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Eunice Kennedy Shriver National Institute of Child Health and Human Development - Program in Cellular Regulation and Metabolism
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Alan Hinnebusch

We study the regulation of amino acid and vitamin biosynthetic genes in budding yeast as a means of dissecting mechanisms of translational and transcriptional control of gene expression. Transcription of these genes is coordinately induced by transcriptional activator Gcn4 in response to amino acid limitation. Gcn4 expression is coupled to amino acid levels through a translational control mechanism involving four short upstream open reading frames (uORFs) in GCN4 mRNA, which repress Gcn4 synthesis in nutrient-replete cells. Paradoxically, GCN4 translation is derepressed in starvation or stress conditions where general protein synthesis is reduced by decreased assembly of the eIF2-GTP-Met-tRNAi ternary complex (TC), which delivers Met-tRNAi to the small (40S) ribosomal subunit for assembly of the 43S preinitiation complex (PIC). TC assembly is reduced by phosphorylation of the alpha subunit of eIF2 (eIF2a-P) by the protein kinase Gcn2, conserved in all eukaryotes, converting eIF2 from substrate to inhibitor of its guanine nucleotide exchange factor (GEF), eIF2B. Hence, GCN4 translation is a sensitive in vivo reporter of impaired TC loading on 40S subunits, a pivotal step of the initiation pathway. We previously exploited this feature to dissect the functions of eIF2B subunits in GDP-GTP exchange, and in mediating the inhibitory effect of eIFa-P on GEF activity. We also implicated particular domains/residues of factors, eIF1, -1A, and 3, and also residues of 18S rRNA located near the "P" decoding site of the 40S subunit, in stimulating TC loading on 40S subunits in vivo.

Recently, we discovered a 10-amino acid repeat in the C-terminal tail of eIF1A that is critical for both TC recruitment and accurate initiation at AUG start codons. Mutations in these repeats, dubbed scanning-enhancers SE1 and SE2, derepress GCN4 translation and reduce TC loading on 40S subunits in vitro, and simultaneously increase initiation at UUG start codons in vivo (the Sui- phenotype). Importantly, all of these defects are suppressed by a mutation in an N-terminal segment of eIF1A, dubbed the scanning inhibitor (SI). These and other findings suggest that the eIF1A SEs (i) promote TC binding to 40S in a conformation conducive to scanning, dubbed POUT; and (ii) block an alternative conformation incompatible with scanning but required for AUG recognition, PIN, with Met-tRNAi fully accommodated in the P site. The SI element antagonizes the SEs to destabilize POUT, promote PIN, and enable selection of AUG codons. Thus, eIF1A regulates AUG selection by controlling distinct modes of Met-tRNAi binding to the 40S P site.

We identified a segment in the largest (a) subunit of eIF3 that is required for attachment of the 43S PIC to mRNA, efficient scanning of long structured mRNA leaders, and efficient start codon recognition. Mutations in this segment impairing these functions also disrupt eIF3a binding to both the N-terminal segment of eIF3b and eIF3j, which bind to one another and also influence scanning and AUG recognition in vivo. Interestingly, the adjacent, extreme C-terminal region of eIF3a binds to the 40S proteins Rps2 and Rps3, which together with other findings, locates the eIF3 a-b-j module near the 40S mRNA entry channel. This location could enable the a-b-j module to regulate 43S attachment to mRNA, efficiency of scanning, and the transition between scanning-conducive and initiation-competent conformations of the PIC.

Previously, we and others showed that start codon recognition depends on dissociation of eIF1 from the 40S, enabling completion of GTP hydrolysis by eIF2 in the TC and stabilizing the closed, scanning-incompatible conformation of the PIC. Thus, mutations that decrease eIF1 affinity for the 40S, dubbed "type I" Sui- mutations, evoke premature dissociation of eIF1 at non-AUG codons and confer the increased ratio of UUG:AUG initiation characteristic of Sui- mutants. Recently, we uncovered a novel class of Sui- mutations in eIF1 ("type II") with the opposite effect of decreasing eIF1 dissociation from the PIC, but only when AUG occupies the P site. Thus, they appear to elevate the UUG:AUG ratio by eliminating the selective advantage of AUG over non-AUG codons, thus implicating eIF1 in distinguishing AUG from non-AUG triplets in the P site. We also obtained evidence that eIF5, the GTPase activating protein for eIF2, stimulates eIF1 dissociation to promote start codon recognition. As the eIF5 N-terminal domain and eIF1 share structural similarity, eIF5 might compete with eIF1 for a common binding site through molecular mimicry. This finding implies a second function for eIF5, beyond GAP activity, in start codon recognition.

