Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA‐sequestering peripheral protein domain

Hoang, Charmaine; Hamilton, C.S.; Mueller, Eugene G.; Ferré-D′Amaré, A.R.

doi:10.1110/ps.051493605

Cited by 26 publications

(41 citation statements)

References 32 publications

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“…TruB was overexpressed containing an N-terminal histidine-tag and purified by affinity and size-exclusion chromatography, similarly to previous reports Hoang and Ferré-D'Amaré 2001). For binding measurements, the active site residue aspartate 48 in TruB was mutated to asparagine (Ramamurthy et al 1999a;Hoang et al 2005). This substitution completely abolishes any catalytic activity as confirmed by a tritium release assay detecting pseudouridine formation (data not shown) and thus prevents tRNA modification and product release while retaining the ability to bind tRNA.…”

Section: Resultsmentioning

confidence: 99%

“…1B; Nurse et al 1995). TruB was the first pseudouridine synthase to be crystallized in complex with its substrate RNA (Hoang and Ferré-D'Amaré 2001), and numerous biochemical investigations have been performed to investigate its substrate specificity and catalytic mechanism Hamilton et al 2005;Hoang et al 2005;Phannachet et al 2005). Furthermore, TruB is of general interest as it is a close homolog and structurally very similar to the eukaryotic pseudouridine synthase Cbf5, the catalytic subunit of H/ACA small nucleolar ribonucleoproteins (Koonin 1996).…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

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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“…How is completion of the pseudouridylation reaction detected by Cbf5, and how is this signaled to the other components of the RNP to enable enzymatic turnover? Structural analysis of E. coli TruB led to the suggestion that the conserved active site arginine (which forms a salt bridge with the catalytic aspartate and hydrogen bonds to the isomerized nucleotide) detects the conversion of uridine to ⌿ and signals the change in the chemical status of the active site to the thumb loop for substrate release (46). In one of the fully assembled H/ACA RNP structures (21), the equivalent arginine (Arg 184 ), along with neighboring residues Ile 183 , Thr 181 , and Gly 180 , interacts with the isomerized nucleotide through the polypeptide backbone.…”

Section: Active Site and Enzymatic Turnovermentioning

confidence: 99%

The Box H/ACA Ribonucleoprotein Complex: Interplay of RNA and Protein Structures in Post-transcriptional RNA Modification

Hamma

Ferré-D′Amaré

2010

Journal of Biological Chemistry

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The box H/ACA ribonucleoproteins (RNPs) are protein-RNA complexes responsible for pseudouridylation, the most abundant post-transcriptional modification of cellular RNAs. Integrity of its box H/ACA domain is also essential for assembly and stability of the human telomerase RNP. The recent publication of the complete box H/ACA RNP structures combined with the previously reported structures of the protein and RNA components makes it possible to deduce the structural accommodation that accompanies assembly of the full particle. This analysis reveals how the protein components distort the RNA component of the RNP, enabling productive docking of the substrate RNA into the enzymatic active site.Cellular RNAs undergo numerous site-specific post-transcriptional modifications. Over 100 chemically distinct modified nucleotides have been identified (the RNA Modification Database (1) and the Modomics Database (2)). The most abundant is ⌿, 2 the C-5 glycoside isomer of uridine. The isomerization of U residues into ⌿ is performed by universally distributed enzymes called ⌿ synthases, which can be classified into two groups depending on their substrate recognition mechanism. First, a number of ⌿ synthases composed of a single polypeptide recognize their substrate RNAs with high specificity and also catalyze the isomerization reaction. Second, the ⌿ synthase Cbf5/dyskerin associates with three additional polypeptides and a guide RNA (which is responsible for site specificity) to form an RNP. These guide RNAs are characterized by a conserved secondary structure and two closely related sequence elements called "box H" and "box ACA," hence the name of the RNP (reviewed in Refs. 3-5).

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“…Both TruB and Cbf5 contain a thumb loop, which plays a crucial role in substrate turnover (Hoang et al 2005;Hamma and Ferré-D'Amaré 2010), but their interactions are somewhat different. The thumb loop of Cbf5, also called the β7_10 loop, is located between its β7 and β10 strands (Li and Ye 2006;Liang et al 2008Liang et al , 2009Duan et al 2009;Hamma and Ferré-D'Amaré 2010;Li et al 2011).…”

Section: Introductionmentioning

confidence: 99%

Structure–function relationships of archaeal Cbf5 during in vivo RNA-guided pseudouridylation

Majumder

Bosmeny

Gupta

2016

RNA

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In Eukarya and Archaea, in addition to protein-only pseudouridine (Ψ) synthases, complexes containing one guide RNA and four proteins can also produce Ψ. Cbf5 protein is the Ψ synthase in the complex. Previously, we showed that Ψ's at positions 1940, 1942, and 2605 of Haloferax volcanii 23S rRNA are absent in a cbf5-deleted strain, and a plasmid-borne copy of cbf5 can rescue the synthesis of these Ψ's. Based on published reports of the structure of archaeal Cbf5 complexed with other proteins and RNAs, we identified several potential residues and structures in H. volcanii Cbf5, which were expected to play important roles in pseudouridylation. We mutated these structures and determined their effects on Ψ production at the three rRNA positions under in vivo conditions. Mutations of several residues in the catalytic domain and certain residues in the thumb loop either abolished Ψ's or produced partial modification; the latter indicates a slower rate of Ψ formation. The universal catalytic aspartate of Ψ synthases could be replaced by glutamate in Cbf5. A conserved histidine, which is common to Cbf5 and TruB is not needed, but another conserved histidine of Cbf5 is required for the in vivo RNA-guided Ψ formation. We also identified a previously unreported novelty in the pseudouridylation activity of Cbf5 where a single stem-loop of a guide H/ACA RNA is used to produce two closely placed Ψ's and mutations of certain residues of Cbf5 abolished one of these two Ψ's. In summary, this first in vivo study identifies several structures of an archaeal Cbf5 protein that are important for its RNAguided pseudouridylation activity.

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Precursor complex structure of pseudouridine synthase TruB suggests coupling of active site perturbations to an RNA‐sequestering peripheral protein domain

Cited by 26 publications

References 32 publications

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

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

The Box H/ACA Ribonucleoprotein Complex: Interplay of RNA and Protein Structures in Post-transcriptional RNA Modification

Structure–function relationships of archaeal Cbf5 during in vivo RNA-guided pseudouridylation

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