Alan Hinnebusch - Academia.edu (original) (raw)

Papers by Alan Hinnebusch

Research paper thumbnail of The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae

Proceedings of The National Academy of Sciences, 2000

The modified nucleoside 1-methyladenosine (m1A) is found at position 58 in the TC loop of many eu... more The modified nucleoside 1-methyladenosine (m1A) is found at position 58 in the TC loop of many eukaryotic tRNAs. The absence of m1A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNAiMet). Gcd10p is found in a complex with Gcd14p, which contains conservedmotifs for binding S-adenosylmethionine (AdoMet). These facts,

Research paper thumbnail of Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex

Molecular Cell, 2015

Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) ... more Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5' end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition.

Research paper thumbnail of Mutations Activating theYeasteIF-2o Kinase GCN2: Isolation ofAlleles Altering theDomainRelated to Histidyl-tRNA Synthetases

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the ... more The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the a subunit of translation initiation factor 2 (eIF-2a) in amino acid-starved cells. Phosphorylation of eIF-2a reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these * Corresponding author. t Present address:

Research paper thumbnail of Homologous Segments in Three Subunits of the Guanine Nucleotide Exchange Factor eIF2B Mediate Translational Regulation by Phosphorylation of eIF2

eIF2B is afive-subunit guanine nucleotide exchange factor that is negatively regulated by phospho... more eIF2B is afive-subunit guanine nucleotide exchange factor that is negatively regulated by phosphorylation of the asubunit of its substrate, eIF2, leading to inhibition of translation initiation. To analyze this regulatory mechanism, we have characterized 29 novel mutations in the homologous eIF2B subunits encoded byGCD2, GCD7, andGCN3that reduce or abolish inhibition of eIF2B activity by eIF2 phosphorylated on its asubunit (eIF2(aP)).

Research paper thumbnail of Interaction of the RNP1 motif in PRT1 with HCR1 promotes 40S binding of eukaryotic initiation factor 3 in yeast

Molecular and cellular biology, 2006

We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation ... more We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation in vivo and reduces 40S-binding of eIF3 to native preinitiation complexes. Several findings indicate that the rnp1 lesion decreases recruitment of eIF3 to the 40S subunit by HCR1: (i) rnp1 strongly impairs the association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (ii) rnp1 impairs the 40S binding of eIF3 more so than the 40S binding of HCR1; (iii) overexpressing HCR1-R215I decreases the Ts(-) phenotype and increases 40S-bound eIF3 in rnp1 cells; (iv) the rnp1 Ts(-) phenotype is exacerbated by tif32-Delta6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1Delta impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also r...

Research paper thumbnail of Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast

Molecular and cellular biology, 2006

Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary com... more Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 d...

Research paper thumbnail of Accumulation of a Threonine Biosynthetic Intermediate Attenuates General Amino Acid Control by Accelerating Degradation of Gcn4 via Pho85 and Cdk8

PLoS Genetics, 2014

Gcn4 is a master transcriptional regulator of amino acid and vitamin biosynthetic enzymes subject... more Gcn4 is a master transcriptional regulator of amino acid and vitamin biosynthetic enzymes subject to the general amino acid control (GAAC), whose expression is upregulated in response to amino acid starvation in Saccharomyces cerevisiae. We found that accumulation of the threonine pathway intermediate β-aspartate semialdehyde (ASA), substrate of homoserine dehydrogenase (Hom6), attenuates the GAAC transcriptional response by accelerating degradation of Gcn4, already an exceedingly unstable protein, in cells starved for isoleucine and valine. The reduction in Gcn4 abundance on ASA accumulation requires Cdk8/Srb10 and Pho85, cyclin-dependent kinases (CDKs) known to mediate rapid turnover of Gcn4 by the proteasome via phosphorylation of the Gcn4 activation domain under nonstarvation conditions. Interestingly, rescue of Gcn4 abundance in hom6 cells by elimination of SRB10 is not accompanied by recovery of transcriptional activation, while equivalent rescue of UAS-bound Gcn4 in hom6 pho85 cells restores greater than wild-type activation of Gcn4 target genes. These and other findings suggest that the two CDKs target different populations of Gcn4 on ASA accumulation, with Srb10 clearing mostly inactive Gcn4 molecules at the promoter that are enriched for sumoylation of the activation domain, and Pho85 clearing molecules unbound to the UAS that include both fully functional and inactive Gcn4 species.