eIF2B is the guanine nucleotide exchange factor (GEF) for eIF2, which stimulates formation of the TC in a manner inhibited by phosphorylated eIF2 (eIF2(aP)). While eIF2B contains five subunits, the e/Gcd6 subunit is sufficient for GEF activity in vitro. The d/Gcd2 and b/Gcd7 subunits function with a/Gcn3 in the eIF2B regulatory subcomplex that mediates tight, inhibitory binding of eIF2(aP)GDP, but the essential functions of d/Gcd2 and b/Gcd7 are not well understood. We showed recently that the depletion of wild-type b/Gcd7 from cells, three lethal b/Gcd7 amino acid substitutions, and a synthetically lethal combination of substitutions in b/Gcd7 and eIF2a all impair eIF2 binding to eIF2B without reducing e/Gcd6 abundance in the native eIF2B eIF2 holocomplex. Additionally, we found that non-lethal b/Gcd7 mutations that impair eIF2B function display extensive allele-specific interactions with mutations in the S1 domain of eIF2a (harboring the phosphorylation site), which binds to eIF2B directly. Consistent with this, we observed that b/Gcd7 can overcome the toxicity of eIF2(aP) and rescue native eIF2B function when overexpressed in cells together with d/Gcd2 or g/Gcd1. In aggregate, these findings provide compelling evidence that b/Gcd7 is crucial for substrate binding by eIF2B in vivo.

Snf1 is the ortholog of mammalian AMP-activated kinase, and is responsible for activation of glucose-repressed genes at low glucose levels in budding yeast. We recently demonstrated that Snf1 promotes formation of phosphorylated eIF2a, by stimulating the kinase activity of Gcn2 during histidine starvation of glucose-grown cells. Thus, eliminating Snf1 or mutating its activation loop lowers Gcn2 kinase activity, reducing autophosphorylation of Thr-882 in the Gcn2 activation loop, and decreases eIF2(aP) levels in starved cells. Consistent with these findings, eliminating Reg1, a negative regulator of Snf1 activity, provokes Snf1-dependent hyperphosphorylation of both Thr-882 and eIF2a. Interestingly, Snf1 also promotes eIF2a phosphorylation in the non-preferred carbon source galactose, but this occurs by inhibition of protein phosphatase (PP) 1a, known as Glc7, and the PP2A-like enzyme Sit4, rather than by activation of Gcn2 function. Both Glc7 and Sit4 were found to physically interact with eIF2a in cell extracts, supporting their direct roles as eIF2a phosphatases. Our results show that Snf1 modulates the level of eIF2a phosphorylation by different mechanisms depending on the kind of nutrient deprivation that exists in cells. When combined with our previously published findings, it can now be stated that two major nutrient-signaling kinases that respond to the availability of different nutrients, Snf1 (carbon) and Tor (nitrogen), make critical contributions to setting the level of eIF2(aP), a key regulator of translation initiation in nutrient-limited cells.

In the arena of transcriptional control, we showed previously that efficient transcriptional activation by Gcn4 depends on recruitment of coactivator complexes Mediator, SAGA, SWI/SNF, and RSC, which collectively mediate nucleosome remodeling and recruitment of general transcription factors and RNA Polymerase II (Pol II) to promoters. We further demonstrated that SAGA is recruited co-transcriptionally to coding sequences by association with the Ser5-phosphorylated CTD (Ser5P) of Pol II, and that the histone acetyltransferase (HAT) subunit of SAGA (Gcn5) promotes increased histone acetylation, histone eviction, Pol II processivity, and histone H3-Lys4 methylation within coding sequences.

Recently, we demonstrated that the histone H4 HAT complex, NuA4, is also recruited co-transcriptionally to coding regions in a manner stimulated by the Ser5 CTD kinase Cdk7/Kin28, and that NuA4 association with nucleosomes further depends on H3 methylation, presumably due to chromodomains and a PHD finger in NuA4 subunits. We also obtained evidence that the HAT activities of NuA4 and SAGA cooperate to enhance co-transcriptional recruitment of the nucleosome remodeler RSC, promote histone eviction from transcribed sequences, and stimulate Pol II elongation rate. Consequently, inactivating Gcn5 and the HAT subunit in NuA4 (Esa1) confers additive reductions in transcript production from long versus short coding sequences in vivo. These findings demonstrate direct, additive roles for the HAT activities of NuA4 and SAGA in promoting the elongation phase of II transcription.

