Komplexní strukturní a funkční analýza jednotlivých podjednotek kvasinkového translačního iniciačního faktoru 3.

5 ABSTRACT Translation initiation in eukaryotes is a complex process promoted by numerous proteins or protein complexes called eukaryotic initiation factors (eIFs). The eukaryotic initiation factor 3 (eIF3) is a multisubunit complex that has been implicated in several steps of the translation initiation pathway. In yeast Saccharomyces cerevisiae, eIF3 is composed of five essential subunits (a/TIF32, b/PRT1, c/NIP1, g/TIF35, i/TIF34) and one nonessential subunit (j/HCR1). It is our long-term goal to understand how eIF3 promotes different stages of translation initiation and which subunits or their domains play a critical role in this process as well as to map the binding sites of eIF3 on 40S ribosomal subunit and to create a structural model of eIF3 complex. Here I present two structural studies showing interactions between the RNA recognition motif of eIF3b and a short peptide of eIF3j subunits of human eIF3 solved by NMR spectroscopy, and a crystal structure of the C-terminal fragment of yeast b/PRT1 in complex with the full length i/TIF34 subunit at 2.2 Å resolution. In the former study, me and my colleagues showed that the critical determinants mediating this eIF3b-eIF3j interaction are evolutionary conserved, since their mutations in yeast proteins reduced cellular growth rate, eliminated j/HCR1...7 ABSTRAKT Eukaryotická iniciace translace je komplexní proces, který je ovlivněn mnoha proteiny a proteinovými komplexy, které nazýváme translační iniciační faktory. Eukaryotický translační iniciační faktor 3 (eIF3) je komplexem více podjednotek a hraje roli hned v několika fázích iniciace translace. Iniciační faktor eIF3 se v kvasince Saccharomyces cerevisiae skládá z pěti esenciálních podjednotek (a/TIF32, b/PRT1, c/NIP1, g/TIF35, i/TIF34) a jedné neesenciální podjednotky (j/HCR1). Naším dlouhodobým cílem je porozumět tomu, jak eIF3 působí v různých fázích iniciace translace, které podjednotky nebo jejich domény hrají při těchto proceses důležitou roli, a také zmapování jeho vazebných míst na malé ribosomální podjednotce a vytvoření strukturního modelu celého komplexu. V této práci prezentuji výsledky dvou strukturních studií. První popisuje interakci objevenou pomocí NMR spektroskopie mezi RNA-rozpoznávajícím motivem lidského faktoru eIF3b a podjednotkou eIF3j. Druhá studie představuje krystalickou strukturu interakce mezi C-koncovým fragmentem kvasinkové podjednotky b/PRT1 a podjednotkou i/TIF34 v rozlišení 2,2 A. V první práci jsme ukázali, že kritické determinanty této interakce jsou konzervovány také v kvasinkách, a že jejich mutace způsobuje zpomalení růstu, eliminuje asociaci j/HCR1 s b/PRT1 in...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

Komplexní strukturní a funkční analýza jednotlivých podjednotek kvasinkového translačního iniciačního faktoru 3.

