자료유형  

 학위논문

Control Number  

0017164024

International Standard Book Number  

9798346567516

Dewey Decimal Classification Number  

575

Main Entry-Personal Name  

Bridgers, Joseph Benjamin.

Publication, Distribution, etc. (Imprint  

[S.l.] : Harvard University., 2024

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2024

Physical Description  

110 p.

General Note  

Source: Dissertations Abstracts International, Volume: 86-05, Section: B.

General Note  

Advisor: Churchman, L. Stirling.

Dissertation Note  

Thesis (Ph.D.)--Harvard University, 2024.

Summary, Etc.  

요약The mitochondrial genome of the budding yeast Saccharomyces cerevisiae encodes seven components of the oxidative phosphorylation (OXPHOS) machinery, which are synthesized by specialized mitochondrial ribosomes. These OXPHOS components are required to generate ATP, the main source of cellular energy. Nuclear-encoded mitochondrial translational regulators activate or repress translation of mitochondrial mRNAs in a transcript-specific manner. Very little is known about the molecular details of how and when these translational regulators interact with the mitoribosome during the translation cycle.I developed selective mitochondrial ribosome profiling (sel-mitoRP) to study the interactions of translational regulators with the mitoribosome at near-codon resolution. I applied sel-mitoRP to several translational activators (TAs). I found that most of the TAs studied are specifically enriched on mitoribosomes translating their target transcript. Within a given target transcript, the TAs exhibit the highest levels of enrichment in the 5' untranslated region (UTR) just upstream of the start codon. I hypothesized that the TAs bind to the 5' UTR of their target transcript to position the mitoribosome at the start codon so that translation initiation can occur. We validated our hypothesis by purifying mitoribosomes stalled at initiation and solving two cryo-EM structures. The first of these two structures revealed a heterotrimeric complex of the three activators of ATP9 (Aep1, Aep2, and Atp25) bound to the small subunit of the mitoribosome at the mRNA exit channel. An mRNA density corresponding to the 5' UTR of ATP9 can be visualized exiting the mitoribosome and wrapping around the ATP9 TA complex. The second class of particles in our stalled initiation complex contained Aep3, the translational activator of ATP8, bound to the small subunit at the mRNA exit channel. We can also resolve mRNA density representing the 5' UTR of ATP8 wrapping around Aep3. Both of these structures validate our hypothesis that TAs bind to the 5' UTR of the target transcript and engage with mitoribosomes at initiation to help position them at the start codon.Finally, I studied the co-translational interactions of Smt1, the translational repressor of ATP6/8, and Oxa1, the mitochondrial inner membrane insertase. I found that Smt1 was enriched during translation elongation of ATP6/8, binding to the ribosome only following the depletion of ATP6/8 TAs. Oxa1 was previously thought to be constitutively bound to the mitoribosome through a C-terminal ribosome binding domain. Sel-mitoRP for Oxa1 revealed that Oxa1 engagement with the mitoribosome is much more dynamic than was previously appreciated. Oxa1 only engaged with the mitoribosome following emergence of the transmembrane domains of its client proteins. Interestingly, Oxa1 enrichment also coincided with the depletion of many TAs from the mitoribosome. We propose that membrane insertion promotes release of TAs from the elongating mitoribosome.These studies offer molecular insight into how translation activation and co-translational protein insertion are regulated in mitochondria. This work establishes sel-mitoRP as a versatile tool to study multiple aspects of mitochondrial translational regulation. Future work will utilize sel-mitoRP to study translational regulators and insertases in human cells.

Subject Added Entry-Topical Term  

Genetics.

Subject Added Entry-Topical Term  

Biochemistry.

Subject Added Entry-Topical Term  

Cellular biology.

Subject Added Entry-Topical Term  

Molecular biology.

Index Term-Uncontrolled  

Mitochondria

Index Term-Uncontrolled  

Ribosome profiling

Index Term-Uncontrolled  

Structural biology

Index Term-Uncontrolled  

Translational activators

Index Term-Uncontrolled  

Translational repressors

Index Term-Uncontrolled  

Yeast

Added Entry-Corporate Name  

Harvard University Medical Sciences

Host Item Entry  

Dissertations Abstracts International. 86-05B.

Electronic Location and Access  

로그인을 한후 보실 수 있는 자료입니다.

Control Number  

joongbu:654416