Structural features of tRNALys favored by anticodon nuclease as inferred from reactivities of anticodon stem and loop substrate analogs.

The bacterial tRNA(Lys)-specific PrrC-anticodon nuclease efficiently cleaved an anticodon stem-loop (ASL) oligoribonucleotide containing the natural modified bases, suggesting this region harbors the specificity determinants. Assays of ASL analogs indicated that the 6-threonylcarbamoyl adenosine modification (t(6)A37) enhances the reactivity. The side chain of the modified wobble base 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) has a weaker positive effect depending on the context of other modifications. The s(2)U34 modification apparently has none and the pseudouridine (psi39) was inhibitory in most modification contexts. GC-rich but not IC-rich stems abolished the activity. Correlating the reported structural effects of the base modifications with their effects on anticodon nuclease activity suggests preference for substrates where the anticodon nucleotides assume a stacked A-RNA conformation and base pairing interactions in the stem are destabilized. Moreover, the proposal that PrrC residue Asp(287) contacts mnm(5)s(2)U34 was reinforced by the observations that the mammalian tRNA(Lys-3) wobble base 5-methoxycarbonyl methyl-2-thiouridine (mcm(5)s(2)U) is inhibitory and that the D287H mutant favors tRNA(Lys-3) over Escherichia coli tRNA(Lys). The detection of this mutation and ability of PrrC to cleave the isolated ASL suggest that anticodon nuclease may be used to cleave tRNA(Lys-3) primer molecules annealed to the genomic RNA template of the human immunodeficiency virus.     

Pus1p-dependent tRNA pseudouridinylation becomes essential when tRNA biogenesis is compromised in yeast

Yeast Pus1p catalyzes the formation of pseudouridine (psi) at specific sites of several tRNAs, but its function is not essential for cell viability. We show here that Pus1p becomes essential when another tRNA:pseudouridine synthase, Pus4p, or the essential minor tRNA for glutamine are mutated. Strikingly, this mutant tRNA, which carries a mismatch in the T psi C arm, displays a nuclear export defect. Furthermore, nuclear export of at least one wild-type tRNA species becomes defective in the absence of Pus1p. Our data, thus, show that the modifications formed by Pus1p are essential when other aspects of tRNA biogenesis or function are compromised and suggest that impairment of nuclear tRNA export in the absence of Pus1p might contribute to this phenotype.     

Partial activity is seen with many substitutions of highly conserved active site residues in human Pseudouridine synthase 1

Pseudouridine synthase 1 (Pus1p) is an enzyme that converts uridine to Pseudouridine (Ψ) in tRNA and other RNAs in eukaryotes. The active site of Pus1p is composed of stretches of amino acids that are highly conserved and it is hypothesized that mutation of select residues would impair the enzyme's ability to catalyze the formation of Ψ. However, most mutagenesis studies have been confined to substitution of the catalytic aspartate, which invariably results in an inactive enzyme in all Ψ synthases tested. To determine the requirements for particular amino acids at certain absolutely conserved positions in Pus1p, three residues (R116, Y173, R267) that correspond to amino acids known to compose the active site of TruA, a bacterial Ψ synthase that is homologous to Pus1p, were mutated in human Pus1p (hPus1p). The effects of those mutations were determined with three different in vitro assays of pseudouridylation and several tRNA substrates. Surprisingly, it was found that each of these components of the hPus1p active site could tolerate certain amino acid substitutions and in fact most mutants exhibited some activity. The most active mutants retained near wild-type activity at positions 27 or 28 in the substrate tRNA, but activity was greatly reduced or absent at other positions in tRNA readily modified by wild-type hPus1p.     

EF-G-catalyzed translocation of anticodon stem-loop analogs of transfer RNA in the ribosome.

