Crystal Structure of an RluF–RNA Complex: A Base-Pair Rearrangement Is the Key to Selectivity of RluF for U2604 of the Ribosome

Alian, Akram; DeGiovanni, Andy; Griner, Sarah L.; Finer-Moore, J.; Stroud, Robert M.

doi:10.1016/j.jmb.2009.03.029

Cited by 18 publications

(37 citation statements)

References 68 publications

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“…In contrast, about 48% of 1500 pmol of RNA (incubated with 0.2 pmol of protein) was cyanoethylated upon treatment with TruA and acrylonitrile. The observed levels of in vitro modification match with the previously reported tritium release assays by other groups (44,45). When TruA or RluF was omitted but the transcript was treated with acrylonitrile, less than 5% of the RNA was cyanoethylated at random positions (data not shown).…”

Section: Lc-ms/ms Analysis Of Rnase T1 Digest Of E Coli Trna Tyr -supporting

confidence: 88%

“…This protein binds to an RNA stem-loop to rearrange the base pair in such a way that it moves the A2602 bulge into the stem, thereby translating the target U2604 by flipping it into the active site. Thus, if a mutation facilitates A2602 refolding into the stem, enzymatic activity increases substantially (44).…”

Section: Discussionmentioning

confidence: 99%

“…The pseudouridine synthase activity assay was performed on the in vitro transcript with recombinant pseudouridine synthase as described (44,45). Briefly, the pseudouridylation assay mixture consisting of purified protein (50 nM for RluF and 2 nM for TruA) and in vitro transcript (0.5 M for RluF and 1.5 M for TruA) in the assay buffer (20 mM Tris-HCl, pH 8.0, 0.1 M NH 4 Cl, 2 mM DTT) was incubated at room temperature for 2 h. Following digestion with RNase T1 (150 units) and BAP (0.02 unit) at 37°C for 2 h, the resulting oligonucleotides were extracted with phenol-chloroform, dried in a SpeedVac, and dissolved in sterile water for derivatization with acrylonitrile.…”

Section: Methodsmentioning

confidence: 99%

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Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

Addepalli

Limbach

2016

Journal of Biological Chemistry

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Pseudouridine is found in almost all cellular ribonucleic acids (RNAs). Of the multiple characteristics attributed to pseudouridine, making messenger RNAs (mRNAs) highly translatable and non-immunogenic is one such feature that directly implicates this modification in protein synthesis. We report the existence of pseudouridine in the anticodon of Escherichia coli tyrosine transfer RNAs (tRNAs) at position 35. Pseudouridine was verified by multiple detection methods, which include pseudouridine-specific chemical derivatization and gas phase dissociation of RNA during liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of total tRNA isolated from E. coli pseudouridine synthase knock-out mutants identified RluF as the enzyme responsible for this modification. Furthermore, the absence of this modification compromises the translational ability of a luciferase reporter gene coding sequence when it is preceded by multiple tyrosine codons. This effect has implications for the translation of mRNAs that are rich in tyrosine codons in bacterial expression systems.Of 150-plus chemical modifications (1, 2) found in cellular RNA, 5-ribosyluridine or pseudouridine (⌿) 3 (3) is the most abundant but still poorly understood (4) modification. Pseudouridine, which has been found in the functionally important regions of RNAs (5), is known to modulate codon-anticodon interactions between mRNA and tRNA (6) and assist assembly of the ribosome (7) and spliceosome (8). The presence of pseudouridine in mRNA makes the message non-immunogenic and highly translatable, thus offering therapeutic opportunities in medical applications (9).Although the exact mechanism of isomerization is still not clear (4), the incorporation of C5 of the uracil base into the glycosidic bond is catalyzed by either standalone proteins (referred to as ⌿ synthases) (10, 11) or RNA-protein complexes (referred to as H/ACA box ribonucleoproteins) (12). Following site-specific recognition, the nitrogen-carbon glycosidic bond is cleaved, and uracil is rotated and then reattached to the sugar, forming a carbon-carbon glycosidic bond on the polyribonucleotide (Fig. 1) by Michael addition-like or glycal mechanisms (13).The ⌿ synthases have been initially classified based on the Escherichia coli enzymes RluA, RsuA, TruA, TruB, and TruD (10, 11). A sixth family with members from archaea and eukaryotes but not from bacteria was added after discovery of Pus10 (14). The enzyme active site carries a catalytically essential aspartate, which is the only absolutely conserved residue in all ⌿ synthases (15, 16). Substrate recognition by the ⌿ synthase is usually in the context of the sequence or structure of the target site in RNA. Site specificity also varies depending on the ⌿ synthase. For example, RsuA isomerizes U516 of 16S rRNA (17) exclusively, whereas the universally conserved ⌿55 of the T⌿C loop in all elongator tRNAs is catalyzed by TruB (18). RluA catalyzes modification of two distinct RNAs, 23S rRNA (position 746) and some tRNAs (position 32) (19). Member...

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Section: Lc-ms/ms Analysis Of Rnase T1 Digest Of E Coli Trna Tyr -supporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

Addepalli

Limbach

2016

Journal of Biological Chemistry

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“…In addition, eukaryotes and archaea use H/ACA small (nucleolar) ribonucleoproteins which catalyze pseudouridylation at many different sites within cellular RNA with the help of many different box H/ACA guide RNAs (Ye 2007). Within the last decade, crystal structures of pseudouridine synthases from all six families have been determined Ferré-D'Amaré 2001, 2004;Ericsson et al 2004;Kaya et al 2004;Hoang et al 2006;Hur and Stroud 2007;McCleverty et al 2007;Alian et al 2009). Despite substantial differences in the primary sequences, all pseudouridine synthases share the same fold in the catalytic domain and very similar active sites containing an essential aspartate residue (Hamma and Ferré-D'Amaré 2006).…”

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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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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“…76 In contrast the specificity of RIuF and RIuB for adjacent sites in the ribosome, is achieved by substrate binding in different conformations. 77,78 This specificity is compromised by a weak activity of RIuF for the substrate position of RIuB. 79 TruB recognizes the shape of the T-stem loop and therewith its substrate position in its single substrate tRNA.…”

Section: Enzymatic Formation Of C Residuesmentioning

confidence: 99%

Pseudouridine: Still mysterious, but never a fake (uridine)!

Spenkuch

Motorin

Helm³

2014

RNA Biology

180

186

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Pseudouridine (C) is the most abundant of >150 nucleoside modifications in RNA. Although C was discovered as the first modified nucleoside more than half a century ago, neither the enzymatic mechanism of its formation, nor the function of this modification are fully elucidated. We present the consistent picture of C synthases, their substrates and their substrate positions in model organisms of all domains of life as it has emerged to date and point out the challenges that remain concerning higher eukaryotes and the elucidation of the enzymatic mechanism.

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Crystal Structure of an RluF–RNA Complex: A Base-Pair Rearrangement Is the Key to Selectivity of RluF for U2604 of the Ribosome

Cited by 18 publications

References 68 publications

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

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

Pseudouridine: Still mysterious, but never a fake (uridine)!

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