7 ABSTRAKT Eukaryotická iniciace translace je komplexní proces, který je ovlivněn mnoha proteiny a proteinovými komplexy, které nazýváme translační iniciační faktory. Eukaryotický translační iniciační faktor 3 (eIF3) je komplexem více podjednotek a hraje roli hned v několika fázích iniciace translace. Iniciační faktor eIF3 se v kvasince Saccharomyces cerevisiae skládá z pěti esenciálních podjednotek (a/TIF32, b/PRT1, c/NIP1, g/TIF35, i/TIF34) a jedné neesenciální podjednotky (j/HCR1). Naším dlouhodobým cílem je porozumět tomu, jak eIF3 působí v různých fázích iniciace translace, které podjednotky nebo jejich domény hrají při těchto proceses důležitou roli, a také zmapování jeho vazebných míst na malé ribosomální podjednotce a vytvoření strukturního modelu celého komplexu. V této práci prezentuji výsledky dvou strukturních studií. První popisuje interakci objevenou pomocí NMR spektroskopie mezi RNA-rozpoznávajícím motivem lidského faktoru eIF3b a podjednotkou eIF3j. Druhá studie představuje krystalickou strukturu interakce mezi C-koncovým fragmentem kvasinkové podjednotky b/PRT1 a podjednotkou i/TIF34 v rozlišení 2,2 A. V první práci jsme ukázali, že kritické determinanty této interakce jsou konzervovány také v kvasinkách, a že jejich mutace způsobuje zpomalení růstu, eliminuje asociaci j/HCR1 s b/PRT1 in...5 ABSTRACT Translation initiation in eukaryotes is a complex process promoted by numerous proteins or protein complexes called eukaryotic initiation factors (eIFs). The eukaryotic initiation factor 3 (eIF3) is a multisubunit complex that has been implicated in several steps of the translation initiation pathway. In yeast Saccharomyces cerevisiae, eIF3 is composed of five essential subunits (a/TIF32, b/PRT1, c/NIP1, g/TIF35, i/TIF34) and one nonessential subunit (j/HCR1). It is our long-term goal to understand how eIF3 promotes different stages of translation initiation and which subunits or their domains play a critical role in this process as well as to map the binding sites of eIF3 on 40S ribosomal subunit and to create a structural model of eIF3 complex. Here I present two structural studies showing interactions between the RNA recognition motif of eIF3b and a short peptide of eIF3j subunits of human eIF3 solved by NMR spectroscopy, and a crystal structure of the C-terminal fragment of yeast b/PRT1 in complex with the full length i/TIF34 subunit at 2.2 Å resolution. In the former study, me and my colleagues showed that the critical determinants mediating this eIF3b-eIF3j interaction are evolutionary conserved, since their mutations in yeast proteins reduced cellular growth rate, eliminated j/HCR1...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyFaculty of SciencePřírodovědecká fakult

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

Complex structural and functional analysis of individual subunits of yeast translation initiation factor 3.

5 ABSTRACT Translation initiation in eukaryotes is a complex process promoted by numerous proteins or protein complexes called eukaryotic initiation factors (eIFs). The eukaryotic initiation factor 3 (eIF3) is a multisubunit complex that has been implicated in several steps of the translation initiation pathway. In yeast Saccharomyces cerevisiae, eIF3 is composed of five essential subunits (a/TIF32, b/PRT1, c/NIP1, g/TIF35, i/TIF34) and one nonessential subunit (j/HCR1). It is our long-term goal to understand how eIF3 promotes different stages of translation initiation and which subunits or their domains play a critical role in this process as well as to map the binding sites of eIF3 on 40S ribosomal subunit and to create a structural model of eIF3 complex. Here I present two structural studies showing interactions between the RNA recognition motif of eIF3b and a short peptide of eIF3j subunits of human eIF3 solved by NMR spectroscopy, and a crystal structure of the C-terminal fragment of yeast b/PRT1 in complex with the full length i/TIF34 subunit at 2.2 Å resolution. In the former study, me and my colleagues showed that the critical determinants mediating this eIF3b-eIF3j interaction are evolutionary conserved, since their mutations in yeast proteins reduced cellular growth rate, eliminated j/HCR1..

Complex structural and functional analysis of individual subunits of yeast translation initiation factor 3.

5 ABSTRACT Translation initiation in eukaryotes is a complex process promoted by numerous proteins or protein complexes called eukaryotic initiation factors (eIFs). The eukaryotic initiation factor 3 (eIF3) is a multisubunit complex that has been implicated in several steps of the translation initiation pathway. In yeast Saccharomyces cerevisiae, eIF3 is composed of five essential subunits (a/TIF32, b/PRT1, c/NIP1, g/TIF35, i/TIF34) and one nonessential subunit (j/HCR1). It is our long-term goal to understand how eIF3 promotes different stages of translation initiation and which subunits or their domains play a critical role in this process as well as to map the binding sites of eIF3 on 40S ribosomal subunit and to create a structural model of eIF3 complex. Here I present two structural studies showing interactions between the RNA recognition motif of eIF3b and a short peptide of eIF3j subunits of human eIF3 solved by NMR spectroscopy, and a crystal structure of the C-terminal fragment of yeast b/PRT1 in complex with the full length i/TIF34 subunit at 2.2 Å resolution. In the former study, me and my colleagues showed that the critical determinants mediating this eIF3b-eIF3j interaction are evolutionary conserved, since their mutations in yeast proteins reduced cellular growth rate, eliminated j/HCR1..