Research paper thumbnail of Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast

Molecular and cellular biology, 2001

Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic ... more Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. In an effort to identify all genes regulated by Gcn4p during amino acid starvation, we performed cDNA microarray analysis. Data from 21 pairs of hybridization experiments using two different strains derived from S288c revealed that more than 1,000 genes were induced, and a similar number were repressed, by a factor of 2 or more in response to histidine starvation imposed by 3-aminotriazole (3AT). Profiling of a gcn4Delta strain and a constitutively induced mutant showed that Gcn4p is required for the full induction by 3AT of at least 539 genes, termed Gcn4p targets. Genes in every amino acid biosynthetic pathway except cysteine and genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, and autophagy proteins were all identified as Gcn4p targets. Unexpectedly, genes involved in amino ac...

Research paper thumbnail of Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain

Molecular and cellular biology, 1998

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by ... more The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized...

Research paper thumbnail of Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5

Molecular and cellular biology, 1998

Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have re... more Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of approximately 600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1 mutant ...

Research paper thumbnail of The Gcn4p activation domain interacts specifically in vitro with RNA polymerase II holoenzyme, TFIID, and the Adap-Gcn5p coactivator complex

Molecular and cellular biology, 1998

The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive co... more The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione S-transferase (GST)-Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive ef...

Research paper thumbnail of The yeast TAF145 inhibitory domain and TFIIA competitively bind to TATA-binding protein

Molecular and cellular biology, 1998

The Drosophila 230-kDa TFIID subunit (dTAF230) interacts with the DNA binding domain of TATA box-... more The Drosophila 230-kDa TFIID subunit (dTAF230) interacts with the DNA binding domain of TATA box-binding protein (TBP) which exists in the same complex. Here, we characterize the inhibitory domain in the yeast TAF145 (yTAF145), which is homologous to dTAF230. Mutation studies show that the N-terminal inhibitory region (residues 10 to 71) can be divided into two subdomains, I (residues 10 to 37) and II (residues 46 to 71). Mutations in either subdomain significantly impair function. Acidic residues in subdomain II are important for the interaction with TBP. In addition, yTAF145 interaction is impaired by mutating the basic residues on the convex surface of TBP, which are crucial for interaction with TFIIA. Consistently, TFIIA and yTAF145 bind competitively to TBP. A deletion of the inhibitory domain of yTAF145 leads to a temperature-sensitive growth phenotype. Importantly, this phenotype is suppressed by overexpression of the TFIIA subunits, indicating that the yTAF145 inhibitory dom...

Research paper thumbnail of Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

PLOS Genetics, 2015

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of transl... more The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.

Research paper thumbnail of The novel ATP-binding cassette protein ARB1 is a shuttling factor that stimulates 40S and 60S ribosome biogenesis

Molecular and cellular biology, 2005

ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding ca... more ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were leth...

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

Molecular Cell, 2000

open reading frames (uORFs) in the GCN4 mRNA leader underlie a specialized reinitiation mechanism... more open reading frames (uORFs) in the GCN4 mRNA leader underlie a specialized reinitiation mechanism that elevates GCN4 translation in response to small decreases in ternary complex levels insufficient to impair general translation. Thus, eIF2␣ phosphorylation in amino acidand Human Development starved yeast cells induces specifically a transcriptional Bethesda, Maryland 20892 activator that can rectify amino acid limitation. Dominant activating alleles of GCN2 have been isolated that derepress GCN4 under nonstarvation conditions, and the Summary most severe of these GCN2 c mutations inhibit general translation and cell growth due to high levels of eIF2␣ Protein kinase GCN2 regulates translation in amino phosphorylation . acid-starved cells by phosphorylating eIF2. GCN2 con-It is thought that GCN2 is activated by uncharged tains a regulatory domain related to histidyl-tRNA syn-tRNAs that accumulate in amino acid-starved cells thetase (HisRS) postulated to bind multiple deacylated partly because mutations in aminoacyl tRNA synthetases (aaRSs) lead to GCN2-dependent derepression of tRNAs as a general sensor of starvation. In accordance GCN4 without any starvation for amino acids (Hinnewith this model, GCN2 bound several deacylated busch, 1996). Additionally, GCN2 contains a region ho-tRNAs with similar affinities, and aminoacylation of mologous to histidyl-tRNA synthetase HisRS) located tRNA Phe weakened its interaction with GCN2. Unex-C-terminal to the kinase domain (Wek et al., 1989) (Figure pectedly, the C-terminal ribosome binding segment of 1A). Mutations in the GCN2 HisRS-like domain in con-GCN2 (C-term) was required in addition to the HisRS served residues required for tRNA binding by authentic domain for strong tRNA binding. A combined HisRSϩ class II aaRSs (the m2 motif) inactivate GCN2 function C-term segment bound to the isolated protein kinase in vivo. These mutations also impair GCN2 kinase activ-(PK) domain in vitro, and tRNA impeded this interacity in cell extracts and destroy binding of tRNA to the tion. An activating mutation (GCN2 c -E803V) that weakisolated HisRS domain (Wek et al., 1995; Zhu et al., ens PK-C-term association greatly enhanced tRNA 1996). As GCN4 translation is derepressed by starvation binding by GCN2. These results provide strong evifor any of several amino acids (Wek et al., 1995; Hinnedence that tRNA stimulates the GCN2 kinase moiety by busch, 1996), the HisRS domain of GCN2 presumably preventing an inhibitory interaction with the bipartite cannot distinguish between different tRNA species and tRNA binding domain.

