Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET

Hengesbach, Martin; Voigts-Hoffmann, Felix; Hofmann, Benjamin J.; Helm, Mark

doi:10.1261/rna.1832510

Cited by 6 publications

(4 citation statements)

References 47 publications

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“…We propose that 5FU containing RNAs may act as mere competitive inhibitors of Pus enzymes in general, without forming stable covalent intermediates. This is in line with our previous studies of Pus1, which failed to convert 5FU on a biochemically relevant time scale, but did show complex dissociation on urea PAGE that was weaker for 5FU- than for U-tRNA, implying that 5FU-RNA forms a non-covalent complex more resistant to denaturants ( 87 ). For several Pus enzymes of E. coli the Mueller group observed turnover of 5FU-RNA ( 8 , 32 ), although not all of them formed SDS-stable complexes ( 32 ).…”

Section: Discussionsupporting

confidence: 92%

Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor

Spenkuch

Hinze

Kellner

et al. 2014

Nucleic Acids Research

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The interest in RNA modification enzymes surges due to their involvement in epigenetic phenomena. Here we present a particularly informative approach to investigate the interaction of dye-labeled RNA with modification enzymes. We investigated pseudouridine (Ψ) synthase TruB interacting with an alleged suicide substrate RNA containing 5-fluorouridine (5FU). A longstanding dogma, stipulating formation of a stable covalent complex was challenged by discrepancies between the time scale of complex formation and enzymatic turnover. Instead of classic mutagenesis, we used differentially positioned fluorescent labels to modulate substrate properties in a range of enzymatic conversion between 6% and 99%. Despite this variegation, formation of SDS-stable complexes occurred instantaneously for all 5FU-substrates. Protein binding was investigated by advanced fluorescence spectroscopy allowing unprecedented simultaneous detection of change in fluorescence lifetime, anisotropy decay, as well as emission and excitation maxima. Determination of Kd values showed that introduction of 5FU into the RNA substrate increased protein affinity by 14× at most. Finally, competition experiments demonstrated reversibility of complex formation for 5FU-RNA. Our results lead us to conclude that the hitherto postulated long-term covalent interaction of TruB with 5FU tRNA is based on the interpretation of artifacts. This is likely true for the entire class of pseudouridine synthases.

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Section: Discussionsupporting

confidence: 92%

Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor

Spenkuch

Hinze

Kellner

et al. 2014

Nucleic Acids Research

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“…Prior to the experiment, folding of tRNA Phe was allowed to occur by heating 2 mM [ 3 H]tRNA Phe in TAKEM 4 buffer to 60°C for 5 min and subsequent slow cooling to 37°C (Hengesbach et al 2010 Phe were incubated with 0-30 mM TruB D48N in TAKEM 4 buffer for 10 min at room temperature. The complete 50 mL reaction mixture was then filtered through a nitrocellulose membrane followed by washing of the membrane with 1 mL cold TAKEM 4 buffer.…”

Section: Nitrocellulose Filtrationmentioning

confidence: 99%

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Wright

Keffer-Wilkes²,

Dobing³

et al. 2011

RNA

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Pseudouridine synthases catalyze formation of the most abundant modification of functional RNAs by site-specifically isomerizing uridines to pseudouridines. While the structure and substrate specificity of these enzymes have been studied in detail, the kinetic and the catalytic mechanism of pseudouridine synthases remain unknown. Here, the first pre-steady-state kinetic analysis of three Escherichia coli pseudouridine synthases is presented. A novel stopped-flow absorbance assay revealed that substrate tRNA binding by TruB takes place in two steps with an overall rate of 6 sec À1 . In order to observe catalysis of pseudouridine formation directly, the traditional tritium release assay was adapted for the quench-flow technique, allowing, for the first time, observation of a single round of pseudouridine formation. Thereby, the single-round rate constant of pseudouridylation (k C ) by TruB was determined to be 0.5 sec À1 . This rate constant is similar to the k cat obtained under multiple-turnover conditions in steady-state experiments, indicating that catalysis is the rate-limiting step for TruB. In order to investigate if pseudouridine synthases are characterized by slow catalysis in general, the rapid kinetic quench-flow analysis was also performed with two other E. coli enzymes, RluA and TruA, which displayed rate constants of pseudouridine formation of 0.7 and 0.35 sec À1 , respectively. Hence, uniformly slow catalysis might be a general feature of pseudouridine synthases that share a conserved catalytic domain and supposedly use the same catalytic mechanism.

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“…With the approach presented here, we have laid the basis for convenient chemical modification and labeling of the 3′-terminus of natural tRNAs. The use of DNA enzymes as a tool for tRNA manipulation has been demonstrated by Helm and coworkers ( 42 , 43 ) for analytical purposes; however, to the best of our knowledge, DNA enzymes have not been integrated into synthetic concepts for tRNA derivatives before. We also mention that our approach is promising to be applicable to many kinds of tRNA, which are of different amino acid specificity and from various organisms.…”

Section: Resultsmentioning

confidence: 99%

Reliable semi-synthesis of hydrolysis-resistant 3′-peptidyl-tRNA conjugates containing genuine tRNA modifications

Graber

Moroder

Steger

et al. 2010

Nucleic Acids Research

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The 3′-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3′-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3′-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5′-fragments carrying the modifications. After dephosphorylation of the 2′,3′-cyclophosphate moieties from these fragments, they are ligated to the respective 3′-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3′-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner.

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Formation of a stalled early intermediate of pseudouridine synthesis monitored by real-time FRET

Cited by 6 publications

References 47 publications

Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor

Dye label interference with RNA modification reveals 5-fluorouridine as non-covalent inhibitor

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

Reliable semi-synthesis of hydrolysis-resistant 3′-peptidyl-tRNA conjugates containing genuine tRNA modifications

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