Translocation, catalyzed by elongation factor EF-G, is the precise movement of the tRNA-mRNA complex within the ribosome following peptide bond formation. Here we examine the structural requirement for A- and P-site tRNAs in EF-G-catalyzed translocation by substituting anticodon stem-loop (ASL) analogs for the respective tRNAs. Translocation of mRNA and tRNA was monitored independently; mRNA movement was assayed by toeprinting, while tRNA and ASL movement was monitored by hydroxyl radical probing by Fe(II) tethered to the ASLs and by chemical footprinting. Translocation depends on occupancy of both A and P sites by tRNA bound in a mRNA-dependent fashion. The requirement for an A-site tRNA can be satisfied by a 15 nucleotide ASL analog comprising only a 4 base pair (bp) stem and a 7 nucleotide anticodon loop. Translocation of the ASL is both EF-G- and GTP-dependent, and is inhibited by the translocational inhibitor thiostrepton. These findings show that the D, T and acceptor stem regions of A-site tRNA are not essential for EF-G-dependent translocation. In contrast, no translocation occurs if the P-site tRNA is substituted with an ASL, indicating that other elements of P-site tRNA structure are required for translocation. We also tested the effect of increasing the A-site ASL stem length from 4 to 33 bp on translocation from A to P site. Translocation efficiency decreases as the ASL stem extends beyond 22 bp, corresponding approximately to the maximum dimension of tRNA along the anticodon-D arm axis. This result suggests that a structural feature of the ribosome between the A and P sites, interferes with movement of tRNA analogs that exceed the normal dimensions of the coaxial tRNA anticodon-D arm.     

Pseudouridine formation in archaeal RNAs: The case of Haloferax volcanii

Pseudouridine (Ψ), the isomer of uridine, is commonly found at various positions of noncoding RNAs of all organisms. Ψ residues are formed by a number of single- or multisite specific Ψ synthases, which generally act as stand-alone proteins. In addition, in Eukarya and Archaea, specific ribonucleoprotein complexes, each containing a distinct box H/ACA guide RNA and four core proteins, can produce Ψ at many sites of different cellular RNAs. Cbf5 is the core Ψ synthase in these complexes. Using Haloferax volcanii as an archaeal model organism, we show that, contrary to eukaryotes, the Cbf5 homolog (HVO_2493) is not essential in this archaeon. The Cbf5-deleted strain of H. volcanii completely lacks Ψ at positions 1940, 1942, 2605, and 2591 (Escherichia coli positions 1915, 1917, 2572, and 2586) of its 23S rRNA, and contains reduced steady-state levels of some box H/ACA RNAs. Archaeal Cbf5 is known to have tRNA Ψ55 synthase activity in vitro but we could not confirm this activity in vivo in H. volcanii. Conversely, the Pus10 (previously PsuX) homolog (HVO_1979), which can produce tRNA Ψ55, as well as Ψ54 in vitro, is shown here to be essential in H. volcanii, whereas the corresponding tRNA Ψ55 synthases, Pus4 and TruB, are not essential in yeast and E. coli, respectively. Finally, we demonstrate that HVO_1852, the TruA/Pus3 homolog, is responsible for the pseudouridylation of position 39 in H. volcanii tRNAs and that the corresponding gene is not essential.     

In Human Pseudouridine Synthase 1 (hPus1), a C-Terminal Helical Insert Blocks tRNA from Binding in the Same Orientation as in the Pus1 Bacterial Homologue TruA,Consistent with Their Different Target Selectivities

Human pseudouridine (Ψ) synthase Pus1 (hPus1) modifies specific uridine residues in several non-coding RNAs: tRNA, U2 spliceosomal RNA, and steroid receptor activator RNA. We report three structures of the catalytic core domain of hPus1 from two crystal forms, at 1.8 Å resolution. The structures are the first of a mammalian Ψ synthase from the set of five Ψ synthase families common to all kingdoms of life. hPus1 adopts a fold similar to bacterial Ψ synthases, with a central antiparallel β-sheet flanked by helices and loops. A flexible hinge at the base of the sheet allows the enzyme to open and close around an electropositive active-site cleft. In one crystal form, a molecule of Mes [2-(N-morpholino)ethane sulfonic acid] mimics the target uridine of an RNA substrate. A positively charged electrostatic surface extends from the active site towards the N-terminus of the catalytic domain, suggesting an extensive binding site specific for target RNAs. Two α-helices C-terminal to the core domain, but unique to hPus1, extend along the back and top of the central β-sheet and form the walls of the RNA binding surface. Docking of tRNA to hPus1 in a productive orientation requires only minor conformational changes to enzyme and tRNA. The docked tRNA is bound by the electropositive surface of the protein employing a completely different binding mode than that seen for the tRNA complex of the Escherichia coli homologue TruA.     