Research paper thumbnail of Gcn5 Promotes Acetylation, Eviction, and Methylation of Nucleosomes in Transcribed Coding Regions

Molecular Cell, 2007

We report that coactivator SAGA, containing the HAT Gcn5p, occupies the GAL1 and ARG1 coding sequ... more We report that coactivator SAGA, containing the HAT Gcn5p, occupies the GAL1 and ARG1 coding sequences during transcriptional induction, dependent on PIC assembly and Ser5 phosphorylation of the Pol II CTD. Induction of GAL1 increases H3 acetylation per nucleosome in the ORF, dependent on SAGA integrity but not the alternative Gcn5p-HAT complex ADA. Unexpectedly, H3 acetylation in ARG1 coding sequences does not increase during induction due to the opposing activities of multiple HDAs associated with the ORF. Remarkably, inactivation of Gcn5p decreases nucleosome eviction from both GAL1 and a long ($8 kb) ORF transcribed from the GAL1 promoter. This is associated with reduced Pol II occupancy at the 3 0 end and decreased mRNA production, selectively, for the long ORF. Gcn5p also enhances H3-K4 trimethylation in the ARG1 ORF and bulk histones. Thus, Gcn5p, most likely in SAGA, stimulates modification and eviction of nucleosomes in transcribed coding sequences and promotes Pol II elongation.

Research paper thumbnail of NuA4 links methylation of histone H3 lysines 4 and 36 to acetylation of histones H4 and H3

The Journal of biological chemistry, Jan 21, 2014

Cotranscriptional methylation of histone H3 lysines 4 and 36 by Set1 and Set2, respectively, stim... more Cotranscriptional methylation of histone H3 lysines 4 and 36 by Set1 and Set2, respectively, stimulates interaction between nucleosomes and histone deacetylase complexes to block cryptic transcription in budding yeast. We previously showed that loss of all H3K4 and H3K36 methylation in a set1Δset2Δ mutant reduces interaction between native nucleosomes and the NuA4 lysine acetyltransferase (KAT) complex. We now provide evidence that NuA4 preferentially binds H3 tails mono- and dimethylated on H3K4 and di- and trimethylated on H3K36, an H3 methylation pattern distinct from that recognized by the RPD3C(S) and Hos2/Set3 histone deacetylase complexes (HDACs). Loss of H3K4 or H3K36 methylation in set1Δ or set2Δ mutants reduces NuA4 interaction with bulk nucleosomes in vitro and in vivo, and reduces NuA4 occupancy of transcribed coding sequences at particular genes. We also provide evidence that NuA4 acetylation of lysine residues in the histone H4 tail stimulates SAGA interaction with nuc...

