Uncharged tRNA Activates GCN2 by Displacing the Protein Kinase Moiety from a Bipartite tRNA-Binding Domain

Protein kinase GCN2 regulates translation in amino acid–starved cells by phosphorylating eIF2. GCN2 contains a regulatory domain related to histidyl-tRNA synthetase (HisRS) postulated to bind multiple deacylated tRNAs as a general sensor of starvation. In accordance with this model, GCN2 bound several deacylated tRNAs with similar affinities, and aminoacylation of tRNAPhe weakened its interaction with GCN2. Unexpectedly, the C-terminal ribosome binding segment of GCN2 (C-term) was required in addition to the HisRS domain for strong tRNA binding. A combined HisRS+C-term segment bound to the isolated protein kinase (PK) domain in vitro, and tRNA impeded this interaction. An activating mutation (GCN2c-E803V) that weakens PK–C-term association greatly enhanced tRNA binding by GCN2. These results provide strong evidence that tRNA stimulates the GCN2 kinase moiety by preventing an inhibitory interaction with the bipartite tRNA binding domain

cpc-3, the Neurospora crassa Homologue of Yeast GCN2, Encodes a Polypeptide with Juxtaposed eIF2α Kinase and Histidyl-tRNA Synthetase-related Domains Required for General Amino Acid Control

Based on characteristic amino acid sequences of kinases that phosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF2α kinases), degenerate oligonucleotide primers were constructed and used to polymerase chain reaction-amplify from genomic DNA of Neurospora crassa a sequence encoding part of a putative protein kinase. With this sequence an open reading frame was identified encoding a predicted polypeptide with juxtaposed eIF2α kinase and histidyl-tRNA synthetase- related domains. The 1646 amino acid sequence of this gene, called cpc-3, showed 35% positional identity over almost the entire sequence with GCN2 of yeast, which stimulates translation of the transcriptional activator of amino acid biosynthetic genes encoded by GCN4. Strains disrupted for cpc-3 were unable to induce increased transcription and derepression of amino acid biosynthetic enzymes in amino acid-deprived cells. The cpc-3 mutation did not affect the ability to up-regulate mRNA levels of cpc-1, encoding the GCN4 homologue and transcriptional activator of amino acid biosynthetic genes in N. crassa, but the mutation abolished the dramatic increase of CPC1 protein level in response to amino acid deprivation. These findings suggest that cpc- 3 is the functional homologue of GCN2, being required for increased translation of cpc-1 mRNA in amino acid-starved cells

Nuclear Surveillance and Degradation of Hypomodified Initiator tRNA\u3csup\u3eMet\u3c/sup\u3e in \u3cem\u3eS. cerevisiae\u3c/em\u3e

The tRNA m1A58 methyltransferase is composed of two subunits encoded by the essential genes TRM6 and TRM61 (formerly GCD10 and GCD14). The trm6-504 mutation results in a defective m1A methyltransferase (Mtase) and a temperature-sensitive growth phenotype that is attributable to the absence of m1A58 and consequential tRNAiMet instability. We used a genetic approach to identify the genes responsible for tRNAiMet degradation in trm6 cells. Three recessive extragenic mutations that suppress trm6-504 mutant phenotypes and restore hypomodified tRNAiMet to near normal levels were identified. The wild-type allele of one suppressor, DIS3/RRP44, encodes a 3′-5′ exoribonuclease and a member of the multisubunit exosome complex. We provide evidence that a functional nuclear exosome is required for the degradation of tRNAiMet lacking m1A58. A second suppressor gene encodes Trf4p, a DNA polymerase (pol σ) with poly(A) polymerase activity. Whereas deletion of TRF4 leads to stabilization of tRNAiMet, overexpression of Trf4p destabilizes the hypomodified tRNAiMet in trm6 cells. The hypomodified, but not wild-type, pre-tRNAiMet accumulates as a polyadenylated species, whose abundance and length distribution both increase upon Trf4p overexpression. These data indicate that a tRNA surveillance pathway exists in yeast that requires Trf4p and the exosome for polyadenylation and degradation of hypomodified pre-tRNAiMet

