We study regulation of amino acid biosynthetic genes in budding yeast as means of dissecting molecular mechanisms of gene regulation 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. 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 mRNA, keeping GCN4 translation repressed. Under starvation conditions, the reinitiating 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), eIF2-GTP-Met-tRNAiMet, which delivers Met-tRNAiMet to the 40S ribosome in assembling the 43S preinitiation complex (PIC). TC abundance is reduced in starved cells by phosphorylation of the alpha subunit of eIF2 by protein kinase GCN2, converting eIF2 from substrate to inhibitor of its guanine nucleotide exchange factor (GEF), eIF2B. Hence, GCN4 translation is a sensitive in vivo indicator of impaired TC loading on 40S subunits. We previously exploited this fact to isolate mutations in subunits of eIF2B that constitutively derepress GCN4 (Gcd- phenotype) by lowering TC assembly in the absence of eIF2 phosphorylation by GCN2.? ? We recently applied the Gcd- selection to obtain the first functional evidence implicating 18S rRNA residues of the 40S subunit in TC loading and AUG selection. Mutations that perturb the location or identity of the bulge G residue in helix 28 impair TC loading during reinitiation on GCN4 mRNA and, uniquely, increase the bypass of AUG start codons during primary initiation (leaky scanning). At least one such mutation impairs the rate and stability of TC loading on mutant 40S subunits in vitro. Site directed mutations in other residues predicted to contact the AUG codon or anticodon loop of tRNAiMet confer dominant Gcd- and recessive-lethal phenotypes, implying that bacterial elongation complexes are relevant to the structure of eukaryotic PICs. ? ? In collaboration with Mercedes Tamame in Salamanca, we identified a Gcd- mutation in 60S ribosomal protein L33A (rpl33a-G76R) that elicits derepression of GCN4 translation and allows scanning PICs to leaky scan uORF4 independent of prior uORF1 translation and reinitiation. At 37C, rpl33a-G76R confers defects in 60S biogenesis comparable to those produced by deletion of RPL33A (∆A). At 28C, however, the 60S biogenesis defect is less severe in rpl33a-G76R than in ∆A cells, yet rpl33a-G76R confers greater derepression of GCN4 and a larger reduction in general translation. Hence, it appears that rpl33a-G76R has a stronger effect on ribosomal subunit joining than does a comparable reduction of wild-type 60S levels conferred by ∆A. We suggested that rpl33a-G76R alters 60S structure in a way that impedes subunit joining to allow 48S PICs to abort initiation at uORF4, resume scanning and initiate downstream at GCN4. Because overexpressing tRNAiMet suppresses the Gcd- phenotype of rpl33a-G76R cells, dissociation of tRNAiMet from the 40S subunit may be responsible for abortive initiation at uORF4 in this mutant.? ? Previously we showed that the NTD of the a/TIF32 subunit of eIF3 interacts with the small ribosomal protein RPS0A located near the mRNA exit channel, where eIF3 is proposed to reside. In collaboration with Leos Valasek in Prague, we found that partial deletion of the RPS0A-binding domain of eIF3a impairs translation initiation and reduces binding of the MFC to 40S subunits. Strikingly, it also severely blocks induction of GCN4 translation (Gcn- phenotype) by a novel mechanism, wherein post-termination 40S ribosomes cannot resume scanning after terminating at uORF1. Genetic analysis revealed a functional interaction between the eIF3a-NTD and sequences 5' of uORF1 that enhance reinitiation. We found that these stimulatory sequences must be positioned precisely relative to the uORF1 stop codon and that reinitiation after uORF1 declines with increasing uORF length. Together, these results suggest that eIF3 is retained on ribosomes throughout uORF1 translation and, upon termination, interacts with the 5' enhancer at the mRNA exit channel to stabilize mRNA association with post-termination 40S subunits and thereby promote the resumption of scanning for reinitiation downstream.? ? 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 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. From detailed analysis of the effects of different coactivator mutations on activation of the ARG1 gene by GCN4, we obtained evidence that SAGA promotes elongation in addition to PIC assembly. Pursuing this observation, we discovered that SAGA is recruited co-transcriptionally to the coding sequences of both ARG1 and GAL1 and obtained evidence that the histone acetyltransferase subunit of SAGA (GCN5) promotes histone eviction, Pol II processivity, and efficient histone H3-Lys4 methylation in the coding sequences. ? ? The SUS1 protein resides in both SAGA and the mRNA export complex TREX2. We recently collaborated with Susana Rodrguez-Navarros group in Valencia to show that SUS1 is associated with the elongating form of RNAP II phosphorylated on Ser5 and Ser2 of the heptad repeats of the RBP1 CTD, and also with essential mRNA export factors (YRA and MEX67) that are recruited to the mRNA cotranscriptionally. We further showed that SUS1 associates with coding sequences dependent on Ser5-CTD phosphorylation, and that SUS1 promotes transcription elongation. Remarkably, we found that SAGA subunit SGF73 is necessary for association of SUS1 with TREX2 as well as SAGA, and its absence dramatically reduces SUS1 targeting to both promoters and coding sequences. Thus, SUS1 functions at the interface between SAGA and TREX2 complexes, in an SGF73-dependent manner, to coordinate transcription elongation and mRNA export.? ? The late endosome (MVB) plays a key role in coordinating vesicular transport of proteins between Golgi, vacuole/lysosome, and plasma membrane. In collaboration with Jiri Hasek in Prague, we found that deleting multiple genes involved in vesicle fusion at the MVB (class C/D vps mutations) impairs transcriptional activation by GCN4 by decreasing the ability of chromatin-bound GCN4 to stimulate PIC assembly. 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, have smaller effects on GCN4 function. Thus, it appears that decreasing cargo proteins in the MVB through impaired delivery or enhanced degradation, and not merely the failure to transport cargo properly to the vacuole or down-regulate plasma membrane proteins by endocytosis, is required to attenuate transcriptional activation by GCN4. Our findings reveal an unexpected coupling of transcriptional activation in the nucleus with vesicular trafficking at the late endosome.
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