Research paper thumbnail of Structural changes enable start codon recognition by the eukaryotic translation initiation complex

Cell, Jan 23, 2014

During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decodin... more During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

Research paper thumbnail of Disrupting vesicular trafficking at the endosome attenuates transcriptional activation by Gcn4

Molecular and cellular biology, 2008

The late endosome (MVB) plays a key role in coordinating vesicular transport of proteins between ... more The late endosome (MVB) plays a key role in coordinating vesicular transport of proteins between the Golgi complex, vacuole/lysosome, and plasma membrane. We found that deleting multiple genes involved in vesicle fusion at the MVB (class C/D vps mutations) impairs transcriptional activation by Gcn4, a global regulator of amino acid biosynthetic genes, by decreasing the ability of chromatin-bound Gcn4 to stimulate preinitiation complex assembly at the promoter. The functions of hybrid activators with Gal4 or VP16 activation domains are diminished in class D mutants as well, suggesting a broader defect in activation. Class E vps mutations, which impair protein sorting at the MVB, also decrease activation by Gcn4, provided they elicit rapid proteolysis of MVB cargo proteins in the aberrant late endosome. By contrast, specifically impairing endocytic trafficking from the plasma membrane, or vesicular transport to the vacuole, has a smaller effect on Gcn4 function. Thus, it appears that ...

Research paper thumbnail of Analysis and reconstitution of translation initiation in vitro

Methods in Enzymology, 2002

... of Translation Initiation in Vitro By KATSURA ASANO, LON PHAN, THANUJA KRISHNAMOORTHY, GRAHAM... more ... of Translation Initiation in Vitro By KATSURA ASANO, LON PHAN, THANUJA KRISHNAMOORTHY, GRAHAM D. PAVITT, EDITH GOMEZ, ERNEST M ... Yeast strain LPY201 [MATa leu2-3, -112 prtl : :kanMX pLPYlO1 (PRT1-His URA3)] is employed, in which chromosomal PRT1 is ...

Research paper thumbnail of The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae

Proceedings of The National Academy of Sciences, 2000

The modified nucleoside 1-methyladenosine (m1A) is found at position 58 in the TC loop of many eu... more The modified nucleoside 1-methyladenosine (m1A) is found at position 58 in the TC loop of many eukaryotic tRNAs. The absence of m1A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNAiMet). Gcd10p is found in a complex with Gcd14p, which contains conservedmotifs for binding S-adenosylmethionine (AdoMet). These facts,

Research paper thumbnail of Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex

Molecular Cell, 2015

Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) ... more Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5' end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition.

Research paper thumbnail of Mutations Activating theYeasteIF-2o Kinase GCN2: Isolation ofAlleles Altering theDomainRelated to Histidyl-tRNA Synthetases

The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the ... more The protein kinase GCN2 stimulates expression of the yeast transcriptional activator GCN4 at the translational level by phosphorylating the a subunit of translation initiation factor 2 (eIF-2a) in amino acid-starved cells. Phosphorylation of eIF-2a reduces its activity, allowing ribosomes to bypass short open reading frames present in the GCN4 mRNA leader and initiate translation at the GCN4 start codon. We describe here 17 dominant GCN2 mutations that lead to derepression of GCN4 expression in the absence of amino acid starvation. Seven of these * Corresponding author. t Present address:

Research paper thumbnail of Homologous Segments in Three Subunits of the Guanine Nucleotide Exchange Factor eIF2B Mediate Translational Regulation by Phosphorylation of eIF2

eIF2B is afive-subunit guanine nucleotide exchange factor that is negatively regulated by phospho... more eIF2B is afive-subunit guanine nucleotide exchange factor that is negatively regulated by phosphorylation of the asubunit of its substrate, eIF2, leading to inhibition of translation initiation. To analyze this regulatory mechanism, we have characterized 29 novel mutations in the homologous eIF2B subunits encoded byGCD2, GCD7, andGCN3that reduce or abolish inhibition of eIF2B activity by eIF2 phosphorylated on its asubunit (eIF2(aP)).

Research paper thumbnail of Interaction of the RNP1 motif in PRT1 with HCR1 promotes 40S binding of eukaryotic initiation factor 3 in yeast

Molecular and cellular biology, 2006

We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation ... more We found that mutating the RNP1 motif in the predicted RRM domain in yeast eukaryotic initiation factor 3 (eIF3) subunit b/PRT1 (prt1-rnp1) impairs its direct interactions in vitro with both eIF3a/TIF32 and eIF3j/HCR1. The rnp1 mutation in PRT1 confers temperature-sensitive translation initiation in vivo and reduces 40S-binding of eIF3 to native preinitiation complexes. Several findings indicate that the rnp1 lesion decreases recruitment of eIF3 to the 40S subunit by HCR1: (i) rnp1 strongly impairs the association of HCR1 with PRT1 without substantially disrupting the eIF3 complex; (ii) rnp1 impairs the 40S binding of eIF3 more so than the 40S binding of HCR1; (iii) overexpressing HCR1-R215I decreases the Ts(-) phenotype and increases 40S-bound eIF3 in rnp1 cells; (iv) the rnp1 Ts(-) phenotype is exacerbated by tif32-Delta6, which eliminates a binding determinant for HCR1 in TIF32; and (v) hcr1Delta impairs 40S binding of eIF3 in otherwise wild-type cells. Interestingly, rnp1 also r...

