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dc.contributor.authorJames, Nathan Rhys
dc.date.accessioned2017-10-09T08:10:21Z
dc.date.available2017-10-09T08:10:21Z
dc.date.issued2017-10-03
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/267783
dc.description.abstractTranslation is the process by which proteins are synthesized from the instructions in the genetic code. Translation is mediated by the ribosome, a large ribonucleoprotein complex, in concert with messenger RNA (mRNA), transfer RNA (tRNA), and a variety of proteins. The canonical mechanism of translation, introduced in Part I of my thesis, is divided into four distinct phases: initiation, elongation, termination, and recycling. Under unusual circumstances, each phase of translation can also proceed via a number of noncanonical mechanisms, many of which are vitally important for cellular growth or viral infectivity. My thesis describes structural insights into two such noncanonical mechanisms. The aim of the first project, described in Part II, was to structurally characterize a noncanonical mechanism of translational termination in bacteria. In the absence of a stop codon, ribosomes arrest at the 3′ end of an mRNA and are unable to terminate. In bacteria, the primary mechanism for rescuing such nonstop complexes is known as trans-translation. In the absence of a functional trans-translation system, however, the small protein ArfA recognizes the empty mRNA channel and recruits the release factor RF2 to the ribosome, enabling termination to occur. Using single-particle electron cryomicroscopy (cryo-EM), I obtained four high-resolution structures of nonstop complexes that reveal the mechanism of ArfA-mediated ribosome rescue and have wider implications for understanding canonical termination in bacteria. The aim of the second project, described in Part III, was to gain structural insights into a noncanonical mechanism of translational initiation in eukaryotes known as internal ribosome entry. Instead of a 5′ cap, many viruses contain intricately structured, cis-acting internal-ribosome-entry sites (IRESs) within their genomes that direct end-independent initiation. The IRES of hepatitis-C virus (HCV), for example, interacts directly with the mammalian ribosome and functionally replaces many of the canonical initiation factors. However, the mechanism by which the HCV IRES coordinates assembly of an initiation complex and progresses through the initiation phase remains poorly understood. I developed a method for purifying native ribosomal complexes from cell lysate that enabled me to obtain multiple cryo-EM maps of the HCV IRES in complex with the 80S ribosome, including a previously unseen conformation of the IRES induced by rotation of the ribosomal small subunit, and to make progress towards capturing earlier steps in the initiation pathway.
dc.description.sponsorshipMedical Research Council
dc.formatAdobe Acrobat
dc.language.isoen
dc.rightsAll Rights Reserveden
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/en
dc.subjectstructure
dc.subjectribosome
dc.subjectcryo-EM
dc.subjectArfA
dc.subjectHCV
dc.subjectIRES
dc.subjecttranslation
dc.subjectinitiation
dc.subjecttermination
dc.subjectribosome rescue
dc.subjectinternal ribosome entry
dc.subjectelectron cryomicroscopy
dc.subjecthepatitis C
dc.titleStructural Insights into Noncanonical Mechanisms of Translation
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.publisher.departmentMRC Laboratory of Molecular Biology
dc.date.updated2017-10-07T11:30:02Z
dc.identifier.doi10.17863/CAM.13713
dc.contributor.orcidJames, Nathan Rhys [0000-0003-1147-6285]
dc.publisher.collegeClare Hall
dc.type.qualificationtitlePhD in Biological Sciences
cam.supervisorRamakrishnan, Venkatraman
cam.supervisor.orcidRamakrishnan, Venkatraman [0000-0002-4699-2194]
rioxxterms.freetoread.startdate2017-10-07


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