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.     

Three modifications in the D and T arms of tRNA influence translation in Escherichia coli and expression of virulence genes in Shigella flexneri

The modified nucleosides 2'-O-methylguanosine, present at position 18 (Gm18), 5-methyluridine, present at position 54 (m(5)U54), and pseudouridine, present at position 55 (Psi55), are located in the D and T arms of tRNAs and are close in space in the three-dimensional (3D) structure of this molecule in the bacterium Escherichia coli. The formation of these modified nucleosides is catalyzed by the products of genes trmH (Gm18), trmA (m(5)U54), and truB (Psi55). The combination of trmH, trmA, and truB mutations resulting in lack of these three modifications reduced the growth rate, especially at high temperature. Moreover, the lack of three modified nucleotides in tRNA induced defects in the translation of certain codons, sensitivity to amino acid analog 3,4-dehydro-DL-proline, and an altered oxidation of some carbon compounds. The results are consistent with the suggestion that these modified nucleosides, two of which directly interact in the 3D structure of tRNA by forming a hydrogen bond between Psi55 and Gm18, stabilize the structure of the tRNA. Moreover, lack of Psi55 in tRNA of human pathogen Shigella flexneri leads to a reduced expression of several virulence-associated genes.     

Differential roles of archaeal box H/ACA proteins in guide RNA-dependent and independent pseudouridine formation.

RNA-guided pseudouridine (Psi) synthesis in Archaea and Eukarya requires a four-protein one-RNA containing box H/ACA ribonucleoprotein (RNP) complex. The proteins in the archaeal RNP are aCbf5, aNop10, aGar1 and L7Ae. Pyrococcus aCbf5-aNop10 is suggested to be the minimal catalytic core in this synthesis and the activity is enhanced by L7Ae and aGar1. The protein aCbf5 is homologous to eukaryal Cbf5 (dyskerin, NAP57) as well as to bacterial TruB and eukaryal Pus4; the last two produce YPsi55 in tRNAs in a guide RNA-independent manner. Here, using recombinant Methanocaldococcus jannaschii proteins, we report that aCbf5 and aGar1 together can function as a tRNA Psi55 synthase in a guide RNA-independent manner. This activity is enhanced by aNop10, but not by L7Ae. The aCbf5 alone can also produce Psi55 in tRNAs that contain the canonical 3'-CCA sequence and this activity is stimulated by aGar1. These results suggest that the roles of accessory proteins are different in guide RNA-dependent and independent Psi synthesis by aCbf5. The presence of conserved C (or U) and A at tRNA positions 56 and 58, respectively, which are required for TruB/Pus4 activity, is not essential for aCbf5-mediated Psi55 formation. Conserved A58 in tRNA normally forms a tertiary reverse Hoogstein base pair with an equally conserved U54. This base pair is recognized by TruB. Apparently aCbf5 does not require this base pair to recognize U55 for conversion to Psi55.     

Formation of the conserved pseudouridine at position 55 in archaeal tRNA

Pseudouridine (Ψ) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Ψ55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are _pfuCbf5, a protein known to play a role in RNA-guided modification of rRNA, and _pfuPsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. _pfuPsuX is hereafter renamed _pfuPus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, _pfuPus10 efficiently complements an Escherichia coli strain deficient in the bacterial Ψ55 synthase TruB. These results indicate that it is probable that _pfuCbf5 or _pfuPus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.     

The spoU gene of Escherichia coli, the fourth gene of the spoT operon, is essential for tRNA (Gm18) 2'-O-methyltransferase activity.

We have evidence that the open reading frame previously denoted spoU is necessary for tRNA (Gm18) 2'-O-methyltransferase activity. The spoU gene is located in the gmk-rpoZ-spoT-spoU-recG operon at 82 minutes on the Escherichia coli chromosome. The deduced amino acid sequence of spoU shows strong similarities to previously characterized 2'-O-methyltransferases. Comparison of the nucleoside modification pattern of hydrolyzed tRNA, 16S rRNA and 23S rRNA from wild-type and spoU null mutants showed that the modified nucleoside 2'-O-methylguanosine (Gm), present in a subset of E. coli tRNAs at residue 18, is completely absent in the spoU mutant, suggesting that spoU encodes tRNA (Gm18) 2'-O-methyltransferase. Nucleoside modification of 16S and 23S rRNA was unaffected in the spoU mutant. Insertions in the downstream recG gene did not affect RNA modification. Absence of Gm18 in tRNA does not influence growth rate under the tested conditions and does not interfere with activity of the SupF amber suppressor, a suppressor tRNA that normally has the Gm18 modification. We suggest that the spoU gene be renamed trmH (tRNA methylation).     