Research paper thumbnail of Eukaryotic translation initiation factor 3 (eIF3) and eIF2 can promote mRNA binding to 40S subunits independently of eIF4G in yeast

Molecular and cellular biology, 2006

Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary com... more Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNAiMet ternary complex to the 40S ribosome is stimulated by multiple initiation factors in vitro, including eIF3, eIF1, eIF5, and eIF1A. Recruitment of mRNA is thought to require the functions of eIF4F and eIF3, with the latter serving as an adaptor between the ribosome and the 4G subunit of eIF4F. To define the factor requirements for these reactions in vivo, we examined the effects of depleting eIF2, eIF3, eIF5, or eIF4G in Saccharomyces cerevisiae cells on binding of the ternary complex, other initiation factors, and RPL41A mRNA to native 43S and 48S preinitiation complexes. Depleting eIF2, eIF3, or eIF5 reduced 40S binding of all constituents of the multifactor complex (MFC), comprised of these three factors and eIF1, supporting a mechanism of coupled 40S binding by MFC components. 40S-bound mRNA strongly accumulated in eIF5-depleted cells, even though MFC binding to 40S subunits was reduced by eIF5 d...

Research paper thumbnail of Accumulation of a Threonine Biosynthetic Intermediate Attenuates General Amino Acid Control by Accelerating Degradation of Gcn4 via Pho85 and Cdk8

PLoS Genetics, 2014

Gcn4 is a master transcriptional regulator of amino acid and vitamin biosynthetic enzymes subject... more Gcn4 is a master transcriptional regulator of amino acid and vitamin biosynthetic enzymes subject to the general amino acid control (GAAC), whose expression is upregulated in response to amino acid starvation in Saccharomyces cerevisiae. We found that accumulation of the threonine pathway intermediate β-aspartate semialdehyde (ASA), substrate of homoserine dehydrogenase (Hom6), attenuates the GAAC transcriptional response by accelerating degradation of Gcn4, already an exceedingly unstable protein, in cells starved for isoleucine and valine. The reduction in Gcn4 abundance on ASA accumulation requires Cdk8/Srb10 and Pho85, cyclin-dependent kinases (CDKs) known to mediate rapid turnover of Gcn4 by the proteasome via phosphorylation of the Gcn4 activation domain under nonstarvation conditions. Interestingly, rescue of Gcn4 abundance in hom6 cells by elimination of SRB10 is not accompanied by recovery of transcriptional activation, while equivalent rescue of UAS-bound Gcn4 in hom6 pho85 cells restores greater than wild-type activation of Gcn4 target genes. These and other findings suggest that the two CDKs target different populations of Gcn4 on ASA accumulation, with Srb10 clearing mostly inactive Gcn4 molecules at the promoter that are enriched for sumoylation of the activation domain, and Pho85 clearing molecules unbound to the UAS that include both fully functional and inactive Gcn4 species.

Research paper thumbnail of Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast

Molecular and cellular biology, 2001

Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic ... more Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. In an effort to identify all genes regulated by Gcn4p during amino acid starvation, we performed cDNA microarray analysis. Data from 21 pairs of hybridization experiments using two different strains derived from S288c revealed that more than 1,000 genes were induced, and a similar number were repressed, by a factor of 2 or more in response to histidine starvation imposed by 3-aminotriazole (3AT). Profiling of a gcn4Delta strain and a constitutively induced mutant showed that Gcn4p is required for the full induction by 3AT of at least 539 genes, termed Gcn4p targets. Genes in every amino acid biosynthetic pathway except cysteine and genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, and autophagy proteins were all identified as Gcn4p targets. Unexpectedly, genes involved in amino ac...

Research paper thumbnail of Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain

Molecular and cellular biology, 1998

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by ... more The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized...

Research paper thumbnail of Identification of a translation initiation factor 3 (eIF3) core complex, conserved in yeast and mammals, that interacts with eIF5

Molecular and cellular biology, 1998

Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have re... more Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of approximately 600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1 mutant ...