Different mechanisms for pseudouridine formation in yeast 5S and 5.8S rRNAs

The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (Psis) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of Psi formation in 5S and 5.8S rRNA in which the unassigned Psis occur. We show that while the formation of the Psi in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the Psi in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the Psi in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis.     

Structural and functional roles of the N1- and N3-protons of psi at tRNA's position 39.

Pseudouridine at position 39 (Psi(39)) of tRNA's anticodon stem and loop domain (ASL) is highly conserved. To determine the physicochemical contributions of Psi(39)to the ASL and to relate these properties to tRNA function in translation, we synthesized the unmodified yeast tRNA(Phe)ASL and ASLs with various derivatives of U(39)and Psi(39). Psi(39)increased the thermal stability of the ASL (Delta T (m)= 1.3 +/- 0.5 degrees C), but did not significantly affect ribosomal binding ( K (d)= 229 +/- 29 nM) compared to that of the unmodified ASL (K (d)= 197 +/- 58 nM). The ASL-Psi(39)P-site fingerprint on the 30S ribosomal subunit was similar to that of the unmodified ASL. The stability, ribosome binding and fingerprint of the ASL with m(1)Psi(39)were comparable to that of the ASL with Psi(39). Thus, the contribution of Psi(39)to ASL stability is not related to N1-H hydrogen bonding, but probably is due to the nucleoside's ability to improve base stacking compared to U. In contrast, substitutions of m(3)Psi(39), the isosteric m(3)U(39)and m(1)m(3)Psi(39)destabilized the ASL by disrupting the A(31)-U(39)base pair in the stem, as confirmed by NMR. N3-methylations of both U and Psi dramatically decreased ribosomal binding ( K (d)= 1060 +/- 189 to 1283 +/- 258 nM). Thus, canonical base pairing of Psi(39)to A(31)through N3-H is important to structure, stability and ribosome binding, whereas the increased stability and the N1-proton afforded by modification of U(39)to Psi(39)may have biological roles other than tRNA's binding to the ribosomal P-site.     

tRNA Binding,Positioning, and Modification by the Pseudouridine Synthase Pus10

Pus10 is the most recently identified pseudouridine synthase found in archaea and higher eukaryotes. It modifies uridine 55 in the TΨC arm of tRNAs. Here, we report the first quantitative biochemical analysis of tRNA binding and pseudouridine formation by Pyrococcus furiosus Pus10. The affinity of Pus10 for both substrate and product tRNA is high (K_d of 30 nM), and product formation occurs with a K_m of 400 nM and a k_cat of 0.9 s^− 1. Site-directed mutagenesis was used to demonstrate that the thumb loop in the catalytic domain is important for efficient catalysis; we propose that the thumb loop positions the tRNA within the active site. Furthermore, a new catalytic arginine residue was identified (arginine 208), which is likely responsible for triggering flipping of the target uridine into the active site of Pus10. Lastly, our data support the proposal that the THUMP-containing domain, found in the N-terminus of Pus10, contributes to binding of tRNA. Together, our findings are consistent with the hypothesis that tRNA binding by Pus10 occurs through an induced-fit mechanism, which is a prerequisite for efficient pseudouridine formation.     

