|M.Sc Student||Yitzhaki Ofer|
|Subject||Studying IRES Mediated Translation in MBF1 mRNA in|
|Department||Department of Biotechnology||Supervisor||PROF. Mordechai Choder|
|Full Thesis text|
Translation of most eukaryotic mRNAs occurs by traditional cap-dependent ribosome scanning. However, alternative cap-independent mechanism was discovered in viral and in cellular mRNAs. The most studied cap-independent mechanism is the Internal Ribosome Entry Site(s) (IRES)-mediated translation. The IRES element is a structured RNA sequence, which enables ribosomes to be recruited onto the mRNA in a cap-independent mechanism during cellular stress. A bicistronic construct is used as a standard assay for demonstrating IRES-mediated initiation, where suspected IRES element is inserted between two reporter genes. We constructed a bicistronic vector, which contains a library of 5' UTR of yeast genes in between the two cistrons, and found potential elements that function as IRESes. We further studied an IRES element whose origin was the MBF1 gene. Mbf1p acts as coactivator and serves as an adaptor between the DNA binding domain of TBP (TATA binding protein) and Gcn4p. We wished to examine whether the MBF1 IRES element is functional within the natural mRNA context and whether it directs translation to the major start codon or to downstream codons. Using a series of control experiments, we ruled out the possibility that the intercistronic sequence (MBF1 IRES) promoted leaky scanning and re-initiation or served as cryptic promoter. In order to study the MBF1 IRES in its natural context, we have cloned the whole genomic MBF1. In spite of the fact that the IRES found by the bicistronic construct was located upstream and downstream of the translation start site, in the natural MBF1 mRNA this IRES promotes translation from the major start site. Based on our results, we suggest that the "ribosome entry window" of MBF1 IRES extends to both sides of the initiation site. The activity of MBF1 IRES in the bicistronic construct was enhanced during the shift from logarithmic phase to early stationary phase (SP). In contrast, Mbf1p is kept constant in log and in SP. We assume that the MBF1 mRNA level decreases during the shift from log to SP, like most of the yeast mRNAs. If MBF1 mRNA decreases during the shift from log to SP, than enhanced IRES-mediated translation maintains almost constant levels of the protein product. Alternatively, the different expression pattern of the natural and the IRES-driven expression from the bicistronic construct suggests that the IRES present in the bicistronic construct is only a portion of the entire IRES. Additional cis-elements in the natural context may be responsible for Mbf1p constant level.