Automated parallel isolation of multiple species of non-coding RNAs by the reciprocal circulating chromatography method

Miyauchi, Katsumi; Ohara, Tomoya; Suzuki, Tsutomu

doi:10.1093/nar/gkl1129

Cited by 74 publications

(75 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Transcribed tRNAs were prepared as described (16) and stored in TE buffer (10 mM Tris-HCl [pH 7.6], 0.1 mM EDTA) at concentrations greater than 100 μM. Native E. coli tRNA Val was purified by hybridization using biotinylated DNA oligonucleotides designed in (47). A 1 mL reaction of 4 mg∕mL unfractionated E. coli tRNA (Roche) was heated to 65°C in 10 mM EDTA for 10 min and 20X SSC buffer was added to a final concentration of 1X SSC (150 mM NaCl, 15 mM NaCitrate pH 7.0).…”

Section: Methodsmentioning

confidence: 99%

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

Schrader

Chapman

Uhlenbeck

2011

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

123

View full text Add to dashboard Cite

To better understand why aminoacyl-tRNAs (aa-tRNAs) have evolved to bind bacterial elongation factor Tu (EF-Tu) with uniform affinities, mutant tRNAs with differing affinities for EF-Tu were assayed for decoding on Escherichia coli ribosomes. At saturating EF-Tu concentrations, weaker-binding aa-tRNAs decode their cognate codons similarly to wild-type tRNAs. However, tighter-binding aa-tRNAs show reduced rates of peptide bond formation due to slow release from EF-Tu•GDP. Thus, the affinities of aa-tRNAs for EF-Tu are constrained to be uniform by their need to bind tightly enough to form the ternary complex but weakly enough to release from EF-Tu during decoding. Consistent with available crystal structures, the identity of the esterified amino acid and three base pairs in the T stem of tRNA combine to define the affinity of each aa-tRNA for EF-Tu, both off and on the ribosome.T he ternary complex of bacterial elongation factor Tu (EF-Tu), GTP and aminoacyl-tRNA (aa-tRNA) binds to the ribosome and participates in a multistep decoding pathway in which GTP is hydrolyzed, EF-Tu•GDP is released, and the aa-tRNA enters the ribosomal A site (1-6). Although all elongator aa-tRNAs bind EF-Tu•GTP with similar affinities (7-9), studies with misacylated tRNAs reveal that the protein shows substantial specificity for both the esterified amino acid and the tRNA body (10-12). The nearly uniform EF-Tu binding affinity observed for tRNAs acylated with their correct (cognate) amino acid occurs because the sequence of each tRNA has evolved to compensate for the variable thermodynamic contribution of the esterified amino acid. Thus, weak-binding esterified amino acids such as glycine and alanine have corresponding tRNAs that bind the protein tightly, while tight-binding amino acids such as tyrosine or glutamine have corresponding tRNAs that bind poorly. The crystal structure of Thermus aquaticus EF-Tu•GTP bound to Saccharomyces cerevisiae Phe-tRNA Phe (13) reveals that the protein primarily forms extensive interactions with the helical phosphodiester backbone of the acceptor and T stems of tRNA Phe . Recent protein (14) and tRNA (15, 16) mutagenesis experiments indicate that much of the specificity is the result of interactions made between three amino acids of EF-Tu and three adjacent base pairs in the T stem (16). Additional mutagenesis experiments indicate that the thermodynamic contribution of each of the three base pairs is independent of the others, making it possible to adjust the affinity of aa-tRNAs to EF-Tu in a predictable manner.Although a detailed structural and thermodynamic understanding of how EF-Tu achieves uniform binding with different aa-tRNAs is beginning to emerge, the underlying selective pressures that lead to uniform binding are less clear. While aa-tRNAs must bind EF-Tu tightly enough to participate in translation, the high intracellular concentration of EF-Tu (17) ensures that they do not significantly compete with one another for the protein.It therefore seems unlikely that a minimum threshold binding af...

