The Bacillus subtilis open reading frame ysgA encodes the SPOUT methyltransferase RlmP forming 2'-O-methylguanosine at position 2553 in the A-loop of 23S rRNA.

A previous bioinformatic analysis predicted that the ysgA open reading frame of Bacillus subtilis encodes an RNA methyltransferase of the SPOUT superfamily. Here we show that YsgA is the 2'-O-methyltransferase that targets position G2553 (Escherichia coli numbering) of the A-loop of 23S rRNA. This was shown by a combination of biochemical and mass spectrometry approaches using both rRNA extracted from B. subtilis wild-type or ΔysgA cells and in vitro synthesized rRNA. When the target G2553 is mutated, YsgA is able to methylate the ribose of adenosine. However, it cannot methylate cytidine nor uridine. The enzyme modifies free 23S rRNA but not the fully assembled ribosome nor the 50S subunit, suggesting that the modification occurs early during ribosome biogenesis. Nevertheless, ribosome subunits assembly is unaffected in a B. subtilis ΔysgA mutant strain. The crystal structure of the recombinant YsgA protein, combined with mutagenesis data, outlined in this article highlights a typical SPOUT fold preceded by an L7Ae/L30 (eL8/eL30 in a new nomenclature) amino-terminal domain.

Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK: insights into tRNA recognition.

1-Methyladenosine (m1A) is a modified nucleoside found at positions 9, 14, 22 and 58 of tRNAs, which arises from the transfer of a methyl group onto the N1-atom of adenosine. The yqfN gene of Bacillus subtilis encodes the methyltransferase TrmK (BsTrmK) responsible for the formation of m1A22 in tRNA. Here, we show that BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of B. subtilis bearing an A22. In addition to a non-Watson-Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. We solved the crystal structure of BsTrmK showing an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. We used NMR chemical shift mapping to drive the docking of BstRNASer to BsTrmK in complex with its methyl-donor cofactor S-adenosyl-L-methionine (SAM). In this model, validated by methyltransferase activity assays on BsTrmK mutants, both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson-Crick R13-A22 base-pair positioning the A22 N1-atom close to the SAM methyl group.

Structural and biochemical analysis of the dual-specificity Trm10 enzyme from Thermococcus kodakaraensis prompts reconsideration of its catalytic mechanism.

tRNA molecules get heavily modified post-transcriptionally. The N-1 methylation of purines at position 9 of eukaryal and archaeal tRNA is catalyzed by the SPOUT methyltranferase Trm10. Remarkably, while certain Trm10 orthologs are specific for either guanosine or adenosine, others show a dual specificity. Structural and functional studies have been performed on guanosine- and adenosine-specific enzymes. Here we report the structure and biochemical analysis of the dual-specificity enzyme from Thermococcus kodakaraensis (TkTrm10). We report the first crystal structure of a construct of this enzyme, consisting of the N-terminal domain and the catalytic SPOUT domain. Moreover, crystal structures of the SPOUT domain, either in the apo form or bound to S-adenosyl-l-methionine or S-adenosyl-l-homocysteine reveal the conformational plasticity of two active site loops upon substrate binding. Kinetic analysis shows that TkTrm10 has a high affinity for its tRNA substrates, while the enzyme on its own has a very low methyltransferase activity. Mutation of either of two active site aspartate residues (Asp206 and Asp245) to Asn or Ala results in only modest effects on the N-1 methylation reaction, with a small shift toward a preference for m1G formation over m1A formation. Only a double D206A/D245A mutation severely impairs activity. These results are in line with the recent finding that the single active-site aspartate was dispensable for activity in the guanosine-specific Trm10 from yeast, and suggest that also dual-specificity Trm10 orthologs use a noncanonical tRNA methyltransferase mechanism without residues acting as general base catalysts.

Structural and functional insights into tRNA binding and adenosine N1-methylation by an archaeal Trm10 homologue.

Purine nucleosides on position 9 of eukaryal and archaeal tRNAs are frequently modified in vivo by the post-transcriptional addition of a methyl group on their N1 atom. The methyltransferase Trm10 is responsible for this modification in both these domains of life. While certain Trm10 orthologues specifically methylate either guanosine or adenosine at position 9 of tRNA, others have a dual specificity. Until now structural information about this enzyme family was only available for the catalytic SPOUT domain of Trm10 proteins that show specificity toward guanosine. Here, we present the first crystal structure of a full length Trm10 orthologue specific for adenosine, revealing next to the catalytic SPOUT domain also N- and C-terminal domains. This structure hence provides crucial insights in the tRNA binding mechanism of this unique monomeric family of SPOUT methyltransferases. Moreover, structural comparison of this adenosine-specific Trm10 orthologue with guanosine-specific Trm10 orthologues suggests that the N1 methylation of adenosine relies on additional catalytic residues.

