Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I.

Kammen, Harold O.; Marvel, Christopher C.; Hardy, Larry W.; Penhoet, Edward E.

doi:10.1016/s0021-9258(18)69199-9

Cited by 124 publications

(37 citation statements)

References 37 publications

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“…Figure 11 shows a comparison of the E. coli 4*516 synthase gene sequence (rsuA) with those of the only other two cloned 4* synthases that are known, the truA (also known as the hisT) gene product, which converts U residues in the anticodon arm of tRNA into 4* (Arps et al" 1985;Kammen et al, 1988), and truB. whose gene product forms 4*55 in tRNA (Nurse et al, 1995).…”

Section: Resultsmentioning

confidence: 99%

Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli

Wrzesiński

Bakin²,

Nurse³

et al. 1995

Biochemistry

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Pseudouridine (psi) is commonly found in both small and large subunit ribosomal RNAs of prokaryotes and eukaryotes. In Escherichia coli small subunit RNA, there is only one psi, at position 516, in a region of the RNA known to be involved in codon recognition [Bakin et al. (1994) Nucleic Acids Res. 22, 3681-3684]. To assess the function of this single psi residue, the enzyme catalyzing its formation was purified and cloned. The enzyme contains 231 amino acids and has a calculated molecular mass of 25,836 Da. It converts U516 in E. coli 16S RNA transcripts into psi but does not modify any other position in this RNA. It does not react with free unmodified 16S RNA at all, and only poorly with 30S particles containing unmodified RNA. The preferred substrate is an RNA fragment from residues 1 to 678 which has been complexed with 30S ribosomal proteins. The yield varied from 0.6 to 1.0 mol of psi/mol of RNA, depending on the preparation. Free RNA(1-678) was inactive, as was RNA(1-526) and the RNP particle made from it. 23S RNA and tRNAVal transcripts were also inactive. These results suggest that psi formation in vivo occurs at an intermediate stage of 30S assembly. The gene is located at 47.1 min immediately 5' to, and oriented in the same direction as, the bicyclomycin resistance gene. The gene was cloned behind a (His)6 leader for affinity purification. Virtually all of the overexpressed protein was found in inclusion bodies but could be purified to homogeneity on a Ni2+(-) containing resin. Over 200 mg of pure protein could be obtained from a liter of cell culture. Amino acid sequence comparison revealed the existence of a gene in Bacillus subtilis with a similar sequence, and psi sequence analysis established that B. subtilis has the equivalent of psi 516 in its small subunit rRNA. On the other hand, no common sequence motifs could be detected among this enzyme and the two tRNA psi synthases which have been cloned up to now.

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Section: Resultsmentioning

confidence: 99%

Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli

Wrzesiński

Bakin²,

Nurse³

et al. 1995

Biochemistry

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“…There was no visible <k spot on the TLC. known to affect the activity of tRNA P synthases (Kammen et al, 1988). P Formation in U5 RNA Mutants.…”

Section: Resultsmentioning

confidence: 99%

Formation of Pseudouridine in U5 Small Nuclear RNA

Patton¹

1994

Biochemistry

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The formation of pseudouridine (psi) on U5 small nuclear RNA (U5 snRNA) was studied using an in vitro modification system. Labeled U5 RNA, synthesized in vitro and therefore unmodified, was incubated in reactions containing S100 and/or nuclear extracts (NE) from HeLa cells, and the levels of psi were determined. There are three psi residues found in human U5 RNA, at positions 43, 46, and 53. Incubation of unmodified U5 RNA in reactions containing either S100 or NE supports psi formation at positions 43 and 46, which are found in a loop in the predicted secondary structure of U5 RNA. However, psi formation at position 53, which is found in a stem, is dependent on the presence of NE during the incubation. The order of extract addition does not have a significant effect on the formation of psi at position 53 as long as NE is present. The most efficient psi formation was observed with a combination of S100 and NE which allowed for efficient small nuclear ribonucleoprotein particle (snRNP) assembly and psi formation. When 9S and 20S U5 snRNPs were isolated by velocity sedimentation gradient centrifugation after incubation in the combined extracts, there was little difference in the psi levels at any of the positions for the two distinct particles. Mutations in the U5 RNA sequence do affect psi formation. U5 RNAs that have mutated Sm binding sites or are truncated prior to the Sm binding site have very low levels of psi formation at positions 43 and 46 and no detectable psi formation at position 53. A deletion of five nucleotides from 39 to 43 abolishes psi formation at positions 43 and 46, but the modification of position 53 is unaffected.

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“…The above mechanism is consistent with most experimental evidence. Kammen et al showed that tRNA Pseudouridine Synthase I (ΨSI) activity was inhibited by sulfhydryl reagents .…”

Section: Introductionmentioning

confidence: 99%

“…Kammen et al showed that tRNA Pseudouridine Synthase I (ΨSI) activity was inhibited by sulfhydryl reagents. 11 5-FUra-RNA, which can form stable 5,6-dihydropyrimidine adducts with enzymes involving a methyltransferase mechanism, has been shown to be an inhibitor of ΨS. 12 However, no covalent intermediates have yet been detected as conclusive evidence in the 5-FUra-RNA interaction with TS.…”

Section: Introductionmentioning

confidence: 99%

Quantum Mechanical Calculations of Nucleophilic Attack in the Pseudouridine Synthesis Reaction

Kollman

1999

J. Am. Chem. Soc.

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Ab initio quantum mechanical calculations at the MP2 level were performed to model nucleophilic attack in the pseudouridine synthesis reaction. The energy profile along the reaction coordinate suggests that the C1‘ attack by Asp may be the first step of the reaction, despite the fact that a COO- is a relatively weak nucleophile. This result supports the new mechanism proposed by Huang et al. for this enzyme. Our calculations also showed that nucleophilic attack by Asp on Cl‘ was stabilized by the uracil ring and that a similar stabilizing effect could exist in other nucleotides.

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Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I.

Cited by 124 publications

References 37 publications

Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli

Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli

Formation of Pseudouridine in U5 Small Nuclear RNA

Quantum Mechanical Calculations of Nucleophilic Attack in the Pseudouridine Synthesis Reaction

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