show abstract

Section: Methodsmentioning

confidence: 99%

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

Schrader

Chapman

Uhlenbeck

2011

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

123

View full text Add to dashboard Cite

show abstract

“…The basic approach for preparing tRNA from overexpressing strains involves insertion of the appropriate tRNA gene into a plasmid with a highly transcribed promoter, purifying the tRNA from intact cells by phenol extraction, fractionation by native polyacrylamide gel electrophoresis (PAGE) and, when necessary, further purification by additional chromatographic steps [12][13][14][15]. Recently, such methods have been refined to allow the separation of tRNAs based on their ability to hybridize to complementary DNA sequences [16].…”

Section: Preparation Of Trna For Kinetic Studiesmentioning

confidence: 99%

“…For example, under saturating amino acid concentrations, the forward rate constant for the amino acid activation reaction, k 3 , and the equilibrium constant for the dissociation of ATP from the E•AA•ATP complex can be determined from the following hyperbolic equation: (14) Similarly, the values of k −3 , K d PPi , k 4 , and K d tRNA can be determined from equations (15) and (16). It should be noted that when the nonvaried substrate is at subsaturating concentrations (i.e.…”

Section: Hyperbolic Kineticsmentioning

confidence: 99%

See 1 more Smart Citation

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Francklyn

First

Perona

et al. 2008

Methods

105

120

View full text Add to dashboard Cite

The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion.

show abstract

“…One particular tRNA, tRNA Lys (UUU), was further purified with reciprocal circulating chromatography, as described previously (4,11 ). The tRNA Lys (UUU) isolated from wild-type (WT) and Cdkal1 KO mice were subjected to qPCRMtR (Fig.…”

Section: Rna Isolationmentioning

confidence: 99%

Quantitative PCR Measurement of tRNA 2-Methylthio Modification for Assessing Type 2 Diabetes Risk

et al. 2013

Self Cite

View full text Add to dashboard Cite

BACKGROUND Genetic variants in the human CDKAL1 (CDK5 regulatory subunit associated protein 1–like 1) gene have been associated with reduced insulin secretion and type 2 diabetes (T2D). CDKAL1 is a methylthiotransferase that catalyzes 2-methylthio (ms2) modification of the adenine at position 37 (A37) of cytoplasmic tRNALys(UUU). We investigated the ms2-modification level of tRNALys(UUU) as a direct readout of CDKAL1 enzyme activity in human samples. METHOD We developed a quantitative PCR (qPCR)-based method to measure ms2 modification. tRNALys(UUU) was reverse-transcribed with 2 unique primers: Reverse primer r1 was designed to anneal to the middle of this tRNA, including the nucleotide at A37, and reverse primer r2 was designed to anneal to the region downstream (3′) of A37. Subsequent qPCR was performed to detect the corresponding transcribed cDNAs. RESULTS The efficiency of reverse transcription of tRNALys(UUU) was ms2-modification dependent. The relative difference in threshold cycle number obtained with the r1 or r2 primer yielded the ms2-modification level in tRNALys(UUU) precisely as predicted by an original mathematical model. The method was capable of measuring ms2-modification levels in tRNALys(UUU) in total RNA isolated from human peripheral blood samples, revealing that the ms2-modification rate in tRNALys(UUU) was decreased in individuals carrying the CDKAL1 genotype associated with T2D. In addition, the ms2-modification level was correlated with insulin secretion. CONCLUSIONS The results point to the critical role of ms2 modification in T2D and to a potential clinical use of a simple and high-throughput method for assessing T2D risk.

show abstract

Automated parallel isolation of multiple species of non-coding RNAs by the reciprocal circulating chromatography method

Cited by 74 publications

References 32 publications

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases

Quantitative PCR Measurement of tRNA 2-Methylthio Modification for Assessing Type 2 Diabetes Risk

Contact Info

Product

Resources

About