We recently extended the two-stage recruitment mechanism, via Ser5P and methylated histones, described above for NuA4 to include the histone deacetylase complex (HDAC) Rpd3C(S).  It was known that methylation of H3 by Set1 and Set2 is required for deacetylation of coding region nucleosomes by Set3C and Rpd3C(S), respectively. We discovered that Set3C and Rpd3C(S) are co-transcriptionally recruited to coding sequences in the absence of both Set1 and Set2, but in a manner stimulated by Cdk7/Kin28. Moreover, Rpd3C(S) and Set3C were shown to interact with both Pol II-Ser5P and histones in extracts, but only the histone interactions require H3 methylation. Moreover, a reconstituted Rpd3C(S) complex bound specifically to Ser5P synthetic peptides. Thus, whereas interaction with methylated H3 is required for Rpd3C(S) and Set3C deacetylation activities, their co-transcriptional recruitment is stimulated by the phosphorylated Pol II CTD. We further demonstrated that the HDAs Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating nucleosomes and limiting co-transcriptional nucleosome eviction.  Moreover, a strong correlation between increased acetylation and lower histone occupancy observed in single, double, and triple HDA mutants supports our contention, based on analysis of HAT mutants, that histone acetylation is a key determinant of co-transcriptional nucleosome eviction.

The Mediator is a multisubunit coactivator required for transcription initiation by Pol II, which is recruited by Gcn4 to its target promoters in vivo.  Previously, we determined that the tail subdomain of Mediator, containing the Gal11/Med15 subunit, is a direct target of Gcn4 in vivo, critical for recruitment by Gcn4 of both intact Mediator and the stable Mediator tail subdomain existing in sin4D cells. Although several Gal11 segments were shown previously to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We demonstrated that interaction of Gcn4 with the Mediator tail in vitro, and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo, involve additive contributions from three different segments in the N-terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we established that the B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrated that Gcn4 binds to the Gal11 KIX domain directly and, in collaboration with Christopher Jaroniec's group at Ohio State University, utilized NMR chemical shift analysis, combined with mutational studies, to identify the likely Gcn4 binding site on the surface of the KIX domain. Together, these results define the physiological mechanism of Mediator recruitment by Gcn4. It appears that Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator to target promoters in vivo.

The hnRNP protein Npl3 of budding yeast has dual functions in promoting transcription elongation and preventing termination at cryptic termination sites by Pol II.  Npl3 was known to be a substrate of arginine methyltransferase Hmt1, but the role of Hmt1 in regulating Npl3's functions in transcription antitermination and elongation were unknown. In collaboration with Chi-Ming Wong and colleagues at the University of Hong Kong, we found that mutants lacking Hmt1 methyltransferase activity exhibit reduced recruitment of Npl3, but elevated recruitment of a component of mRNA cleavage/termination factor CFI, to the transcriptionally activated GAL10-GAL7 locus. Consistent with this, hmt1 mutants displayed increased termination at the defective gal10-D56 terminator, indicating a reduction in antitermination activity. Remarkably, hmt1 cells also exhibit diminished recruitment of transcription elongation factor Tho2 (a component of the THO complex) and a reduced rate of transcription elongation in vivo. Importantly, the defects in recruitment of Npl3 and Tho2, in antitermination, and in transcription elongation in hmt1D cells were all mitigated by substitutions in the "RGG" repeats of Npl3 that functionally mimic arginine methylation by Hmt1. These findings indicate that Hmt1 promotes transcription elongation and suppresses utilization of cryptic termination sites by methylating the RGG repeats in Npl3. As Hmt1 stimulates dissociation of Tho2 from an Npl3-mRNP complex, it might act to recycle these factors back to sites of ongoing transcription as the means of promoting their functions in transcription elongation and antitermination.

