Regulation of human 48S initiation complex formation
by Natalie Schmidt
Date of Examination:2024-04-17
Date of issue:2024-04-23
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Ralf Ficner
Persistent Address:
http://dx.doi.org/10.53846/goediss-10458
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Abstract
English
During the initiation phase of protein synthesis in humans, the small ribosome subunit interacts with initiator tRNA (tRNAi) and at least ten different initiation factors (eIFs) to form the initiation complex. The initiation complex is then recruited on the mRNA 5’ end and proceeds to mRNA scanning, i.e. the progression in 3’ direction towards the start codon. The scanning ends when the complex encounters a start codon. Start codon recognition by base pairing with tRNAi results in structural rearrangements of the small subunit and initiation factors that lock the initiation complex on the start codon of the mRNA, allowing the recruitment of the large ribosome subunit, mediated by eIF5 and eIF5B. However, the detailed structural and compositional dynamics of the initiation complex that promote scanning, mediate start codon recognition and recruit the large subunit on the initiation complex are not yet known. In this study, we employ single molecule Förster resonance energy transfer (smFRET) to follow the dynamics of eIF1A on individual initiation complexes during scanning, start codon recognition, and large subunit joining. eIF1A plays an important role throughout the entire process, while its intrinsically disordered N-terminal tail (NTT) is involved in regulation at all steps of initiation. We observe that eIF1A NTT interconverts between multiple conformations. However, as the initiation proceeds from step to step, different conformations of eIF1A NTT are stabilized. During scanning, eIF1A NTT is stably bound on the complex adopting predominantly a scanning-like conformation. However, upon start codon recognition, the NTT becomes highly dynamic showing fluctuations between different conformations. Additionally, in the late stages of initiation, the binding of eIF5 and eIF5B drive eIF1A into a novel conformation that notably precedes its dissociation right before large subunit joining. My data show, for the first time and in real time, how the regulatory eIF1A NTT interconverts between stable and dynamic modes to modulate translation initiation. The results suggest that the heterogeneity of eIF1A conformations in complex with the ribosome is crucial to monitor and regulate translation initiation at different steps. I anticipate this study to elucidate how the structural dynamics of key components of the initiation complex are regulated by the wide spectrum of start site contexts on human mRNAs.
Keywords: Human Translation Initiation; Single-Molecule FRET; Ribosome; eIF1A