Alterations in the Ure2 αCap domain elicit different GATA factor responses to rapamycin treatment and nitrogen limitation.

Ure2 is a phosphoprotein and central negative regulator of nitrogen-responsive Gln3/Gat1 localization and their ability to activate transcription. This negative regulation is achieved by the formation of Ure2-Gln3 and -Gat1 complexes that are thought to sequester these GATA factors in the cytoplasm of cells cultured in excess nitrogen. Ure2 itself is a dimer the monomer of which consists of two core domains and a flexible protruding αcap. Here, we show that alterations in this αcap abolish rapamycin-elicited nuclear Gln3 and, to a more limited extent, Gat1 localization. In contrast, these alterations have little demonstrable effect on the Gln3 and Gat1 responses to nitrogen limitation. Using two-dimensional PAGE we resolved eight rather than the two previously reported Ure2 isoforms and demonstrated Ure2 dephosphorylation to be stimulus-specific, occurring after rapamycin treatment but only minimally if at all in nitrogen-limited cells. Alteration of the αcap significantly diminished the response of Ure2 dephosphorylation to the TorC1 inhibitor, rapamycin. Furthermore, in contrast to Gln3, rapamycin-elicited Ure2 dephosphorylation occurred independently of Sit4 and Pph21/22 (PP2A) as well as Siw14, Ptc1, and Ppz1. Together, our data suggest that distinct regions of Ure2 are associated with the receipt and/or implementation of signals calling for cessation of GATA factor sequestration in the cytoplasm. This in turn is more consistent with the existence of distinct pathways for TorC1- and nitrogen limitation-dependent control than it is with these stimuli representing sequential steps in a single regulatory pathway.

Intranuclear function for protein phosphatase 2A: Pph21 and Pph22 are required for rapamycin-induced GATA factor binding to the DAL5 promoter in yeast.

Protein phosphatase 2A (PP2A), a central Tor pathway phosphatase consisting of a catalytic subunit (Pph21 or Pph22), a scaffold subunit (Tpd3), and one of two regulatory subunits (Cdc55 or Rts1), has been repeatedly shown to play important roles in cytoplasmically localized signal transduction activities. In contrast, its involvement in intranuclear control of mRNA production has heretofore not been reported. Here, we demonstrate for the first time that binding of the nitrogen catabolite repression-responsive GATA transcription activators (Gln3 and Gat1) to the DAL5 promoter and DAL5 expression require Pph21/22-Tpd3-Cdc55/Rts1 in rapamycin-treated glutamine-grown cells. This conclusion is supported by the following observations. (i) Rapamycin-induced DAL5 expression along with Gln3 and Gat1 binding to the DAL5 promoter fails to occur in pph21Δ pph22Δ, tpd3Δ, and cdc55Δ rts1Δ mutants. (ii) The Pph21/22 requirement persists even when Gat1 and Gln3 are rendered constitutively nuclear, thus dissociating the intranuclear requirement of PP2A from its partial requirement for rapamycin-induced nuclear Gat1 localization. (iii) Pph21-Myc(13) (Ppp21 tagged at the C terminus with 13 copies of the Myc epitope) weakly associates with the DAL5 promoter in a Gat1-dependent manner, whereas a similar Pph22-Myc(13) association requires both Gln3 and Gat1. Finally, we demonstrate that a pph21Δ pph22Δ double mutant is epistatic to ure2Δ for nuclear Gat1 localization in untreated glutamine-grown cells, whereas for Gln3, just the opposite occurs: i.e., ure2Δ is epistatic to pph21Δ pph22Δ. This final observation adds additional support to our previous conclusion that the Gln3 and Gat1 GATA factor localizations are predominantly controlled by different regulatory pathways.

The yeast GATA factor Gat1 occupies a central position in nitrogen catabolite repression-sensitive gene activation.

Saccharomyces cerevisiae cells are able to adapt their metabolism according to the quality of the nitrogen sources available in the environment. Nitrogen catabolite repression (NCR) restrains the yeast's capacity to use poor nitrogen sources when rich ones are available. NCR-sensitive expression is modulated by the synchronized action of four DNA-binding GATA factors. Although the first identified GATA factor, Gln3, was considered the major activator of NCR-sensitive gene expression, our work positions Gat1 as a key factor for the integrated control of NCR in yeast for the following reasons: (i) Gat1 appeared to be the limiting factor for NCR gene expression, (ii) GAT1 expression was regulated by the four GATA factors in response to nitrogen availability, (iii) the two negative GATA factors Dal80 and Gzf3 interfered with Gat1 binding to DNA, and (iv) Gln3 binding to some NCR promoters required Gat1. Our study also provides mechanistic insights into the mode of action of the two negative GATA factors. Gzf3 interfered with Gat1 by nuclear sequestration and by competition at its own promoter. Dal80-dependent repression of NCR-sensitive gene expression occurred at three possible levels: Dal80 represses GAT1 expression, it competes with Gat1 for binding, and it directly represses NCR gene transcription.