Publications

  • Govind, C.K., Zhang, F., Qiu, H., Hofmeyer, K., Hinnebusch, A.G. Gcn5 promotes acetylation, eviction and methylation of nucleosomes in transcribed coding regions. Mol. Cell 25: 31-42, 2007.
  • Fekete, C.A, Mitchell, S.F., Cherkasova, V.A., Applefield, D., Algire, M.A., Maag, D., Saini, A.K., Lorsch, J.R., Hinnebusch, A.G. N- and C-terminal residues of eIF1A have opposing effects on the fidelity of start codon selection. EMBO J. 26:1602-1614, 2007.
  • Cheung,Y.N., Maag, D., Mitchell, S.F., Fekete C.A., Algire, M.A., Takacs, J.E., Shirokikh, N., Pestova, T. Lorsch, J.R. Hinnebusch, A.G. Dissociation of eIF1 from the 40S ribosomal subunit is a key step in start codon selection in vivo. Genes Dev. 21:1217-1230, 2007.
  • Wong, C.M., Qiu, H., Hu, C., Dong, J., Hinnebusch, A.G. Yeast cap binding complex (CBC) impedes recruitment of cleavage factor IA to weak termination sites. Mol. Cell. Biol. 27: 6520-6531, 2007.
  • Dong, J., Nanda, J.S., Rahman, H., Pruitt, M.R., Shin, B.S., Wong, C.M., Lorsch, J.R., Hinnebusch, A.G., Genetic identification of yeast 18S rRNA residues required for efficient recruitment of initiator tRNAMet and AUG selection. Genes Dev. 22: 2242-2255, 2008.
  • Pascual-García, P., Govind, C.K., Queralt, E., Cuenca-Bono, B., Llopis, A., Chavez, S., Hinnebusch, A.G. and Rodríguez-Navarro, S., Sus1 is recruited to coding regions and functions during transcription elongation in association with SAGA and TREX2. Genes Dev. 22: 2811-22, 2008.
  • Zhang F, Gaur N.A, Hasek J, Kim S.J., Qiu H., Swanson M.J., Hinnebusch A.G. Disrupting Vesicular Trafficking at the Endosome Attenuates Transcriptional Activation by Gcn4. Mol. Cell. Biol. 28: 6796-818, 2008.
  • Gárriz, A., Qiu, H., Dey, M., Seo, E.J., Dever, T.E., Hinnebusch, A.G. A network of hydrophobic residues impeding helix alphaC rotation maintains latency of kinase Gcn2, which phosphorylates the alpha subunit of translation initiation factor 2. Mol. Cell. Biol. 29: 1592-607, 2008.
  • Qiu, H., Hu, C., Hinnebusch, A.G. Phosphorylation of the Po1 II CTD by KIN28 enhances BUR1/BUR2 recruitment and Ser2 CTD phosphorylation near promoters. Mol Cell 33: 752-62, 2009.
  • Nanda JS, Cheung YN, Takacs JE, Martin-Marcos P, Saini AK, Hinnebusch AG, Lorsch JR. eIF1 Controls Multiple Steps in Start Codon Recognition during Eukaryotic Translation Initiation. J Mol Biol. 394: 268-285, 2009.
  • Dev K, Santangelo TJ, Rothenburg S, Neculai D, Dey M, Sicheri F, Dever TE, Reeve JN, Hinnebusch AG. Archaeal aIF2B interacts with eukaryotic translation initiation factors eIF2alpha and eIF2Balpha: Implications for aIF2B function and eIF2B regulation. J Mol Biol. 392: 701-722, 2009.
  • Ginsburg DS, Govind CK, Hinnebusch AG. NuA4 lysine acetyltransferase Esa1 is targeted to coding regions and stimulates transcription elongation with Gcn5. Mol Cell Biol. 24: 6473-6487, 2009.
  • Jedidi I, Zhang F, Qiu H, Stahl SJ, Palmer I, Kaufman JD, Nadaud PS, Mukherjee S, Wingfield PT, Jaroniec CP, Hinnebusch AG. Activator Gcn4 employs multiple segments of Med15/Gal11, including the KIX domain, to recruit Mediator to target genes in vivo. J Biol Chem. 285: 2438-2455, 2010.
  • Saini AK, Nanda JS, Lorsch JR, Hinnebusch AG. Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNAiMet binding to the ribosome. Genes Dev. 24: 97-110, 2010.
  • Govind CK, Qiu H, Ginsburg DS, Ruan C, Hofmeyer K, Hu C, Swaminathan V, Workman JL, Li B, Hinnebusch AG. Phosphorylated Pol II CTD recruits multiple HDACs, including Rpd3C(S), for methylation-dependent deacetylation of ORF nucleosomes. Mol Cell. 39: 234-246, 2010.
  • Chiu WL, Wagner S, Herrmannová A, Burela L, Zhang F, Saini AK, Valasek L, Hinnebusch AG. The C-terminal region of eIF3a promotes mRNA recruitment, scanning and, together with eIF3j and the eIF3b RRM, selection of AUG start codons. Mol Cell Biol. 30: 4415-4434, 2010.
