title: Structural integrity of the PCI domain of eIF3a/TIF32 is required for mRNA recruitment to the 43S pre-initiation complexes.
abstract: Transfer of genetic information from genes into proteins is mediated by messenger RNA (mRNA) that must be first recruited to ribosomal pre-initiation complexes (PICs) by a mechanism that is still poorly understood. Recent studies showed that besides eIF4F and poly(A)-binding protein, eIF3 also plays a critical role in this process, yet the molecular mechanism of its action is unknown. We showed previously that the PCI domain of the eIF3c/NIP1 subunit of yeast eIF3 is involved in RNA binding. To assess the role of the second PCI domain of eIF3 present in eIF3a/TIF32, we performed its mutational analysis and identified a 10-Ala-substitution (Box37) that severely reduces amounts of model mRNA in the 43-48S PICs in vivo as the major, if not the only, detectable defect. Crystal structure analysis of the a/TIF32-PCI domain at 2.65-Å resolution showed that it is required for integrity of the eIF3 core and, similarly to the c/NIP1-PCI, is capable of RNA binding. The putative RNA-binding surface defined by positively charged areas contains two Box37 residues, R363 and K364. Their substitutions with alanines severely impair the mRNA recruitment step in vivo suggesting that a/TIF32-PCI represents one of the key domains ensuring stable and efficient mRNA delivery to the PICs.
authors.affiliation: Department of Molecular Structural Biology, Institute for Microbiology and Genetics, George-August University, Goettingen, Germany, 37077 and Laboratory of Regulation of Gene Expression, Institute of Microbiology ASCR, Videnska 1083, 142 20 Prague, the Czech Republic.
authors.forename: Sohail
authors.initials: S
authors.lastname: Khoshnevis
authors.forename: Stanislava
authors.initials: S
authors.lastname: Gunišová
authors.forename: Vladislava
authors.initials: V
authors.lastname: Vlčková
authors.forename: Tomáš
authors.initials: T
authors.lastname: Kouba
authors.forename: Piotr
authors.initials: P
authors.lastname: Neumann
authors.forename: Petra
authors.initials: P
authors.lastname: Beznosková
authors.forename: Ralf
authors.initials: R
authors.lastname: Ficner
authors.forename: Leoš Shivaya
authors.initials: LS
authors.lastname: Valášek
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics; D039621:Eukaryotic Initiation Factor-3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010442:Peptide Chain Initiation, Translational; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D039621:Eukaryotic Initiation Factor-3; C501391:GCN4 protein, S cerevisiae; C077915:NIP1 protein, S cerevisiae; D012333:RNA, Messenger; C111889:RPG1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000409:Alanine