Nitrogen catabolite repression-sensitive transcription as a readout of Tor pathway regulation: the genetic background, reporter gene and GATA factor assayed determine the outcomes.

Nitrogen catabolite repression (NCR)-sensitive genes, whose expression is highly repressed when provided with excess nitrogen and derepressed when nitrogen is limited or cells are treated with rapamycin, are routinely used as reporters in mechanistic studies of the Tor signal transduction pathway in Saccharomyces cerevisiae. Two GATA factors, Gln3 and Gat1, are responsible for NCR-sensitive transcription, but recent evidence demonstrates that Tor pathway regulation of NCR-sensitive transcription bifurcates at the level of GATA factor localization. Gln3 requires Sit4 phosphatase for nuclear localization and NCR-sensitive transcription while Gat1 does not. In this article, we demonstrate that the extent to which Sit4 plays a role in NCR-sensitive transcription depends upon whether or not (i) Gzf3, a GATA repressor homologous to Dal80, is active in the genetic background assayed; (ii) Gat1 is able to activate transcription of the assayed gene in the absence of Gln3 in that genetic background; and (iii) the gene chosen as a reporter is able to be transcribed by Gln3 or Gat1 in the absence of the other GATA factor. Together, the data indicate that in the absence of these three pieces of information, overall NCR-sensitive gene transcription data are unreliable as Tor pathway readouts.

Rapamycin-induced Gln3 dephosphorylation is insufficient for nuclear localization: Sit4 and PP2A phosphatases are regulated and function differently.

Gln3, the major activator of nitrogen catabolite repression (NCR)-sensitive transcription, is often used as an assay of Tor pathway regulation in Saccharomyces cerevisiae. Gln3 is cytoplasmic in cells cultured with repressive nitrogen sources (Gln) and nuclear with derepressive ones (Pro) or after treating Gln-grown cells with the Tor inhibitor, rapamycin (Rap). In Raptreated or Pro-grown cells, Sit4 is posited to dephosphorylate Gln3, which then dissociates from a Gln3-Ure2 complex and enters the nucleus. However, in contrast with this view, Sit4-dependent Gln3 dephosphorylation is greater in Gln than Pro. Investigating this paradox, we show that PP2A (another Tor pathway phosphatase)-dependent Gln3 dephosphorylation is regulated oppositely to that of Sit4, being greatest in Pro- and least in Gln-grown cells. It thus parallels nuclear Gln3 localization and NCR-sensitive transcription. However, because PP2A is not required for nuclear Gln3 localization in Pro, PP2A-dependent Gln3 dephosphorylation and nuclear localization are likely parallel responses to derepressive nitrogen sources. In contrast, Rap-induced nuclear Gln3 localization absolutely requires all four PP2A components (Pph21/22, Tpd3, Cdc55, and Rts1). In pph21Delta22Delta, tpd3Delta, or cdc55Delta cells, however, Gln3 is dephosphorylated to the same level as in Rap-treated wild-type cells, indicating Rap-induced Gln3 dephosphorylation is insufficient to achieve nuclear localization. Finally, PP2A-dependent Gln3 dephosphorylation parallels conditions where Gln3 is mostly nuclear, while Sit4-dependent and Rap-induced dephosphorylation parallels those where Gln3 is mostly cytoplasmic, suggesting the effects of these phosphatases on Gln3 may occur in different cellular compartments.

Saccharomyces cerevisiae Sit4 phosphatase is active irrespective of the nitrogen source provided, and Gln3 phosphorylation levels become nitrogen source-responsive in a sit4-deleted strain.

Tor1,2 control of type 2A-related phosphatase activities in Saccharomyces cerevisiae has been reported to be responsible for the regulation of Gln3 phosphorylation and intracellular localization in response to the nature of the nitrogen source available. According to the model, excess nitrogen stimulates Tor1,2 to phosphorylate Tip41 and/or Tap42. Tap42 then complexes with and inactivates Sit4 phosphatase, thereby preventing it from dephosphorylating Gln3. Phosphorylated Gln3 complexes with Ure2 and is sequestered in the cytoplasm. When Tor1,2 kinase activities are inhibited by limiting nitrogen, or rapamycin-treatment, Tap42 can no longer complex with Sit4. Active Sit4 dephosphorylates Gln3, which can then localize to the nucleus and activate transcription. The paucity of experimental data directly correlating active Sit4 and Pph3 with Gln3 regulation prompted us to assay Gln3-Myc(13) phosphorylation and intracellular localization in isogenic wild type, sit4, pph3, and sit4pph3 deletion strains. We found that Sit4 actively brought about Gln3-Myc(13) dephosphorylation in both good (glutamine or ammonia) and poor (proline) nitrogen sources. This Sit4 activity masked nitrogen source-dependent changes in Gln3-Myc(13) phosphorylation which were clearly visible when SIT4 was deleted. The extent of Sit4 requirement for Gln3 nuclear localization was both nitrogen source- and strain-dependent. In some strains, Sit4 was not even required for Gln3 nuclear localization in untreated or rapamycin-treated, proline-grown cells or Msx-treated, ammonia-grown cells.

