Crystal structure of eukaryotic ribosome functional complexes – CSERFC

The mechanism of translation in eukaryotes is very complex, particularly at the stage of initiation, which requires as many as 9 initiation factors, which can be a target for regulation of gene expression. During initiation, the 40S ribosomal subunit with initiation factors (eIF) 1, 1A, 2 and 3 attach to the 5'-end of mRNA (43S complex) and then scan to the initiation codon. Capped 5'-end of mRNA is recognized by initiation factors 4A, 4B, 4E and 4F. Subsequent joining of a 60S subunit requires in addition the initiation factors 5 and 5B. Despite the fact that all factors required for assembly of elongation-competent 80S ribosomes have been identified, and their principal role in this process have been determined, the molecular mechanism of even the most fundamental steps in initiation (e.g. ribosomal attachment to mRNA and subsequent ribosomal scanning) remain unknown, largely because of the lack of high resolution structures of the eukaryotic ribosome as well as of architectural models of initiation complexes. In contrast to the 40S subunit scanning for initiation of translation, some viruses including hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. Initiation on the hepatitis C virus IRES is well studied: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit. The primary aim of this proposal is to obtain the X-ray structural data that are required for detailed understanding of the mechanism of eukaryotic translation, particularly its initiation stage. The specific aims of the proposed research include: 1) crystallization of 80S ribosomes and 40S subunits in parallel from yeast and from chicken embryos for determination of their structures at medium to high resolution, 2) crystallization and determination of the structure of 80S and 40S ribosomal complexes with functional ligands (e.g. mRNA and tRNAs), 3) crystallization and structure determination of ribosome complex containing mRNA with a viral internal ribosome entry site (IRES). In summary, these experiments are designed to provide a foundation for understanding the molecular architecture of eukaryotic ribosome and to develop a method for detailed investigation of the mechanism of initiation, the most complex stage of eukaryotic translation.