Publications

• A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe).

  Wrzesinski J., Nurse K., Bakin A., Lane B.G., Ofengand J.

  An Escherichia coli pseudouridine (psi) synthase, which forms both psi 746 in E. coli 23S ribosomal RNA and psi 32 in tRNA(Phe), has been isolated and cloned. The enzyme contains 219 amino acids and has a calculated MW of 24,432 Da. Amino acid sequence comparison with the three other psi synthases ... >> More

  An Escherichia coli pseudouridine (psi) synthase, which forms both psi 746 in E. coli 23S ribosomal RNA and psi 32 in tRNA(Phe), has been isolated and cloned. The enzyme contains 219 amino acids and has a calculated MW of 24,432 Da. Amino acid sequence comparison with the three other psi synthases that have been cloned to date, two for tRNA and one for 16S RNA, did not reveal any common sequence motifs, despite the catalysis of a common reaction. The gene was cloned behind a (His)6 leader for affinity purification. Upon overexpression, most of the enzyme remained soluble in the cell cytoplasm and could be purified to homogeneity on a Ni(2+)-containing resin. The enzyme reacted with both full-length 23S RNA or a fragment from residues 1-847, forming 1 mol psi/mol RNA at position 746, a normal site for psi. The enzyme has no dependence on Mg2+. The same yield was obtained in 1 mM EDTA as in 10 mM Mg2+, and the rate was faster in EDTA than in Mg2+. Full-length 16S RNA or fragments 1-526 or 1-678, as well as tRNA(Val) transcripts, were not modified in either EDTA or Mg2+. tRNA(Phe) transcripts, however, were modified with a yield of 1 mol psi/mol transcript at a rate in EDTA like that of 23S RNA. Sequencing showed all of the psi to be at position 32, a normal site for psi in this tRNA. Both 23S rRNA psi 746 and tRNA psi 32 occur in single-stranded segments of the same sequence, psi UGAAAA, closed by a stem. Therefore, this synthase may require for recognition only a short stretch of primary sequence 3' to the site of pseudouridylation. This is the first example of a dual-specificity modifying enzyme for RNA, that is, one which is specific for a single site in one RNA, and equally site-specific in a second class of RNA. The essentiality of these psi residues can now be assessed by disruption of the synthase gene. << Less

  RNA 1:437-448(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Pseudouridylation at position 32 of mitochondrial and cytoplasmic tRNAs requires two distinct enzymes in Saccharomyces cerevisiae.

  Behm-Ansmant I., Grosjean H., Massenet S., Motorin Y., Branlant C.

  Cytoplasmic and mitochondrial tRNAs contain several pseudouridylation sites, and the tRNA:Psi-synthase acting at position 32 had not been identified in Saccharomyces cerevisiae. By combining genetic and biochemical analyses, we demonstrate that two enzymes, Rib2/Pus8p and Pus9p, are required for P ... >> More

  Cytoplasmic and mitochondrial tRNAs contain several pseudouridylation sites, and the tRNA:Psi-synthase acting at position 32 had not been identified in Saccharomyces cerevisiae. By combining genetic and biochemical analyses, we demonstrate that two enzymes, Rib2/Pus8p and Pus9p, are required for Psi32 formation in cytoplasmic and mitochondrial tRNAs, respectively. Pus9p acts mostly in mitochondria, and Rib2/Pus8p is strictly cytoplasmic. This is the first case reported so far of two distinct tRNA modification enzymes acting at the same position but present in two different compartments. This peculiarity may be the consequence of a gene fusion that occurred during yeast evolution. Indeed, Rib2/Pus8p displays two distinct catalytic activities involved in completely unrelated metabolism: its C-terminal domain has a DRAP-deaminase activity required for riboflavin biogenesis in the cytoplasm, whereas its N-terminal domain carries the tRNA:Psi32-synthase activity. Pus9p has only a tRNA:Psi32-synthase activity and contains a characteristic mitochondrial targeting sequence at its N terminus. These results are discussed in terms of RNA:Psi-synthase evolution. << Less

  J. Biol. Chem. 279:52998-53006(2004) [PubMed] [EuropePMC]

• Crystal structure of pseudouridine synthase RluA: indirect sequence readout through protein-induced RNA structure.

  Hoang C., Chen J., Vizthum C.A., Kandel J.M., Hamilton C.S., Mueller E.G., Ferre-D'Amare A.R.

  RluA is a dual-specificity enzyme responsible for pseudouridylating 23S rRNA and several tRNAs. The 2.05 A resolution structure of RluA bound to a substrate RNA comprising the anticodon stem loop of tRNA(Phe) reveals that enzyme binding induces a dramatic reorganization of the RNA. Instead of adop ... >> More

  RluA is a dual-specificity enzyme responsible for pseudouridylating 23S rRNA and several tRNAs. The 2.05 A resolution structure of RluA bound to a substrate RNA comprising the anticodon stem loop of tRNA(Phe) reveals that enzyme binding induces a dramatic reorganization of the RNA. Instead of adopting its canonical U turn conformation, the anticodon loop folds into a new structure with a reverse-Hoogsteen base pair and three flipped-out nucleotides. Sequence conservation, the cocrystal structure, and the results of structure-guided mutagenesis suggest that RluA recognizes its substrates indirectly by probing RNA loops for their ability to adopt the reorganized fold. The planar, cationic side chain of an arginine intercalates between the reverse-Hoogsteen base pair and the bottom pair of the anticodon stem, flipping the nucleotide to be modified into the active site of RluA. Sequence and structural comparisons suggest that pseudouridine synthases of the RluA, RsuA, and TruA families employ an equivalent arginine for base flipping. << Less

  Mol Cell 24:535-545(2006) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.