Identification of direct and indirect targets of the Gln3 and Gat1 activators by transcriptional profiling in response to nitrogen availability in the short and long term.

Nitrogen catabolite repression (NCR) consists in the specific inhibition of transcriptional activation of genes encoding the permeases and catabolic enzymes needed to degrade poor nitrogen sources. Under nitrogen limitation or rapamycin treatment, NCR genes are activated by Gln3 or Gat1, or by both factors. To compare the sets of genes responding to rapamycin or to nitrogen limitation, we used DNA microarrays to establishing the expression profiles of a wild type strain, and of a double gln3Delta-gat1Delta strain, grown on glutamine, after addition of rapamycin, on proline, or after a shift from glutamine to proline. Analysis of microarray data revealed 392 genes whose expression was dependent on the nitrogen source quality. 91 genes were activated in a GATA factor-dependent manner in all growth conditions, suggesting a direct role of Gln3 and Gat1 in their expression. Other genes were only transiently up-regulated (stress-responsive genes) or down-regulated (genes encoding ribosomal proteins and translational factors) upon nitrogen limitation, and this regulation was delayed in a gln3Delta-gat1Delta strain. Repression of amino acid and nucleotide biosynthetic genes after a nitrogen shift did not depend on Gcn4. Several transporter genes were repressed as a consequence of enhanced levels of NCR-responsive permeases present at the plasma membrane.

Transduction of the nitrogen signal activating Gln3-mediated transcription is independent of Npr1 kinase and Rsp5-Bul1/2 ubiquitin ligase in Saccharomyces cerevisiae.

Nitrogen Catabolite Repression (NCR) allows the adaptation of yeast cells to the quality of nitrogen supply by inhibiting the transcription of genes encoding proteins involved in transport and degradation of nonpreferred nitrogen sources. In cells using ammonium or glutamine, the GATA transcription factor Gln3 is sequestered in the cytoplasm by Ure2 whereas it enters the nucleus after a shift to a nonpreferred nitrogen source like proline or upon addition of rapamycin, the TOR complex inhibitor. Recently, the Npr1 kinase and the Rsp5, Bul1/2 ubiquitin ligase complex were reported to have antagonistic roles in the nuclear import and Gln3-mediated activation. The Npr1 kinase controls the activity of various permeases including transporters for nitrogen sources that stimulate NCR such as the Mep ammonium transport systems. Combining data from growth tests, Northern blot analysis and Gln3 immunolocalization, we show that the Npr1 kinase is not a direct negative regulator of Gln3-dependent transcription. The derepression of Gln3-activated genes in ammonium-grown npr1 cells results from the reduced uptake of the nitrogen-repressing compound because NCR could be restored in npr1 cells by repairing ammonium-uptake defects through different means. Finally, we show that the impairment of the ubiquitin ligase complex does not prevent induction of NCR genes under nonpreferred nitrogen conditions. The apparent Rsp5-, Bul1/2-dependent Gln3 activation keeps to the cellular status, as it is only observed in cells having left the balanced phase of exponential growth.

The proper folding of a long C-terminal segment of the yeast Lys14p regulator is required for activation of LYS genes in response to the metabolic effector.

Transcription of lysine genes in Saccharomyces cerevisiae is dependent on Lys14p and on alpha-aminoadipate semialdehyde (alphaAASA), an intermediate of the pathway. The two-thirds C-terminal end of Lys14p is sufficient to ensure the activation function of the protein and its modulation by alphaAASA. Here, we show that no single discrete domain of Lys14p is able to activate transcription and that most of the deleted LexA-Lys14p proteins are inactive even in the presence of a high alphaAASA concentration. The point mutations abolishing the activation capacity of Lys14p are distributed all over the entire C-terminal segment. Although the deletion of 20 residues rich in leucine and located downstream of the DNA-binding domain converts Lys14p to a constitutive transcriptional activator, our analysis provides evidence that the modulation process of Lys14p activity does not involve an effector-dependent masking/unmasking mechanism. Furthermore, we show that the protein chaperone Hsp82p is required for full activation of LYS genes by the alphaAASA-activated Lys14p as well as by the constitutive Lys14p. Our results suggest that the proper folding of the two-thirds C-terminal portion of Lys14p is essential not only to activate transcription but also to modulate it according to alphaAASA concentration.