Research paper thumbnail of The Gcn4p activation domain interacts specifically in vitro with RNA polymerase II holoenzyme, TFIID, and the Adap-Gcn5p coactivator complex

Molecular and cellular biology, 1998

The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive co... more The Gcn4p activation domain contains seven clusters of hydrophobic residues that make additive contributions to transcriptional activation in vivo. We observed efficient binding of a glutathione S-transferase (GST)-Gcn4p fusion protein to components of three different coactivator complexes in Saccharomyces cerevisiae cell extracts, including subunits of transcription factor IID (TFIID) (yeast TAFII20 [yTAFII20], yTAFII60, and yTAFII90), the holoenzyme mediator (Srb2p, Srb4p, and Srb7p), and the Adap-Gcn5p complex (Ada2p and Ada3p). The binding to these coactivator subunits was completely dependent on the hydrophobic clusters in the Gcn4p activation domain. Alanine substitutions in single clusters led to moderate reductions in binding, double-cluster substitutions generally led to greater reductions in binding than the corresponding single-cluster mutations, and mutations in four or more clusters reduced binding to all of the coactivator proteins to background levels. The additive ef...

Research paper thumbnail of The yeast TAF145 inhibitory domain and TFIIA competitively bind to TATA-binding protein

Molecular and cellular biology, 1998

The Drosophila 230-kDa TFIID subunit (dTAF230) interacts with the DNA binding domain of TATA box-... more The Drosophila 230-kDa TFIID subunit (dTAF230) interacts with the DNA binding domain of TATA box-binding protein (TBP) which exists in the same complex. Here, we characterize the inhibitory domain in the yeast TAF145 (yTAF145), which is homologous to dTAF230. Mutation studies show that the N-terminal inhibitory region (residues 10 to 71) can be divided into two subdomains, I (residues 10 to 37) and II (residues 46 to 71). Mutations in either subdomain significantly impair function. Acidic residues in subdomain II are important for the interaction with TBP. In addition, yTAF145 interaction is impaired by mutating the basic residues on the convex surface of TBP, which are crucial for interaction with TFIIA. Consistently, TFIIA and yTAF145 bind competitively to TBP. A deletion of the inhibitory domain of yTAF145 leads to a temperature-sensitive growth phenotype. Importantly, this phenotype is suppressed by overexpression of the TFIIA subunits, indicating that the yTAF145 inhibitory dom...

Research paper thumbnail of Interaction between the tRNA-Binding and C-Terminal Domains of Yeast Gcn2 Regulates Kinase Activity In Vivo

PLOS Genetics, 2015

The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of transl... more The stress-activated protein kinase Gcn2 regulates protein synthesis by phosphorylation of translation initiation factor eIF2α. Gcn2 is activated in amino acid-deprived cells by binding of uncharged tRNA to the regulatory domain related to histidyl-tRNA synthetase, but the molecular mechanism of activation is unclear. We used a genetic approach to identify a key regulatory surface in Gcn2 that is proximal to the predicted active site of the HisRS domain and likely remodeled by tRNA binding. Mutations leading to amino acid substitutions on this surface were identified that activate Gcn2 at low levels of tRNA binding (Gcd- phenotype), while other substitutions block kinase activation (Gcn- phenotype), in some cases without altering tRNA binding by Gcn2 in vitro. Remarkably, the Gcn- substitutions increase affinity of the HisRS domain for the C-terminal domain (CTD), previously implicated as a kinase autoinhibitory segment, in a manner dampened by HisRS domain Gcd- substitutions and by amino acid starvation in vivo. Moreover, tRNA specifically antagonizes HisRS/CTD association in vitro. These findings support a model wherein HisRS-CTD interaction facilitates the autoinhibitory function of the CTD in nonstarvation conditions, with tRNA binding eliciting kinase activation by weakening HisRS-CTD association with attendant disruption of the autoinhibitory KD-CTD interaction.

