Kinetic Analysis of  Mammalian Translation Initiation

Yi, Sung-Hui

dc.contributor.advisor	Rodnina, Marina Prof. Dr.
dc.contributor.author	Yi, Sung-Hui
dc.date.accessioned	2021-01-22T11:58:56Z
dc.date.available	2021-12-13T00:50:03Z
dc.date.issued	2021-01-22
dc.identifier.uri	http://hdl.handle.net/21.11130/00-1735-0000-0005-1558-1
dc.identifier.uri	http://dx.doi.org/10.53846/goediss-8411
dc.language.iso	eng	de
dc.rights.uri	http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc	572	de
dc.title	Kinetic Analysis of Mammalian Translation Initiation	de
dc.type	doctoralThesis	de
dc.contributor.referee	Adio, Sarah Dr.
dc.date.examination	2020-12-15
dc.description.abstracteng	During translation initiation in mammalian cells, 80S ribosomes are assembled at the translation start site on mRNA via a cap-dependent scanning mechanism. The formation of the 80S initiation complex (IC) is essential for proper selection of the start codon and requires concerted action of at least 10 eukaryotic initiation factors (eIFs). This process can be divided into five stages: binding of eIFs to the 40S subunit to assemble the 43S pre-IC (PIC), mRNA recruitment to the 43S PIC, mRNA scanning, 48S IC formation upon start codon recognition, and finally, 80S IC formation after 60S subunit joining. Genetic and thermodynamic studies of translation initiation in lower and higher eukaryotes have revealed the major functions of individual eIFs. However, due to the lack of kinetic analysis, the precise order of events and the regulatory mechanisms underlying IC formation in mammals remain unknown. Here, we show the assembly of human 48S IC in real time using fluorescence-based rapid kinetic techniques in an in vitro reconstituted mammalian translation system. We find that eIF1A binds rapidly to two distinct states of the 40S subunit and stabilizes one of them by a two-population binding-equilibrium model. We also observe that the dissociation of eIF1A from the 40S subunit is slower in the presence of eIF1. By monitoring eIF1A dissociation rates, we are able to systematically analyze the kinetic stability of the partial initiation complexes en route to the 43S PIC or 48S IC. Depending on the combinations of initiation factors bound to the 40S subunit, the complexes take on one or two kinetically distinct states. The 43S PIC and scanning 48S PIC are single-population, thermodynamically and kinetically stable complexes. Upon start codon recognition, the 48S IC resumes its dynamic behavior with two kinetically distinct populations: one dissociating rapidly and the other slowly. Our results uncover key kinetic checkpoints that drive the thermodynamic equilibrium toward complete assembly of the 48S IC by the individual and combined action of initiation factors. This mechanistic understanding of the kinetically favored routes guiding initiation complex formation sets a milestone in deciphering the regulation of protein synthesis in mammalian cells.	de
dc.contributor.coReferee	Bohnsack, Markus Prof. Dr.
dc.contributor.thirdReferee	Tittmann, Kai Prof. Dr.
dc.contributor.thirdReferee	Stark, Holger Prof. Dr.
dc.contributor.thirdReferee	Urlaub, Henning Prof. Dr.
dc.subject.eng	Protein Biosynthesis	de
dc.subject.eng	Eukaryotic Translation	de
dc.subject.eng	Translation Initiation	de
dc.subject.eng	in vitro Reconstituted Translation System	de
dc.subject.eng	Human Ribosome	de
dc.subject.eng	48S IC	de
dc.subject.eng	80S IC	de
dc.subject.eng	eIF1A	de
dc.subject.eng	Kinetics	de
dc.subject.eng	Fluorescence-Based Kinetic Assay	de
dc.subject.eng	Stopped-Flow	de
dc.identifier.urn	urn:nbn:de:gbv:7-21.11130/00-1735-0000-0005-1558-1-1
dc.affiliation.institute	Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)	de
dc.subject.gokfull	Biologie (PPN619462639)	de
dc.description.embargoed	2021-12-13
dc.identifier.ppn	1745522751

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GGNB - Göttinger Graduiertenzentrum für Neurowissenschaften, Biophysik und molekulare Biowissenschaften [1112]
GGNB - Göttingen Graduate Center for Neurosciences, Biophysics and Molecular Biosciences

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