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Charakterizace dvou nejmensich podjednotek eIF3 a jejich úloh v translaci.
Cuchalová, Lucie ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Pospíšek, Martin (referee)
Protein synthesis or mRNA translation is a complex and highly conserved process. Translation consists of initiation, elongation, termination, and ribosome recycling stages. Since most regulation occurs during initiation, its mechanism is being studied intensively to elucidate the molecular basis of every potential control point. The initiation factor eIF3, which in yeast consists of five essential core subunits (eIF3a/TIF32, b/PRT1, c/NIP1, g/TIF35, and i/TIF34) and one transiently associated, non-essential subunit (j/HCR1), is undisputedly one of the key promoters of initiation. In addition, it has also been implicated in playing a critical role during ribosomal recycling, reinitiation, signal transduction, NMD etc. We have focused on determining the molecular mechanism of the roles of eIF3 and its associated eIFs not only in translation initiation but also in termination and in reinitiation. This included the biochemical and genetic mapping of yeast eIF3 binding site on the small ribosomal subunit, among others. We showed that the interaction between the residues 200-400 of a/TIF32-NTD and flexible C-terminal tail RPS0A significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo, thus a/TIF32-NTD together with the recently published PCI (proteasome...
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Complex structural and functional analysis of individual subunits of yeast translation initiation factor 3.
Herrmannová, Anna ; Valášek, Leoš (advisor) ; Krásný, Libor (referee) ; Pospíšek, Martin (referee)
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...
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Translational control in immune response.
Hlaváček, Adam ; Valášek, Leoš (advisor) ; Čáp, Michal (referee)
Immune reaction often requires a prompt modification of gene expression that in turn alters cellular physiology. There are an increasing number of articles supporting a critical role of translational control in this aspect of cellular biology. The aim of this work is to present some of cellular and molecular mechanisms that connect translational control and immune reaction in immune and somatic cells and can be possibly misused by some viruses. Perhaps not surprisingly, many immunologically relevant translational control mechanisms are similar to those acting during the stress response. Over the years it has been documented that the T cells, dendrocytes, Natural killer cells and macrophages utilize translational control for their immunological activation following stimulation. Combination of general and gene-specific translational control mechanisms enables fast changes in proteome and physiology that are characteristic for immune cell activation. The overall impact of translational control on immune response is further illustrated by the fact that it acts upon each stage of life of immune cells - from their activation, through survival, to a programmed cell death. Even in some non-immune cells the translational control plays an important role with respect to immunity, as these cells are known to have an...
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The role of the N-terminal domain of the a/TIF32 subunit of eIF3 in mRNA recruitment to the 43S pre-initiation complexes.
Vlčková, Vladislava ; Valášek, Leoš (advisor) ; Mašek, Tomáš (referee)
Translation initiation is a complex process which results in the assembly of the elongation competent 80S ribosome from the 40S and 60S ribosomal subunits, the initiator tRNA and mRNA, and is orchestrated by numerous eukaryotic initiation factors (eIFs). Although it represents one of the most regulated processes of gene expression, the exact mechanism of one of the key steps of translation initiation - mRNA recruitment to the 43S pre-initiation complex (PIC) - is still only poorly understood. Recent studies indicated that besides eIF4F and poly(A)-binding protein, also eIF3 might play an important, if not crucial, role in this step. In our laboratory, we recently identified a 10 Ala substitution (Box37) in the a/TIF32 subunit of Saccharomyces cerevisiae eIF3, which interfered with translation initiation rates. Detailed analysis showed that this mutation significantly reduces the amounts of model mRNA in the gradient fractions containing 48S PICs as the only detectable effect in vivo. Moreover, a recently solved crystal structure of the N-terminal part of a/TIF32 pointed to two Box37 residues, Arg363 and Lys364, both proposed to contribute to one of the positive, potentially RNA-binding areas on the a/TIF32 surface. The fact that also their substitutions with alanines severely impaired the mRNA recruitment...