The Saccharomyces cerevisiae Pus2 protein encoded by YGL063w ORF is a mitochondrial tRNA:Psi27/28-synthase

The RNA:pseudouridine (Psi)-synthase family is one of the most complex families of RNA modification enzymes. Ten genes encoding putative RNA:Psi-synthases have been identified in S. cerevisiae. Most of the encoded enzymes have been characterized experimentally. Only the putative RNA:Psi-synthase Pus2p (encoded by the YGL063w ORF) had no identified substrate. Here, we analyzed Psi residues in cytoplasmic and mitochondrial tRNAs extracted from S. cerevisiae strains, carrying disruptions in the PUS1 and/or PUS2 ORFs. Our results demonstrate that Pus2p is a mitochondrial-specific tRNA:Psi-synthase acting at positions 27 and 28 in tRNAs. The importance of the Asp56 residue in the conserved ARTD motif of the Pus2p catalytic site is demonstrated in vivo. Interestingly, in spite of the absence of a characteristic N-terminal targeting signal, our data strongly suggest an efficient and rapid targeting of Pus2p in yeast mitochondria. In contradiction with the commonly held idea that a unique nuclear gene encodes the enzyme required for both cytoplasmic and mitochondrial tRNA modifications, here we show the existence of an enzyme specifically dedicated to mitochondrial tRNA modification (Pus2p), the corresponding modification in cytoplasmic tRNAs being catalyzed by another protein (Pus1p).     

A base-paired hairpin structure essential for the functional priming signal for DNA replication of the broad host range plasmid RSF1010.

The two single-strand DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010) of the broad host-range plasmid RSF1010 contain proposed stem-loop structures. Nine single base-change mutations in the stem of the ssiA structure, each of which destroyed a relevant base pairing, damaged the ssiA activity. A second single-base change was introduced into each of the nine ssiA mutants in such a way that the base pairing was restored. Only three out of nine second base changes that restored the base pairing restored the ssiA activity up to the wild-type level. Thus, the three are intramolecular suppressors. The results strongly suggested that, in the area of the stem of ssiA where the suppressor mutations fell, base pairing was the most important structural parameter for the ssiA activity. By contrast, it is most probable that, in the other part of the stem of ssiA, both base-pairing and the intrinsic base sequence were the major determinants of the ssiA activity.     

tRNA regulation of gene expression: interactions of an mRNA 5'-UTR with a regulatory tRNA

Many genes encoding aminoacyl-tRNA synthetases and other amino acid-related products in Gram-positive bacteria, including important pathogens, are regulated through interaction of unacylated tRNA with the 5'-untranslated region (5'-UTR) of the mRNA. Each gene regulated by this mechanism responds specifically to the cognate tRNA, and specificity is determined by pairing of the anticodon of the tRNA with a codon sequence in the "Specifier Loop" of the 5'-UTR. For the 5'-UTR to function in gene regulation, the mRNA folding interactions must be sufficiently stable to present the codon sequence for productive binding to the anticodon of the matching tRNA. A model bimolecular system was developed in which the interaction between two half molecules ("Common" and "Specifier") would reconstitute the Specifier Loop region of the 5'-UTR of the Bacillus subtilis glyQS gene, encoding GlyRS mRNA. Gel mobility shift analysis and fluorescence spectroscopy yielded experimental Kds of 27.6 +/- 1.0 microM and 10.5 +/- 0.7 microM, respectively, for complex formation between Common and Specifier half molecules. The reconstituted 5'-UTR of the glyQS mRNA bound the anticodon stem and loop of tRNA(Gly) (ASL(Gly)(GCC)) specifically and with a significant affinity (Kd = 20.2 +/- 1.4 microM). Thus, the bimolecular 5'-UTR and ASL(Gly)(GCC) models mimic the RNA-RNA interaction required for T box gene regulation in vivo.     

rRNA pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells

How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked dyskeratosis congenita (X-DC) and Hoyeraal-Hreidarsson (HH) syndrome. Here, we characterize ribosomes isolated from?a yeast strain in which Cbf5p, the yeast homolog of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A). Ribosomes from cbf5-D95A cells display decreased affinities for tRNA binding to the A and P sites as well as the cricket paralysis virus internal ribosome entry site (IRES), which interacts with both the P and the E sites of the ribosome. This biochemical impairment in ribosome activity manifests as decreased translational fidelity and IRES-dependent translational initiation, which are also evident in mouse and human cells deficient for DKC1 activity. These findings uncover specific roles for Ψ modification in ribosome-ligand interactions that are conserved in yeast, mouse, and humans.