The β-hairpin of 40S exit channel protein Rps5/uS7 promotes efficient and accurate translation initiation in vivo

Abstract The eukaryotic 43S pre-initiation complex bearing tRNA i Met scans the mRNA leader for an AUG start codon in favorable context. Structural analyses revealed that the β-hairpin of 40S protein Rps5/uS7 protrudes into the 40S mRNA exit-channel, contacting the eIF2•GTP•Met-tRNA i ternary complex (TC) and mRNA context nucleotides; but its importance in AUG selection was unknown. We identified substitutions in β-strand-1 and C-terminal residues of yeast Rps5 that reduced bulk initiation, conferred 'leaky-scanning' of AUGs; and lowered initiation fidelity by exacerbating the effect of poor context of the eIF1 AUG codon to reduce eIF1 abundance. Consistently, the β-strand-1 substitution greatly destabilized the 'P IN ' conformation of TC binding to reconstituted 43S·mRNA complexes in vitro. Other substitutions in β-hairpin loop residues increased initiation fidelity and destabilized P IN at UUG, but not AUG start codons. We conclude that the Rps5 β-hairpin is as crucial as soluble initiation factors for efficient and accurate start codon recognition

The C-terminal domain of eukaryotic initiation factor 5 promotes start codon recognition by its dynamic interplay with eIF1 and eIF2 beta

Recognition of the proper start codon on mRNAs is essential for protein synthesis, which requires scanning and involves eukaryotic initiation factors (eIFs) eIF1, eIF1A, eIF2, and eIF5. The carboxyl terminal domain (CTD) of eIF5 stimulates 43S preinitiation complex (PIC) assembly; however, its precise role in scanning and start codon selection has remained unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we identified the binding sites of eIF1 and eIF2β on eIF5-CTD and found that they partially overlapped. Mutating select eIF5 residues in the common interface specifically disrupts interaction with both factors. Genetic and biochemical evidence indicates that these eIF5-CTD mutations impair start codon recognition and impede eIF1 release from the PIC by abrogating eIF5-CTD binding to eIF2β. This study provides mechanistic insight into the role of eIF5-CTD's dynamic interplay with eIF1 and eIF2β in switching PICs from an open to a closed state at start codons.publishedVersio

Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p

The heterogeneous nuclear ribonucleoprotein Npl3p of budding yeast is a substrate of arginine methyltransferase Hmt1p, but the role of Hmt1p in regulating Npl3p’s functions in transcription antitermination and elongation were unknown. We found that mutants lacking Hmt1p methyltransferase activity exhibit reduced recruitment of Npl3p, but elevated recruitment of a component of mRNA cleavage/termination factor CFI, to the activated GAL10-GAL7 locus. Consistent with this, hmt1 mutants displayed increased termination at the defective gal10-Δ56 terminator. Remarkably, hmt1Δ cells also exhibit diminished recruitment of elongation factor Tho2p and a reduced rate of transcription elongation in vivo. Importantly, the defects in Npl3p and Tho2p recruitment, antitermination and elongation in hmt1Δ cells all were mitigated by substitutions in Npl3p RGG repeats that functionally mimic arginine methylation by Hmt1p. Thus, Hmt1p promotes elongation and suppresses termination at cryptic terminators by methylating RGG repeats in Npl3p. As Hmt1p stimulates dissociation of Tho2p from an Npl3p-mRNP complex, it could act to recycle these elongation and antitermination factors back to sites of ongoing transcription

Active destruction of defective ribosomes by a ubiquitin ligase involved in DNA repair

Progression of DNA replication forks through damaged DNA requires a ubiquitin ligase comprised of the cullin Rtt101, the RING finger protein Hrt1, and the adaptor protein Mms1. Rtt101 and Mms1 were implicated recently by Fujii and colleagues (pp. 963–974) in the degradation of catalytically inactive mutant 25S ribosomal RNAS (rRNAs) in mature 60S ribosomal subunits, a process that requires ubiquitin and is accompanied by ubiquitination of 60S components. It now seems likely that the same ubiquitin ligase is enlisted to deal with defective rRNA and damaged DNA