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Bacillus subtilis ribosomes: regulation of ribosomal RNA biosynthesis and identification of the new ribosomal protein YbxF
Sojka, Luděk ; Jonák, Jiří (advisor) ; Valášek, Leoš (referee) ; Lichá, Irena (referee)
1 Abstract The biology of the bacterial ribosome of gram positive bacterium Bacillus subtilis is the central point of this thesis that includes studies of both ribosomal components - ribosomal RNA (rRNA) and one of ribosomal proteins. The first part of the thesis focuses on the regulation of rRNA synthesis and the second part focuses on the identification and characterization of a new ribosomal protein, YbxF. rRNA synthesis is mostly regulated at the level of transcription initiation. Initiating nucleoside triphosphates (iNTPs) are important molecule effectors that regulate this process. Varying iNTP concentration in the cell directly affects RNA polymerase (RNAP) at rRNA promoters as these promoters are sensitive to [iNTP] in vivo. Most of the knowledge about this regulation is derived from Escherichia coli, where the rRNA promoter sequence is key for this regulation. Nevertheless, sequence characteristics of [iNTP]-regulated rRNA promoters from gram positive bacterium B. subtilis do not emulate the sequence characteristics derived from [iNTP]-regulated rRNA promoters from gram negative bacterium E. coli. Using a combination of in vitro and in vivo approaches, we determined promoter DNA elements that are responsible for [iNTP] sensitivity of ribosomal and non ribosomal promoters in B. subtilis. The second...
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Dissection of eIF3 functional domains promoting the 48S pre-initiation complex assembly
Beznosková, Petra ; Valášek, Leoš (advisor) ; Novotný, Marian (referee)
In eukaryotes, translation initiation is guided by up to twelve protein initiation factors (eIFs) and begins with the formation of the 43S pre-initiation complex (PIC) composed of the small ribosomal subunit (40S), eIF2.GTP/Met-tRNAi Met ternary complex, and eIFs 1, 1A, 3 and 5. The 43S PIC subsequently interacts with the 5'end of an mRNA (an mRNA recruitment step) and thus formed 48S PIC travels in 5' to 3' direction along the mRNA leader sequence to locate the AUG start codon (this presumably linear movement is generally known as scanning). Start site selection results in the dissociation of the initiation factors and joining of the large (60S) ribosomal subunit to form the 80S initiation complex poised for elongation. Eukaryotic initiation factor 3 (eIF3) plays a critical role in most of these events; however, the molecular details of most of its contributions are still unknown to us. Previous in vivo studies generated numerous mutations in all eIF3 subunits with specific defects either in the PICs assembly or in the following steps such as scanning, AUG recognition, etc. To understand the exact role of eIF3 in this intriguing process at the molecular level, we have embarked on a study that aims to dissect the individual functions of each eIF3 subunit in translation initiation using the purified...
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Translation reinitiation mechanism on mRNA of trascriptional activator GCN4.
Vlčková, Vladislava ; Valášek, Leoš (advisor) ; Strachotová, Dita (referee)
Translation reinitiation is a gene-specific translational control mechanism exploiting the ability of some short upstream open reading frames (uORFs) to retain post-termination 40S ribosomal subunit on the mRNA. Reinitiation efficiency depends on cis-acting sequences surrounding the uORF, translation elongation rates on the uORF, selected initiation factors, and the intercistronic distance of the short uORF from the main ORF. Although the precise mechanism of reinitiation is still not known, great progress in elucidating some of its details has been recently made with help of the GCN4 translational control model system. Among them, involvement of eIF3 was shown to play a critical role for efficiency of this process. In particular, it was proposed that eIF3 specifically interacts with sequences located upstream of a reinitiation-permissive uORF upon termination, and that this step is instrumental in stabilizing the 40S ribosomal subunit on the mRNA to allow subsequent resumption of scanning for reinitiation downstream. In this thesis, the current knowledge of the translation reinitiation mechanism is summarized. As a typical example, the yeast transcriptional activator GCN4 has been chosen, the mRNA of which is subjected to a tight translational control via the very reinitiation mechanism.
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The role of eIF3 in mRNA recruitment to the 43S pre-initiation complex
Beznosková, Petra ; Mašek, Tomáš (referee) ; Valášek, Leoš (advisor)
Translation initiation in eukaryotes plays an important role in gene expression and relies on interactions between many eukaryotic initiation factors, small and large ribosomal subunits, mRNA and initiator tRNA. The largest of these initiation factors, the eukaryotic initiation factor 3 (eIF3), participates in the most of translation initiation reactions. In yeast, eIF3 occurs together with eIF1, eIF5 and ternary complex (TC, eIF2-GTP-Met-tRNAi Met complex) in the multifactor complex (MFC) and promotes formation of the 43S preinitiation complexes. eIF3 also stimulates mRNA recruitment to the 43S preinitiation complexes, scanning the 5' untranslated region of mRNA for AUG recognition and has an important role in the gene-specific translational control mechanism called reinitiation involving short upstream open reading frames (uORFs).
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Functional analysis of the TIF32-HLD-PRT1-RRM-HCR1 module of the yeast translation initiation factor 3
Herrmannová, Anna ; Valášek, Leoš (advisor) ; Mašek, Tomáš (referee)
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....
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