Research paper thumbnail of The novel ATP-binding cassette protein ARB1 is a shuttling factor that stimulates 40S and 60S ribosome biogenesis

Molecular and cellular biology, 2005

ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding ca... more ARB1 is an essential yeast protein closely related to members of a subclass of the ATP-binding cassette (ABC) superfamily of proteins that are known to interact with ribosomes and function in protein synthesis or ribosome biogenesis. We show that depletion of ARB1 from Saccharomyces cerevisiae cells leads to a deficit in 18S rRNA and 40S subunits that can be attributed to slower cleavage at the A0, A1, and A2 processing sites in 35S pre-rRNA, delayed processing of 20S rRNA to mature 18S rRNA, and a possible defect in nuclear export of pre-40S subunits. Depletion of ARB1 also delays rRNA processing events in the 60S biogenesis pathway. We further demonstrate that ARB1 shuttles from nucleus to cytoplasm, cosediments with 40S, 60S, and 80S/90S ribosomal species, and is physically associated in vivo with TIF6, LSG1, and other proteins implicated previously in different aspects of 60S or 40S biogenesis. Mutations of conserved ARB1 residues expected to function in ATP hydrolysis were leth...

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

Molecular Cell, 2000

open reading frames (uORFs) in the GCN4 mRNA leader underlie a specialized reinitiation mechanism... more open reading frames (uORFs) in the GCN4 mRNA leader underlie a specialized reinitiation mechanism that elevates GCN4 translation in response to small decreases in ternary complex levels insufficient to impair general translation. Thus, eIF2␣ phosphorylation in amino acidand Human Development starved yeast cells induces specifically a transcriptional Bethesda, Maryland 20892 activator that can rectify amino acid limitation. Dominant activating alleles of GCN2 have been isolated that derepress GCN4 under nonstarvation conditions, and the Summary most severe of these GCN2 c mutations inhibit general translation and cell growth due to high levels of eIF2␣ Protein kinase GCN2 regulates translation in amino phosphorylation . acid-starved cells by phosphorylating eIF2. GCN2 con-It is thought that GCN2 is activated by uncharged tains a regulatory domain related to histidyl-tRNA syn-tRNAs that accumulate in amino acid-starved cells thetase (HisRS) postulated to bind multiple deacylated partly because mutations in aminoacyl tRNA synthetases (aaRSs) lead to GCN2-dependent derepression of tRNAs as a general sensor of starvation. In accordance GCN4 without any starvation for amino acids (Hinnewith this model, GCN2 bound several deacylated busch, 1996). Additionally, GCN2 contains a region ho-tRNAs with similar affinities, and aminoacylation of mologous to histidyl-tRNA synthetase HisRS) located tRNA Phe weakened its interaction with GCN2. Unex-C-terminal to the kinase domain (Wek et al., 1989) (Figure pectedly, the C-terminal ribosome binding segment of 1A). Mutations in the GCN2 HisRS-like domain in con-GCN2 (C-term) was required in addition to the HisRS served residues required for tRNA binding by authentic domain for strong tRNA binding. A combined HisRSϩ class II aaRSs (the m2 motif) inactivate GCN2 function C-term segment bound to the isolated protein kinase in vivo. These mutations also impair GCN2 kinase activ-(PK) domain in vitro, and tRNA impeded this interacity in cell extracts and destroy binding of tRNA to the tion. An activating mutation (GCN2 c -E803V) that weakisolated HisRS domain (Wek et al., 1995; Zhu et al., ens PK-C-term association greatly enhanced tRNA 1996). As GCN4 translation is derepressed by starvation binding by GCN2. These results provide strong evifor any of several amino acids (Wek et al., 1995; Hinnedence that tRNA stimulates the GCN2 kinase moiety by busch, 1996), the HisRS domain of GCN2 presumably preventing an inhibitory interaction with the bipartite cannot distinguish between different tRNA species and tRNA binding domain.

Research paper thumbnail of Gcn5 Promotes Acetylation, Eviction, and Methylation of Nucleosomes in Transcribed Coding Regions

Molecular Cell, 2007

We report that coactivator SAGA, containing the HAT Gcn5p, occupies the GAL1 and ARG1 coding sequ... more We report that coactivator SAGA, containing the HAT Gcn5p, occupies the GAL1 and ARG1 coding sequences during transcriptional induction, dependent on PIC assembly and Ser5 phosphorylation of the Pol II CTD. Induction of GAL1 increases H3 acetylation per nucleosome in the ORF, dependent on SAGA integrity but not the alternative Gcn5p-HAT complex ADA. Unexpectedly, H3 acetylation in ARG1 coding sequences does not increase during induction due to the opposing activities of multiple HDAs associated with the ORF. Remarkably, inactivation of Gcn5p decreases nucleosome eviction from both GAL1 and a long ($8 kb) ORF transcribed from the GAL1 promoter. This is associated with reduced Pol II occupancy at the 3 0 end and decreased mRNA production, selectively, for the long ORF. Gcn5p also enhances H3-K4 trimethylation in the ARG1 ORF and bulk histones. Thus, Gcn5p, most likely in SAGA, stimulates modification and eviction of nucleosomes in transcribed coding sequences and promotes Pol II elongation.

