We study the regulation of amino acid biosynthetic genes in budding yeast to probe basic mechanisms of gene control at the translational and transcriptional levels. Transcription of these unlinked genes is coordinately induced by the transcriptional activator GCN4 in response to starvation for any amino acid (general amino acid control). Expression of GCN4 is coupled to amino acid levels by a translational control mechanism involving four short upstream open reading frames (uORFs) in GCN4 mRNA. Ribosomes translate the 5-most uORF (uORF1) resume scanning downstream, and under nonstarvation conditions, reinitiate translation at uORFs 2, 3, or 4 and then dissociate from the mRNAkeeping GCN4 translation repressed. Under starvation conditions, the re-scanning ribosomes bypass uORFs 2-4 and reinitiate at the GCN4 start codon instead. This bypass is triggered by decreased formation of the ternary complex (TC) comprised of initiation factor 2 (eIF2), GTP, and Met-tRNAiMet , which delivers Met-tRNAiMet to the 40S ribosome to form the 43S preinitiation complex (PIC). TC abundance is reduced in starved cells by phosphorylation of the 61537;61472;subunit of eIF2 by protein kinase GCN2, converting eIF2 from substrate to inhibitor of its guanine nucleotide exchange factor (GEF), eIF2B. By isolating and characterizing mutants that are uninducible (Gcn-) or constitutively derepressed (Gcd-) for expression of GCN4 and its target genes, we previously identified all five subunits of eIF2B. In addition, we provided evidence that the GCD2, GCD7, and GCN3 subunits of eIF2B form a regulatory subcomplex that binds the phosphorylated eIF2 alpha subunit and impedes the GEF activity of the GCD6 subunit of eIF2B. ? ? Biochemical analysis of eIF1, eIF1A and eIF3 indicates that these factors promote 40S binding of TC in vitro, but their importance for 43S PIC assembly in vivo was unknown. We previously obtained genetic evidence that the C-terminal tail (CTT) of eIF1A contributes to TC loading and, hence, is required for GCN4 translational repression. We also discovered that eIFs 1 and 3 reside with TC and its GTPase activating protein, eIF5, in a multifactor complex (MFC), and that destabilizing multiple eIF2-eIF3 contacts within this complex reduces TC loading in vitro, thus supporting the notion that MFC formation promotes PIC assembly. We recently provided molecular insights into the overlapping functions of eIFs 1, 1A, 3, and 5 in 43S PIC assembly in vivo. We developed a new assay for measuring 43S PIC levels in living cells and used it to provide direct evidence that multiple eIF2-eIF3 contacts in the MFC, specific residues in eIF1 (pinpointed by Gcd- mutations), and residues in the eIF1A CTT, all enhance 43S assembly in vivo. Collaborating with Jon Lorschs group at Johns Hopkins we showed that the critical eIF1A and eIF1 residues also accelerate TC loading in a reconstituted yeast system. We implicated the eIF3j subunit and its binding to the eIF3b RRM in recruiting eIF3 to the 40S subunit. We also showed that the novel ATP-binding-cassette (ABC) protein RLI1 stimulates MFC binding to 40S subunits, both in yeast and (in collaboration with Michael Deans group in NCI) mammalian cells. In collaboration with Mercedes Tamames group in Salamanca, we implicated the large (60S) subunit protein RPL33 in subunit joining, the last step of initiation. ? ? Another area of progress was in dissecting functions of initiation factors in scanning and AUG selection. Our genetic analysis implicated the eIF3b subunit, a domain in eIF3c which binds eIFs 1 and 5 within the MFC, and the eIF1A CTT in the efficiency of scanning or discrimination against non-AUG codons. We confirmed the scanning defects for eIF1A mutations in a reconstituted mammalian system in collaboration with Tatyana Pestovas group at SUNY-Brooklyn. With Lorschs group, we showed that eIF1 acts as a gate-keeper to block non-AUG selection by promoting continued scanning and inhibiting the completion of GTP hydrolysis by the TC. These gate-keeper functions are eliminated at AUG codons when eIF1 dissociates from its 40S binding site. We also showed that the eIF1A CTT promotes an open configuration of the PIC conducive to scanning, while the N-terminal tail (NTT) of this factor acts oppositely to promote eIF1 release and optimum eIF1A-PIC association at AUG codons. These findings suggest a molecular model for the conformational transitions in the PIC that ensure stringent AUG selection. ? ? In the arena of transcriptional control, we showed previously that efficient activation by GCN4 in vivo depends on coactivator complexes known as SAGA, SWISNF, mediator, RSC, and CCR4-NOT, which collectively mediate nucleosome remodeling and recruitment of general transcription factors and RNA Polymerase II (Pol II) to promoters, and that GCN4 interacts with these coactivators in vitro via critical hydrophobic clusters in its activation domain. We had also demonstrated by chromatin immunoprecipitation that GCN4 recruits all of these coactivators to its upstream activation sequences (UASs) at several target genes in living cells. We have recently provided new insights into the mechanism of coactivator recruitment and function at GCN4 target genes. We showed that recruitment of SAGA, SWISNF, and mediator to the ARG1 UAS occurs simultaneously on GCN4 induction, but is nevertheless highly interdependent, and that it precedes PIC assembly at the TATA element. We also implicated these coactivators (and RSC too) in stimulating TBP and Pol II recruitment to the PIC and in promoter clearance or elongation. We then established that the elongation factor complex Paf1C is recruited to coding sequences at GCN4 target genes dependent on Ser-5 phosphorylation of the heptad repeats in the CTD of Pol II subunit RPB1 (by TFIIH), and also on the SPT4 subunit of elongation factor DSIF, revealing a new function for SPT4 in supporting Paf1C-dependent methylation of histone H3 in coding sequences. Pursuing the functions of coactivators in elongation, we discovered that SAGA is also recruited co-transcriptionally to ARG1 and GAL1 coding sequences and provided evidence that the histone acetyltransferase subunit in SAGA (GCN5) promotes several elongation-related functions, including histone eviction, Pol II processivity, and efficient H3-Lys4 methylation. We observed that GCN4 also recruits histone deacetylase complexes, presumably to counteract SAGA and establish an optimum level of histone acetylation in the coding sequence. Finally, we showed recently that the nuclear mRNA m7G-cap-binding complex (CBC) is recruited directly to the m7G-cap of the nascent transcript and participates with the NPL3 protein (with whom it interacts) in blocking the utilization of weak or defective termination signals in the mRNA, thus identifying a novel anti-termination function for the CBC in vivo.
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