Complex structural and functional analysis of individual subunits of yeast translation initiation factor 3.

5 ABSTRACT Translation initiation in eukaryotes is a complex process promoted by numerous proteins or protein complexes called eukaryotic initiation factors (eIFs). The eukaryotic initiation factor 3 (eIF3) is a multisubunit complex that has been implicated in several steps of the translation initiation pathway. In yeast Saccharomyces cerevisiae, eIF3 is composed of five essential subunits (a/TIF32, b/PRT1, c/NIP1, g/TIF35, i/TIF34) and one nonessential subunit (j/HCR1). It is our long-term goal to understand how eIF3 promotes different stages of translation initiation and which subunits or their domains play a critical role in this process as well as to map the binding sites of eIF3 on 40S ribosomal subunit and to create a structural model of eIF3 complex. Here I present two structural studies showing interactions between the RNA recognition motif of eIF3b and a short peptide of eIF3j subunits of human eIF3 solved by NMR spectroscopy, and a crystal structure of the C-terminal fragment of yeast b/PRT1 in complex with the full length i/TIF34 subunit at 2.2 Å resolution. In the former study, me and my colleagues showed that the critical determinants mediating this eIF3b-eIF3j interaction are evolutionary conserved, since their mutations in yeast proteins reduced cellular growth rate, eliminated j/HCR1..

Complex structural and functional analysis of individual subunits of yeast translation initiation factor 3.

5 ABSTRACT Translation initiation in eukaryotes is a complex process promoted by numerous proteins or protein complexes called eukaryotic initiation factors (eIFs). The eukaryotic initiation factor 3 (eIF3) is a multisubunit complex that has been implicated in several steps of the translation initiation pathway. In yeast Saccharomyces cerevisiae, eIF3 is composed of five essential subunits (a/TIF32, b/PRT1, c/NIP1, g/TIF35, i/TIF34) and one nonessential subunit (j/HCR1). It is our long-term goal to understand how eIF3 promotes different stages of translation initiation and which subunits or their domains play a critical role in this process as well as to map the binding sites of eIF3 on 40S ribosomal subunit and to create a structural model of eIF3 complex. Here I present two structural studies showing interactions between the RNA recognition motif of eIF3b and a short peptide of eIF3j subunits of human eIF3 solved by NMR spectroscopy, and a crystal structure of the C-terminal fragment of yeast b/PRT1 in complex with the full length i/TIF34 subunit at 2.2 Å resolution. In the former study, me and my colleagues showed that the critical determinants mediating this eIF3b-eIF3j interaction are evolutionary conserved, since their mutations in yeast proteins reduced cellular growth rate, eliminated j/HCR1..

Analýza úlohy podjednotek TIF32 a HCR1 eukaryotního iniciačního faktoru 3 (eIF3) v asociaci multifaktorového komplexu s malou podjednotkou ribosomu.

Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyPřírodovědecká fakultaFaculty of Scienc

Functional analysis of the TIF32-HLD-PRT1-RRM-HCR1 module of the yeast translation initiation factor 3

The eIF3 is in yeast S. cerevisiae composed of five core essential subunits (TIF32, NIP1, PRT1, TIF34 and TIF35) and one nonessential substoichiometric subunit (HCR1), and as such represents the most complex initiation factor among all. Perhaps owing to that, it was shown to stimulate nearly all steps of the initiation pathway culminating in the formation of the 80S initiation complex at the AUG start codon on mRNA. Yeast eIF3 was also demonstrated to assemble together with the ternary complex, eIF1 and eIF5 into so called Multifactor complex that can exist free of ribosomes and whose formation greatly stimulates initiation efficiency. TIF32, the largest eIF3 subunit, was shown to make at least two critical contacts with the 40S ribosomal subunit and its middle domain, designated as the HLD, to share a significant sequence similarity with the HCR1 subunit. Experiments conducted here indicate that the TIF32-HLD and HCR1 share also some functional similarity as the recombinant HLD expressed under control of the HCR1 promoter in a domain- swapping experiment partially suppressed the slow growth phenotype of cells deleted for HCR1. In addition to the HLD, HCR1 also simultaneously interacts with the RRM domain of PRT1, which is considered to be the main scaffolding subunit of eIF3. The group of Dr. P.J...