Modified nucleotides in Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli.

In the present study, modified nucleotides in the B. subtilis tRNA(Trp) cloned and hyperexpressed in E. coli have been identified by TLC and HPLC analyses. The modification patterns of the two isoacceptors of cloned B. subtilis tRNA(Trp) have been compared with those of native tRNA(Trp) from B. subtilis and from E. coli. The modifications of the A73 mutant of B. subtilis tRNA(Trp), which is inactive toward its cognate TrpRS, were also investigated. The results indicate the formation of the modified nucleotides S4U8, Gm18, D20, Cm32, i6A/ms2i6A37, T54 and psi 55 on cloned B. subtilis tRNA(Trp). This modification pattern resembles the pattern of E. coli tRNA(Trp), except that m7G is missing from the cloned tRNA(Trp), probably on account of its short extra loop. In contrast, the pattern departs substantially from that of native B. subtilis tRNA(Trp). Therefore, the cloned B. subtilis tRNA(Trp) has taken on largely the modification pattern of E. coli tRNA(Trp) despite the 26% sequence difference between the two species of tRNA, gaining in particular the Cm32 and Gm18 modifications from the E. coli host. A notable difference between the isoacceptors of the cloned tRNA(Trp) was seen in the extent of modification of A37, which occurred as either the hypomodified i6A or the hypermodified ms2i6A form. Surprisingly, base substitution of guanosine by adenosine at position 73 of the cloned tRNA(Trp) has led to the abolition of the 2'-O-methylation modification of the remote G18 residue.     

An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation

Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.     

Production and characterization of two N-terminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: effect of N-terminus in thermal activity and stability

Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP_004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the ΔN16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 °C for ΔN16 and ΔN26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 °C, and maximal stability at pH 8.1 and 65 °C. KLEST-5A (ΔN26) did not show an absolute maximum of activity. However, best results were obtained at 40 °C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 °C (t(?)< 5 min) than the wild-type enzyme (t(?)=135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(?)=230 min, at 85 °C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability.     

Transfer RNA modifications that alter +1 frameshifting in general fail to affect -1 frameshifting

Using mutants (tgt, mnmA(asuE, trmU), mnmE(trmE), miaA, miaB, miaE, truA(hisT), truB) of either Escherichia coli or Salmonella enterica serovar Typhimurium and the trm5 mutant of Saccharomyces cerevisiae, we have analyzed the influence by the modified nucleosides Q34, mnm(5)s(2)U34, ms(2)io(6)A37, Psi39, Psi55, m(1)G37, and yW37 on -1 frameshifts errors at various heptameric sequences, at which at least one codon is decoded by tRNAs having these modified nucleosides. The frequency of -1 frameshifting was the same in congenic strains only differing in the allelic state of the various tRNA modification genes. In fact, in one case (deficiency of mnm(5)s(2)U34), we observed a reduced ability of the undermodified tRNA to make a -1 frameshift error. These results are in sharp contrast to earlier observations that tRNA modification prevents +1 frameshifting suggesting that the mechanisms by which -1 and +1 frameshift errors occur are different. Possible mechanisms explaining these results are discussed.     

Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.     

Mutational analysis of YgfZ, a folate-dependent protein implicated in iron/sulphur cluster metabolism

Proteins of the YgfZ family occur in all domains of life and are characterized by the conserved dodecapeptide motif KGC[Y/F]-x-GQE-x(3) -[R/K]. YgfZ proteins are known to participate in assembly or repair of iron/sulphur clusters, and to require folate for biological activity, but their mechanism of action is unknown. To assess the importance of individual residues in the conserved motif, Escherichia coli Ygf?Z was expressed from a plasmid in a ΔygfZ strain and subjected to alanine-scanning mutagenesis. The impacts on YgfZ functionality were evaluated by assays of growth and of the in vivo activity of the iron/sulphur enzyme MiaB, which modifies tRNA. By these criteria, the motif's tyrosine residue (Y229) had a detectable influence but only the cysteine residue (C228) was critical, for only the C228A mutant failed to complement the growth and MiaB activity phenotypes of the ΔygfZ strain. Immunoblots confirmed that the latter result was not simply because of a low level of the C228A mutant protein. Collectively, these data demonstrate a pivotal role for the Ygf?Z motif's cysteine residue and a subsidiary one for the adjacent tyrosine, and help formulate a hypothesis about the folate requirement of Ygf?Z proteins.     

