Charakterizace RPG1, velké podjednotky kvasinek S. cerevisiae iniciačního translačního faktoru 3 (elF3)

Characterization of RPG1, the Large Subunit of the Yeast S. cerevisiae Initiation Translation Factor 3 (elF3). Dissertation for the attainment of the academic degree Doctor of Natural Sciences at the Formal and Natural Sciences Faculty of the University of Vienna submitted by Mag. Leoš Valášek Vienna, September 1998 ABSTRACT Eukaryotic initiation factor 3 (eIF3) consists of at least eight subunits and plays a key role in the formation of the 43S preinitiation complex by dissociating 40S and 60S ribosomal subunits, stabilizing the ternary complex and promoting mRNA binding to 40S ribosomal subunits. The product of the S. cerevisiae RPG1 gene has been described to encode a protein required for passage through the G1 phase of the cell cycle and to exhibit significant sequence similarity to the largest subunit ofhuman eIF3. Here I show that under non-denaturing conditions, Rpg1p copurifies with a known yeast eIF3 subunit, Prt1p. An anti-Rpg1p antibody co-immunoprecipitates Prt1p, and an antibody directed against the Myc-tag of a tagged version of Prt1p co- immunoprecipitates Rpg1p demonstrating that both proteins are present in the same complex. The integrity of this complex is not influenced by the increased temperature in the background of a rpg1-1 temperature sensitive mutant strain. A cell-free translation...Charakterizace RPG1, velké podjednotky kvasinek S. cerevisiae iniciačního translačního faktoru 3 (elF3). Disertační práce pro získání akademického titulu Doktor přírodních věd na Fakulta formálních a přírodních věd Vídeňské univerzity předložil Mag. Leoš Valášek Vídeň, září 1998 ABSTRAKT Eukaryotický iniciační faktor 3 (eIF3) se skládá z nejméně osmi podjednotek a hraje klíčovou roli při tvorbě preiniciačního komplexu 43S tím, že disociuje ribozomální podjednotky 40S a 60S, stabilizuje ternární komplex a podporuje vazbu mRNA na ribozomální podjednotky 40S. Bylo popsáno, že produkt genu RPG1 S. cerevisiae kóduje protein potřebný pro průchod fází G1 buněčného cyklu a vykazuje významnou sekvenční podobnost s největší podjednotkou lidského eIF3. Zde ukazuji, že za nedenaturačních podmínek Rpg1p ko-purifikuje se známou kvasinkovou podjednotkou eIF3, Prt1p. Protilátka proti Rpg1p ko-imunoprecipituje Prt1p a protilátka namířená proti Myc-značce označené verze Prt1p ko-imunoprecipituje Rpg1p, což dokazuje, že oba proteiny jsou přítomny ve stejném komplexu. Integrita tohoto komplexu není ovlivněna zvýšenou teplotou na pozadí teplotně citlivého mutantního kmene rpg1-1. Bezbuněčný translační systém odvozený z tohoto teplotně citlivého mutantního kmene rpg1-1 se inkubací při 37 řC inaktivuje a jeho aktivitu lze obnovit...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyFaculty of SciencePřírodovědecká fakult

Translation Reinitiation Relies on the Interaction between eIF3a/TIF32 and Progressively Folded cis-Acting mRNA Elements Preceding Short uORFs

Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 5′ and 3′ cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5′ cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 5′ sequences making up the 5′ enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/TIF32-NTD were identified that stimulate reinitiation in concert with the 5′ enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5′ enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5′ enhancer's stimulatory activity is strictly dependent on and thus follows the 3′ enhancer's activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 5′ leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved

Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly

Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1G107R but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs

Characterization of RPG1, the large subunit of the yeast S. cerevisiae initiation translation factor 3 (eIF3)

Characterization of RPG1, the Large Subunit of the Yeast S. cerevisiae Initiation Translation Factor 3 (elF3). Dissertation for the attainment of the academic degree Doctor of Natural Sciences at the Formal and Natural Sciences Faculty of the University of Vienna submitted by Mag. Leoš Valášek Vienna, September 1998 ABSTRACT Eukaryotic initiation factor 3 (eIF3) consists of at least eight subunits and plays a key role in the formation of the 43S preinitiation complex by dissociating 40S and 60S ribosomal subunits, stabilizing the ternary complex and promoting mRNA binding to 40S ribosomal subunits. The product of the S. cerevisiae RPG1 gene has been described to encode a protein required for passage through the G1 phase of the cell cycle and to exhibit significant sequence similarity to the largest subunit ofhuman eIF3. Here I show that under non-denaturing conditions, Rpg1p copurifies with a known yeast eIF3 subunit, Prt1p. An anti-Rpg1p antibody co-immunoprecipitates Prt1p, and an antibody directed against the Myc-tag of a tagged version of Prt1p co- immunoprecipitates Rpg1p demonstrating that both proteins are present in the same complex. The integrity of this complex is not influenced by the increased temperature in the background of a rpg1-1 temperature sensitive mutant strain. A cell-free translation..

Increased expression of tryptophan and tyrosine tRNAs elevates stop codon readthrough of reporter systems in human cell lines

International audienceRegulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens

Eukaryotic Translation Initiation Factor 3 (eIF3) and eIF2 Can Promote mRNA Binding to 40S Subunits Independently of eIF4G in Yeast

Recruitment of the eukaryotic translation initiation factor 2 (eIF2)-GTP-Met-tRNA(i)(Met) 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 depletion. Hence, stimulation of the GTPase activity of the ternary complex, a prerequisite for 60S subunit joining in vitro, is likely the rate-limiting function of eIF5 in vivo. Depleting eIF2 or eIF3 impaired mRNA binding to free 40S subunits, but depleting eIF4G led unexpectedly to accumulation of mRNA on 40S subunits. Thus, it appears that eIF3 and eIF2 are more critically required than eIF4G for stable binding of at least some mRNAs to native preinitiation complexes and that eIF4G has a rate-limiting function at a step downstream of 48S complex assembly in vivo