Research paper thumbnail of NuA4 links methylation of histone H3 lysines 4 and 36 to acetylation of histones H4 and H3

The Journal of biological chemistry, Jan 21, 2014

Cotranscriptional methylation of histone H3 lysines 4 and 36 by Set1 and Set2, respectively, stim... more Cotranscriptional methylation of histone H3 lysines 4 and 36 by Set1 and Set2, respectively, stimulates interaction between nucleosomes and histone deacetylase complexes to block cryptic transcription in budding yeast. We previously showed that loss of all H3K4 and H3K36 methylation in a set1Δset2Δ mutant reduces interaction between native nucleosomes and the NuA4 lysine acetyltransferase (KAT) complex. We now provide evidence that NuA4 preferentially binds H3 tails mono- and dimethylated on H3K4 and di- and trimethylated on H3K36, an H3 methylation pattern distinct from that recognized by the RPD3C(S) and Hos2/Set3 histone deacetylase complexes (HDACs). Loss of H3K4 or H3K36 methylation in set1Δ or set2Δ mutants reduces NuA4 interaction with bulk nucleosomes in vitro and in vivo, and reduces NuA4 occupancy of transcribed coding sequences at particular genes. We also provide evidence that NuA4 acetylation of lysine residues in the histone H4 tail stimulates SAGA interaction with nuc...

Research paper thumbnail of Structural changes enable start codon recognition by the eukaryotic translation initiation complex

Cell, Jan 23, 2014

During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decodin... more During eukaryotic translation initiation, initiator tRNA does not insert fully into the P decoding site on the 40S ribosomal subunit. This conformation (POUT) is compatible with scanning mRNA for the AUG start codon. Base pairing with AUG is thought to promote isomerization to a more stable conformation (PIN) that arrests scanning and promotes dissociation of eIF1 from the 40S subunit. Here, we present a cryoEM reconstruction of a yeast preinitiation complex at 4.0 Å resolution with initiator tRNA in the PIN state, prior to eIF1 release. The structure reveals stabilization of the codon-anticodon duplex by the N-terminal tail of eIF1A, changes in the structure of eIF1 likely instrumental in its subsequent release, and changes in the conformation of eIF2. The mRNA traverses the entire mRNA cleft and makes connections to the regulatory domain of eIF2?, eIF1A, and ribosomal elements that allow recognition of context nucleotides surrounding the AUG codon.

Research paper thumbnail of Disrupting vesicular trafficking at the endosome attenuates transcriptional activation by Gcn4

Molecular and cellular biology, 2008

The late endosome (MVB) plays a key role in coordinating vesicular transport of proteins between ... more The late endosome (MVB) plays a key role in coordinating vesicular transport of proteins between the Golgi complex, vacuole/lysosome, and plasma membrane. We found that deleting multiple genes involved in vesicle fusion at the MVB (class C/D vps mutations) impairs transcriptional activation by Gcn4, a global regulator of amino acid biosynthetic genes, by decreasing the ability of chromatin-bound Gcn4 to stimulate preinitiation complex assembly at the promoter. The functions of hybrid activators with Gal4 or VP16 activation domains are diminished in class D mutants as well, suggesting a broader defect in activation. Class E vps mutations, which impair protein sorting at the MVB, also decrease activation by Gcn4, provided they elicit rapid proteolysis of MVB cargo proteins in the aberrant late endosome. By contrast, specifically impairing endocytic trafficking from the plasma membrane, or vesicular transport to the vacuole, has a smaller effect on Gcn4 function. Thus, it appears that ...

Research paper thumbnail of Analysis and reconstitution of translation initiation in vitro

Methods in Enzymology, 2002

... of Translation Initiation in Vitro By KATSURA ASANO, LON PHAN, THANUJA KRISHNAMOORTHY, GRAHAM... more ... of Translation Initiation in Vitro By KATSURA ASANO, LON PHAN, THANUJA KRISHNAMOORTHY, GRAHAM D. PAVITT, EDITH GOMEZ, ERNEST M ... Yeast strain LPY201 [MATa leu2-3, -112 prtl : :kanMX pLPYlO1 (PRT1-His URA3)] is employed, in which chromosomal PRT1 is ...