tRNA Pseudouridine Synthase Research Articles

A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst.

tRNA pseudouridine synthase I catalyzes the conversion of uridine to pseudouridine at positions 38, 39, and/or 40 in the anticodon loop of many tRNAs. Pseudouridine synthase I was cloned behind a T7 promoter and expressed in Escherichia coli to about 20% of total soluble proteins. Fluorouracil-substituted tRNA caused a time-dependent inactivation of pseudouridine synthase I and formed a covalent complex with the enzyme that involved the FUMP at position 39. Asp60, conserved in all known and putative pseudouridine synthases, was mutated to amino acids with diverse side chains. All Asp60 mutants bound tRNA but were catalytically inactive and failed to form covalent complexes with fluorouracil-substituted tRNA. We conclude that the conserved Asp60 is essential for pseudouridine synthase activity and propose mechanisms which involve this residue in important catalytic roles.

The Role of Cysteine Residues in the Rearrangement of Uridine to Pseudouridine Catalyzed by Pseudouridine Synthase I

Escherichia coli tRNA pseudouridine synthase I (PSUI) catalyzes the conversion of uridine residues to pseudouridine in positions 38, 39, and 40 of various tRNA molecules. In previous biochemical studies with this enzyme (Kammen, H. O., Marvel, C. C., Hardy, L., and Penhoet, E. E. (1988) J. Biol. Chem. 263, 2255-2263) it was reported that cysteine residues are important in maintaining the active structure of the enzyme and are possibly involved in the catalytic reaction mechanism via a covalent cysteine intermediate. In order to further investigate the biochemical properties of PSUI, a high level expression and purification system for the enzyme and its corresponding mutants was developed. PSUI has three cysteine residues among 270 amino acids. In the present investigation, each cysteine residue was individually changed to serine and alanine. In addition, a triple mutant was prepared wherein all three cysteine residues were replaced by alanine. Surprisingly, while two of the three cysteine to serine mutants were inactive, all alanine mutants exhibited near wild-type levels of activity, including the triple mutant. These results provide the first direct and unambiguous chemical evidence against a covalent cysteine intermediate in the rearrangement mechanism of uridine to pseudouridine.

Nuclear pore proteins are involved in the biogenesis of functional tRNA.

Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide-specific and intron-dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre-tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(-) pus1(-) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA.

Proton-nitrogen-15 NMR studies of Escherichia coli tRNAPhe from HisT mutants: a structural role for pseudouridine

Escherichia coli tRNA(Phe)U39 was isolated from a specially constructed bacterial strain (DD1003/pRK3) carrying mutations in the hisT gene (the structural gene for tRNA pseudouridine synthase I) and in the pyrB gene (uracil auxotrophy). The pheU gene for tRNA(Phe) under control of the native tRNA promoter was on a multicopy plasmid and gave up to 40-fold overproduction of tRNA(Phe)U39. The double mutant permitted efficient incorporation of [3-15N]uracil, resulting in greater than 95% 15N enrichment of uracil-derived bases. 1H and 1H-15N NMR experiments were used to assign the low-field proton resonances to specific hydrogen-bonding interactions. 1H NMR assignments indicate that tRNA(Phe)U39 has a structure similar to that of native tRNA(Phe) except in the anticodon region where replacement of pseudouridine (psi) at position 39 with uridine (U) destabilizes hydrogen-bonding interactions at the base of the anticodon stem. We propose that U----psi modifications further stabilize interactions normally available to U by providing an additional locus for hydrogen bonding to the pyrimidine ring.

A gene tightly linked to CEN6 is important for growth of Saccharomyces cerevisiae

Transcriptional analysis of the region flanking the left boundary of the centromere of chromosome VI revealed the presence of a gene immediately adjacent to CEN6. The transcription of the gene is directed toward the centromere, and nucleotide sequence analysis showed that the coding region terminates only 50 bp away from CEN6. Our results extend to chromosome VI the observation that centromere-flanking regions of S. cerevisiae are transcriptionally active. Disruption of the coding region of the gene showed that its product, whilst not essential for cell viability, is important for normal cell growth. The gene has been termed DEG1 (DEpressed Growth rate). Comparison of the deduced amino acid sequence of DEG1 with a protein sequence databank revealed homology with the enzyme tRNA pseudouridine synthase I of E. coli.

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

The RNA modification enzyme, tRNA pseudouridine synthase I has been isolated in 95% purity from an Escherichia coli strain harboring a multicopy plasmid with a 2.3-kilobase pair insert from the hisT operon. Its molecular size, amino acid composition, and amino-terminal sequence correspond to those predicted by the structure and expression of the hisT gene. Enzyme activity, as measured by a 3H release assay, is unaffected by pretreatment of tRNA pseudouridine synthase I with micrococcal nuclease and is optimized by the addition of a monovalent cation and thiol reductant. The activity is inhibited by all tRNA species tested, including substrates, modified tRNAs, nonsubstrates, or tRNAs containing 5-fluorouridine. Binding of tRNA pseudouridine synthase I occurs with both substrate and nonsubstrate tRNAs and does not require a monovalent cation. Our findings are consistent with a multistep mechanism whereby tRNA pseudouridine synthase I first binds nonspecifically and then forms transient covalent adducts with tRNA substrates. In the absence of other proteins, purified tRNA pseudouridine synthase I forms psi at all three modification sites known to be affected in hisT mutants. The 36.4-kDa polypeptide product of the gene adjacent to hisT, whose translation is linked to that of tRNA pseudouridine synthase I, is not a functional subunit for tRNA pseudouridine synthase I activity, nor is it a separate synthase acting at one of the three loci.

HisT is part of a multigene operon in Escherichia coli K-12.

The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene. Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site. Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon.

Purification and properties of a mammalian tRNA pseudouridine synthase.

A tRNA pseudouridine synthase has been extensively purified from steer thymus extracts, using undermodified tRNA from hisT- mutants of Salmonella typhimurium as a substrate. The enzyme synthesizes a group of psi residues in the anticodon region of various hisT- isoacceptors and behaves like a eukaryotic homologue of Salmonella tRNA psi synthase I. The thymus enzyme requires a thiol and a monovalent cation (NH4+ or K+) for optimum activity; no energy sources or cofactors are required. The activity is inhibited by single tRNAs or bulk tRNA from all sources tested, and by ribosomal RNAs, various polyribonucleotides, and DNA. The enzyme modifies the two hisT- tRNAPhe isoacceptors, both tRNATyr acceptors and at least five of the tRNALeu isoacceptors to products that coelute with the respective wild type species on reverse-phase columns. With pure hisT- tRNA2Phe as substrate, the enzyme specifically converts residue U39 to psi. Interestingly, a psi residue is still present at position 32, in the anticodon loop of hisT- tRNA2Phe, indicating the existence of other uncharacterized pseudouridylation enzymes in S. typhimurium. These composite results show that the thymus enzyme can form psi at residues 38, 39, and 40 in the anticodon region of appropriate hisT- isoacceptors. During the enzyme purification, a second activity is partially resolved, which releases 3H from wild type S. typhimurium [pyrimidine-5-3H]tRNA. This activity may be associated with an enzyme that pseudouridylates sites that are uniquely modified in eukaryotic tRNAs, but not in Salmonella tRNAs. Our observations support the view that the psi residues in tRNA are synthesized by a family of enzymes, whose members act on uridine residues in specific regions of the molecule.