Alan G. Hinnebusch, Ph.D.
NIH Distinguished Investigator
Section on Nutrient Control of Gene Expression
Building 6, Room 230
6 Center Drive
Bethesda, MD 20892
We study the regulation of amino acid biosynthetic genes in budding yeast as a means of dissecting molecular mechanisms of gene regulation at the translational and transcriptional levels. Transcription of these and many other 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 conserved translational control mechanism involving upstream open reading frames (uORFs) in GCN4 mRNA. Ribosomes translate the 5′-most uORF (uORF1), and, under non-starvation conditions, re-initiate translation at uORFs 2, 3, or 4 and then dissociate from the mRNA, keeping GCN4 translation repressed. In starvation conditions, the re-initiating ribosomes instead bypass uORFs 2–4 and re-initiate at GCN4, owing to decreased availability of the ternary complex (TC)—comprised of initiation factor 2 (eIF2), GTP, and initiator Met-tRNAi—which binds to the small (40S) ribosomal subunit to assemble a 43S pre-initiation complex (PIC). TC abundance is reduced in starved cells by phosphorylation of the alpha subunit of eIF2 (eIF2a) by Gcn2, a protein kinase conserved in all eukaryotes, converting eIF2 from substrate to inhibitor of its guanine nucleotide exchange factor (GEF) eIF2B. Hence, GCN4 translation is an in vivo indicator of impaired TC loading on 40S subunits. We previously exploited this fact to isolate mutations in subunits of eIF2B that constitutively de-repress GCN4 (Gcd− phenotype) by lowering TC assembly in the absence of eIF2 phosphorylation. More recently, we used the Gcd− selection to identify domains/residues in eIF1, eIF1A, and eIF3 as well as residues of 18S rRNA located near the “P” decoding site of the 40S subunit, all of which participate in rapid TC recruitment in vivo. In collaboration with Jon Lorsch's group, we demonstrated that segments/residues in eIF1, eIF1A, and 18S rRNA, which are implicated genetically in TC recruitment, also stimulate the same reaction in a fully reconstituted in vitro system.
We also investigate the roles of various eIFs and the 40S subunit in scanning the mRNA 5′ untranslated region and accurately identifying the AUG initiation codon. Our studies exploit a genetic selection for mutations that either elevate initiation at near-cognate UUG start codons (Sui− phenotype) or suppress this aberrant initiation event (Ssu− phenotype). In this way, we provided the first evidence that eIF1A and the c-subunit of eIF3 make critical contributions to accurate AUG recognition, and we localized these functions to the unstructured N- and C-tails of eIF1A and the N-terminal domain of eIF3c. In collaboration with Lorsch's group, we also provided strong evidence that dissociation of eIF1 from the PIC is a critical step in AUG recognition, indicating that this factor functions as a "gate-keeper" to suppress initiation at non–AUG triplets. Biochemical analysis revealed that eIF1 stabilizes an "open" conformation of the 40S subunit that is conducive to scanning and loading of the TC in a metastable state, permitting inspection of successive triplets as they enter the P site for complementarity with the anticodon of Met-tRNAi. However, eIF1 must dissociate from the PIC for AUG selection, as the absence of eIF1 favors the closed, scanning-arrested conformation of the 40S subunit to which TC is more tightly bound...
Cherkasova VA, Hinnebusch AG. Translational control by TOR and TAP42 through dephosphorylation of eIF2alpha kinase GCN2. Genes Dev. 2003;17(7):859-72.
Dong J, Munoz A, Kolitz SE, Saini AK, Chiu WL, Rahman H, Lorsch JR, Hinnebusch AG. Conserved residues in yeast initiator tRNA calibrate initiation accuracy by regulating preinitiation complex stability at the start codon. Genes Dev. 2014;28(5):502-20.
Qiu H, Hu C, Gaur NA, Hinnebusch AG. Pol II CTD kinases Bur1 and Kin28 promote Spt5 CTR-independent recruitment of Paf1 complex. EMBO J. 2012;31(16):3494-505.
Saini AK, Nanda JS, Lorsch JR, Hinnebusch AG. Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNA(i)(Met) binding to the ribosome. Genes Dev. 2010;24(1):97-110.