Assembly and control of the eukaryotic translation initiation complex

The information contained in the genes of living cells has to be converted into cellular components that form structures and enable biochemical reactions to take place. This process is called gene expression and it is vital to all life. Gene expression comprises two main steps, called transcription and translation. In transcription, the information in the DNA sequences of the genes is converted into equivalent sequences in so-called messenger RNA (mRNA) molecules. In translation, the mRNA molecules are 'read' by a large molecular structure called the ribosome, which uses the information to dictate the synthesis of proteins. In previous work, we have established that the ribosome undergoes large conformational changes in order to become competent for recruitment of mRNA molecules. These conformational changes are induced by the binding of a set of so-called initiation factors, the eIFs. We wish to capitalise on our exciting preliminary data in order to elucidate further details of the changes in the ribosome that enable protein synthesis to initiate and to understand how the interactions with the eIFs bring these changes about.

Recent studies of complexes between mammalian 40S and two distinct IRES RNAs have yielded informative structures of probable initiation-like complexes, indicating that the hepatitis C virus (HCV) and cricket paralysis virus (CrPV) IRESs induce conformational changes that may render the channel in the rabbit 40S subunit more accessible to RNA. The CrPV IRES interacts with the intersubunit side of the 40S close to the tRNA binding sites. In contrast, the HCV IRES was observed draped over the platform of the 40S subunit (with the majority on its solvent face) despite the fact that its binding is also thought to enhance access to the mRNA channel. The implication is that these IRESs utilise different sets of specific interactions with the head and platform domains of the 40S subunit to promote access to the mRNA channel. These results have led to the suggestion that each IRES may act like an RNA-based translation factor. However, our preliminary data indicate that the respective mechanisms of action of IRES RNAs and of eIFs are quite distinct. Moreover, the 40S-IRES structures raise a topological issue related to the threading of the RNA through the mRNA channel. This is especially important because, although initiation-like, these complexes are likely to represent steps on the translation initiation pathway subsequent to the loading of mRNA onto the ribosome. The aim of our work is to understand the structural changes leading up to accommodation of mRNA in the channel. The general aim of the present proposal is to obtain structural information about the fully assembled ribosomal 43S and 48S complexes of S.cerevisiae. The latter state represents the stage where a 40S subunit plus eIFs (43S complex) has been recruited to an mRNA and is 'scanning-enabled'. This information is vital to our understanding of the functional interactions between the eIFs and the eukaryotic ribosome.