  • Cherkasova V, Qiu H, Hinnebusch AG. Snf1 promotes phosphorylation of the alpha ssubunit of eukaryotic translation initiation factor 2 by activating Gcn2 and inhibiting phosphatases Glc7 and Sit4. Mol Cell Biol. 30(12):2862-2873, 2010.
  • Wong CM, Tang HM, Kong KY, Wong GW, Qiu H, Jin DY, Hinnebusch AG. Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p. Nucleic Acids Res. 38(7):2217-2228, 2010.
  • Dev K, Qiu H, Dong J, Zhang F, Barthlme D, Hinnebusch AG. The beta/Gcd7 subunit of eukaryotic translation initiation factor 2B (eIF2B), a guanine nucleotide exchange factor, is crucial for binding eIF2 in vivo.. Mol Cell Biol. 30(21):5218-5233, 2010. Highlighted as an article of significant interest selected from the issue by the editors.
  • Mitchell SF, Walker SE, Algire MA, Park EH, Hinnebusch AG, Lorsch JR. The 5'7methylguanosine cap on eukaryotic mRNAs serves both to stimulate canonical translation initiation and to block an alternative pathway. Mol Cell. 39(6):950-962, 2010.
  • Park EH, Walker SE, Lee JM, Rothenburg S, Lorsch JR, Hinnebusch AG. Multiple elements in the eIF4G1 N-terminus promote assembly of eIF4G1•PABP mRNPs in vivo. EMBO J. 30(2):302-316, 2011.
  • Takacs JE, Neary TB, Ingolia NT, Saini AK, Martin-Marcos P, Pelletier J, Hinnebusch AG, Lorsch JR. Identification of compounds that decrease the fidelity of start codon recognition by the eukaryotic translational machinery. RNA. 17(3):439-452, 2011.
  • Zhang F, Hinnebusch AG. An upstream ORF with non-AUG start codon is translated in vivo but dispensable for translational control of GCN4 mRNA. Nucleic Acids Res. 39(18):3128-40, 2011.
  • Park EH, Zhang F, Warringer J, Sunnerhagen P, Hinnebusch AG. Depletion of eIF4G from yeast cells narrows the range of translational efficiencies genome-wide. BMC Genomics. 12:68, 2011.
  • Visweswaraiah J, Lageix S, Castilho BA, Izotova L, Kinzy TG, Hinnebusch AG, Sattlegger E. Evidence that eukaryotic translation elongation factor 1A (eEF1A) binds the Gcn2 protein C terminus and inhibits Gcn2 activity. J Biol Chem. 286(42):36568-79, 2011.
  • Martin-Marcos P, Cheung YN, Hinnebusch, AG. Functional elements in initiation factors 1, 1A, and 2 discriminate against poor AIG context and non-AUG start codons. Mol Cell Biol. 31(23):4814-31, 2011.
  • Mousley CJ, Yuan P, Gaur NA, Trettin KD, Nile AH, Deminoff SJ, Dewar BJ, Wolpert M, Macdonald JM, Herman PK, Hinnebusch AG, Bankaitis VA. A sterol-binding protein integrates endosomal lipid metabolism with TOR signaling and nitrogen sensing. Cell. 17;148(4):702-15, 2012.
  • Rajagopal V, Park EH, Hinnebusch AG, Lorsh JR. Specific domains in yeast elF4G bias RNA unwinding activity of the elF4F complex towards duplexes with 5’-overhangs. J Biol Chem. 2012.
  • Luna RE, Arthanari H, Hiraishi H, Nanda J, Martin-Marcos P, Markus MA, Akabayov B, Milbradt AG, Luna LE, Seo HC, Hyberts SG, Fahmy A, Reibarkh M, Miles D, Hagner PR, O’Day EM, Yi T, Marintchev A, Hinnebusch AG, Lorsch JR, Asano K, Wagner G. The C-terminal domain of eukaryotic initiation factor 5 promotes start codon recognition by its dynamic interplay with elF1 and elF2β. Cell Rep. 1(6):689-702, 2012.
  • Qiu H, HuC, Gaur NA, Hinnebusch AG. Poll II CTD kinases Bur1 and Kin28 promote Spt5 CTR-independent recruitment of Paf1 complex. EMBO J. 13;31(16):3494-505, 2012.
  • Sonenberg, N., Hinnebusch, A.G. New Modes of Translational Control in Development, Behavior and Disease. Mol. Cell, 28: 721-729, 2008.
  • Sonenberg, N., Hinnebusch, A.G. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136: 731-45, 2009.
  • Hinnebusch AG. Molecular mechanism of scanning and start codon selection in eukaryotes. Microbiol Mol Biol. 75(3):434-67, 2011.
  • Hinnebusch AG, Lorsh JR. The Mechanism of Eukaryotic Translation Initation: New Insights and Challenges. Hershey, J.W.B., Sonenberg, N., Mathews, M (eds). Cold Spring Harb. Perspect. Biol., Cold Spring Harbor Laboratory Press: NY, pp. 29-54, 2012.

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