N7-Methylguanine at position 46 (m7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network

N⁷-methylguanine at position 46 (m⁷G46) in tRNA is produced by tRNA (m⁷G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (ΔtrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the ΔtrmB strain and the lack of the m⁷G46 modification in tRNA^Phe were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the ΔtrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m⁷G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m¹G37, suggesting that the m⁷G46 positively affects their formations. Although the lack of the m⁷G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA^Phe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNA^Phe and tRNA^Ile. ³⁵S-Met incorporation into proteins revealed that protein synthesis in ΔtrmB cells is depressed above 70°C. At 80°C, the ΔtrmB strain exhibits a severe growth defect. Thus, the m⁷G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m⁷G46 modification supports introduction of other modifications.     

Two types of tRNA(Gm)methylase found in extreme thermophile, Thermus thermophilus strains HB 8 and HB 27

There are two distinct strains HB 8 and HB 27 in an extreme thermophile, Thermus thermophilus, and both strains have their own tRNA(Gm)methylases, which specifically methylates the 2'-OH of the ribose ring in the D loop of tRNA. The Gm-methylases are very similar with respect to the recognition mechanism of substrate tRNA and the molecular weight, but differ in the temperature dependency of the enzyme activity. Gm-methylase from strain HB 8 possesses its activity even at low temperature (40 degrees C), whereas that of strain HB 27 shows very low activity at the temperature and increases the activity as the incubation temperature is raised. Amino acid compositions of both the enzymes are very similar except for Glx and Asx, but the content of secondary structure is very different as judged by circular dichroism.     

Indole-3-glycerol-phosphate synthase is recognized by a cold-inducible group II chaperonin in Thermococcus kodakarensis

Thermococcus kodakarensis optimally grows at 85°C and possesses two chaperonins, cold-inducible CpkA and heat-inducible CpkB. Gene disruptants DA1 (ΔcpkA) and DB1 (ΔcpkB) showed decreased cell growth at 60°C and 93°C, respectively. The DB2 mutant (ΔcpkAcpkB ΔcpkB), whose cpkB gene was expressed under the control of the cpkA promoter, did not grow at 60°C, and the DB3 mutant [ΔcpkA(1-524)cpkB(1-524) ΔcpkB], whose CpkA amino acid residues 1 to 524 were replaced with corresponding CpkB residues that maintained the C-terminal region intact, grew at 60°C, implying that the CpkA C-terminal region plays a key role in cell growth at 60°C. To screen for specific CpkA target proteins, comparative pulldown studies with anti-Cpk were performed using cytoplasmic fractions from DA1 cells cultivated at 93°C and DB1 cells cultivated at 60°C. Among the proteins coprecipitated with anti-Cpk, TK0252, encoding indole-3-glycerol-phosphate synthase (TrpC), showed the highest Mascot score. Counter-pulldown experiments were also performed on DA1 and DB1 extracts using anti-TrpC. CpkA coimmunoprecipitated with anti-TrpC while CpkB did not. The results obtained indicate that TrpC is a specific target for CpkA. The effects of Cpks on denatured TrpC were then examined. The refolding of partially denatured TrpC was accelerated by the addition of CpkA but not by adding CpkB. DA1 cells grew optimally in minimal medium only in the presence of tryptophan but hardly grew in the absence of tryptophan at 60°C. It has been suggested that a lesion of functional TrpC is caused by cpkA disruption, resulting in tryptophan auxotrophy.     

The Catalytic Domain of Topological Knot tRNA Methyltransferase (TrmH) Discriminates between Substrate tRNA and Nonsubstrate tRNA via an Induced-fit Process

A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2′-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity. Type I TrmH, including Thermus thermophilus TrmH, can modify all tRNA species, whereas type II TrmH, for example Escherichia coli TrmH, modifies only a subset of tRNA species. Our previous crystal study showed that T. thermophilus TrmH is a class IV S-adenosyl-l-methionine-dependent methyltransferase, which maintains a topological knot structure in the catalytic domain. Because TrmH enzymes have short stretches at the N and C termini instead of a clear RNA binding domain, these stretches are believed to be involved in tRNA recognition. In this study, we demonstrate by site-directed mutagenesis that both N- and C-terminal regions function in tRNA binding. However, in vitro and in vivo chimera protein studies, in which four chimeric proteins of type I and II TrmHs were used, demonstrated that the catalytic domain discriminates substrate tRNAs from nonsubstrate tRNAs. Thus, the N- and C-terminal regions do not function in the substrate tRNA discrimination process. Pre-steady state analysis of complex formation between mutant TrmH proteins and tRNA by stopped-flow fluorescence measurement revealed that the C-terminal region works in the initial binding process, in which nonsubstrate tRNA is not excluded, and that structural movement of the motif 2 region of the catalytic domain in an induced-fit process is involved in substrate tRNA discrimination.