Structural and functional investigation of the protein synthesis in saccharomyces cerevisiae

Abstract

Protein synthesis is an important house-keeping activity of all the cells. It follows the same concepts in all three domains of life: the genetic code is deciphered and transcribed into the mRNA, which in turn is translated into the polypeptide by the help of the ribosome. Translation of proceeds through four steps: initiation, elongation, termination and ribosome recycling. Despite following the same concepts, different steps of translation show certain domain-specificities. One of the hall-marks of eukaryotic initiation, is the complex initiation step, requiring intricate interaction of several protein factors called eukaryotic initiation factors (eIFs). The largest eukaryotic initiation factor, eIF3, is a multi-subunit complex which serves as a scaffold to which other initiation factors bind. It facilitates mRNA recruitment and assembly of other initiation factors on the 40S ribosomal subunit. Its complex and flexible nature hinders its purification for structural and biochemical studies. The major part of the present work, which is presented in chapter two, was to establish a protocol for recombinant purification and in vitro assembly of Saccharomyces cerevisiae eIF3. This complex was subjected to structural studies by single-particle electron microscopy. Initial results were obtained regarding positioning of eIF3 on the 40S subunit, showing its binding to the solvent exposed side of the small ribosome subunit. In vitro reconstitution of eIF3, in combination with Limited proteolysis and mass-spectrometry, allowed the formation and analysis of its different subcomplexes and stable fragments which form the core of eIF3 complex. In addition, fluorescence-labeling of eIF3 introduced new strategy for studying the kinetics of translation initiation and order of complex formation. One of the large subunits of eIF3, eIF3b/Prt1, serves as a scaffold within eIF3 as it interacts with several other subunits. It harbors an RNA Recognition Motif (RRM), which is shown to be a non-canonical RRM in human as it is not capable to interact with oligonucleotides, but rather interacts with eIF3j/Hcr1, a sub-stoichiometric subunit of eIF3. In chapter three, the high-resolution crystal structure of the eIF3b RRM domain from yeast is presented. It exhibits the same fold as its human ortholog. Thermodynamic analysis of the interaction between yeast eIF3b-RRM and eIF3j as well the conservation of the eIF3j binding site between human and yeast eIF3b-RRM suggested that the same mode of interaction between eIF3b and eIF3j in both organisms. However, analysis of the surface charge distribution of the putative RNA-binding â-sheet as well as the conservation of its RNA binding elements, suggested that in contrast to its human ortholog, yeast eIF3b-RRM could potentially bind oligonucleotides. Interaction studies with yeast total RNA extract confirmed the proposed RNA binding activity of yeast eIF3b-RRM. eIF3j/Hcr1, a loosely associated subunit of eIF3 has been previously shown to interact with Rli1, an iron-sulfur-cluster containing member of the super-family of ABC ATPases. In addition to translation initiation, Rli1 plays roles in ribosomal subunit maturation and transport of both ribosomal subunits into the cytoplasm. In chapter four, a novel function for Rli1 in translation termination is presented. Rli1 was shown to physically interact with the translation termination factors eRF1/Sup45 and eRF3/Sup35 in Saccharomyces cerevisiae. Genetic interactions were uncovered between a strain depleted for Rli1 and sup35-21 or sup45-2. Further, down regulation of the RLI1 expression was shown to cause defects in the recognition of a stop codon, as seen in mutants of other termination factors. In chapter five, results obtained from different projects are discussed in a broad perspective. Furthermore, an expansion of the presented data is provided which should shed light on the herein presented results. The future perspective of the projects as well as suggestions for further experiments are presented in chapter six.