Structural Studies of Translation

Cellular translation is one of the fundamental processes that make life possible. It is the point at which stored information, coded by the nucleic acid, is read and converted into three-dimensional functional proteins. By linking together the correct sequence of amino acids, spontaneous order is created. It is truly a remarkable process. The plechanism by which this is achieved can be understood in physical terms, by the structure of the molecular machinery involved. These structures are dynamic and elegant and we can observe them in action. Through the application of the techniques of structural biology I undertake some structural studies of the translation process, focussing on the mechanism in eukaryotes. In the first instance I use X-ray crystallography to determine the structure of an enzyme that demonstrates a novel function in translation, Nhm1. This protein is an enzyme that removes the cap structure from messenger RNA, which is an important determinant of the cellular stability of this molecule. Usually this class of enzyme works only on free cap structures, after the message has been degraded. Four forms of the molecule are investigated: an apo form, a mutant form, a GTP-bound form and a cap-bound form. The structure is compared to other decapping enzymes to try and identify the basis of its novel function. Next I use cryo-electron microscopy to investigate how viral RNA can manipulate its host's ribosomes during translation. Many viral RNAs use RNA secondary structure to physically shift the ribosome back one base (-1) between reading frames. By visualising the apo mammalian ribosome, as well as a complex with a viral pseudoknot structure and a complex with a stemloop structure formed from deletion of loops in the pseudoknot I have been able to dissect out the changes induced by a functional pseudoknot structure. This has revealed a mechanistic explanation of pseudoknot function in -1 frameshifting and provided the first insights into the functional interaction of the ribosomal translocase with the tRNA during translocation. In addition I have helped develop a reconstruction method that can deal with the problems associated with preferred particle orientations on a cryo-EM grid and begun investigations into the structures of eukaryotic initiation factor 3 (eIF3) and a kinase important in regulating translation initiation, Mnkl.EThOS - Electronic Theses Online ServiceGBUnited Kingdo