the large and small subunit (Fig. 1). Each tRNA is separated from its neighbor at the elbow region (the site of Ϸ90°b ending) by 25-45 Å (5, 6). The anticodon stem loops of A-and P-site tRNA form Watson-Crick base pairs with adjacent mRNA codons on the 30S subunit (5, 7), whereas the 3Ј CCA terminal residues of A-and P-site tRNAs base-pair with conserved ribosomal RNA (rRNA) loops (8-10) within the 50S subunit peptidyltransferase center, the site of peptide bond formation (11). Additional interactions with rRNA and ribosomal proteins position tRNA molecules on the ribosome (5).The essential components and principal steps of translation have been delineated through genetic, biochemical, and kinetic methods (12). The process of polypeptide elongation has been most extensively characterized (13). Binding of aminoacyl-tRNA (aa-tRNA) to the A site occurs as a ''ternary complex'' with the GTPase elongation ...

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“…Reduction of enzyme disulfide bonds is mostly seen around 0.02 A4 2-mercaptocthanol and it seems unlikely for concentrations under lo-:$ M . The formation of mixed disulfidcs between tRNA and 2-mercaptoethanol is meaningless since it occurs in the presence of iodine and does not have any inhibitory effect on arninoacylation (9). The greater effect in the presence of a given buffer would indicate certain interactions that would inhibit the enzyme activity as has been proposed for cacodylate since no interaction was found with Tris, which was the sther comparing buffer (4).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Aminoacyl-tRNA Synthetases From Rat Liver by Reducing Agents

Vargas¹,

Castañeda²

1973

Can. J. Biochem.

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Commonly used reducing agents 2-mercaptoethanol and dithiothreitol inhibit in vitro homologous aminoacylation of rat liver systems. The extent of inhibition depends on the buffer and is greater in Tris than in N-2-hydroxyethyl piperazine-N′-2-ethanesulfonic acid (HEPES). Variations in the values of pH and the concentrations of Mg2+, ATP, tRNA, enzyme preparation, and buffers were ineffective to reverse the inhibition produced by these reducing agents. Enzyme activity with no reducing agent is, in general, the same or higher in Tris than in HEPES, although it depends on the pH values and the concentrations of buffer and Mg2+ in the reaction mixture.

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Thionucleotides in transfer ribonucleic acid. Addition of N-ethylmaleimide and formation of mixed disulfides with thiol compounds

Cited by 40 publications

References 14 publications

Site-specific labeling of the ribosome for single-molecule spectroscopy

Site-specific labeling of the ribosome for single-molecule spectroscopy

tRNA dynamics on the ribosome during translation

Inhibition of Aminoacyl-tRNA Synthetases From Rat Liver by Reducing Agents

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