자료유형  

 학위논문

Control Number  

0017162209

International Standard Book Number  

9798383164310

Dewey Decimal Classification Number  

574

Main Entry-Personal Name  

Sheng, Kai.

Publication, Distribution, etc. (Imprint  

[S.l.] : The Scripps Research Institute., 2024

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2024

Physical Description  

297 p.

General Note  

Source: Dissertations Abstracts International, Volume: 85-12, Section: B.

General Note  

Advisor: Williamson, James R.

Dissertation Note  

Thesis (Ph.D.)--The Scripps Research Institute, 2024.

Summary, Etc.  

요약Ribosomes play a crucial role in protein translation, and their assembly is both intricate and rapid within cells. For E. coli, it takes approximately two minutes to produce a functional ribosome from scratch. Investigating the complex and rapid folding mechanism of large RNA-protein complexes, such as the ribosome, remains a formidable challenge in structural biology.In Chapter 2, a streamlined pipeline was developed for solving ribosomal large subunit (LSU) assembly intermediates in genetically manipulated strains, such as those depleted of ribosomal proteins and assembly factors. This pipeline, including heterogeneous reconstruction methods, cooperativity assembly block identification, construction of networks between assembly blocks, and delineation of putative assembly pathways, was applied to three datasets: ∆deaD, ∆srmB, and a bL17-depletion strain. This novel approach facilitated the robust discovery of the smallest assembly core containing only 600 nucleotides and four ribosomal proteins, as well as the assembly pathway for early events in LSU solvent-side maturation at the early stage.Chapter 3 utilized this pipeline to discover 14 distinct LSU intermediates ranging from early to late stages in wild-type E. coli grown at 12 ˚C, providing insights into detailed assembly dependencies, including the formation of the central protuberance, stalks, and peptidyl transferase center. Additionally, the serendipitous trapping of an early intermediate by a pseudouridine synthase provided novel insights into early-stage assembly factor binding and substrate recognition.In Chapter 4, specific anti-sense oligonucleotides (ASOs) and their analogs were employed to disrupt native interactions of bacterial ribosomal RNA, capturing assembly intermediates in a near-physiological in vitro reconstitution platform. A screen identified 10 peptide nucleic acid (PNA) hits, with 6 progressing to structure characterization. This effort led to the discovery of 37 structures never reported before, revealing the smallest consensus core comprising 9 helices and 2 proteins. Notably, a major conformational rearrangement for domain III and VI rRNA was observed in a PNA-inhibited library, indicating that intra-domain folding can precede inter-domain docking in LSU assembly. Moreover, this approach further dissected the LSU into 158 structural smaller segments, enabling detailed assembly at single helix resolution, following a template-directed RNA foldon docking mechanism.Subsequently, Chapter 5 established a high-throughput dCas13/sgRNA two-plasmid system for in vivo rRNA targeting. The screening resulted in potential competitors in rRNA folding within the cellular environment. Two of the hits were cloned out and the disruption of LSU assembly has been verified by Cryo-EM single particle analysis. The platform also eluded possible assembly mechanisms in wild type and assembly factor deletion strains.In summary, this thesis successfully streamlined the generation of bacterial LSU assembly intermediates by various perturbations, solved heterogeneous assembly structures using Cryo-EM single particle analysis, and performed dependency and pathway analysis, thereby unveiling a landscape for bacterial LSU assembly.

Subject Added Entry-Topical Term  

Biology.

Subject Added Entry-Topical Term  

Biochemistry.

Subject Added Entry-Topical Term  

Biophysics.

Index Term-Uncontrolled  

Ribosome

Index Term-Uncontrolled  

Nucleotides

Index Term-Uncontrolled  

Anti-sense oligonucleotides

Index Term-Uncontrolled  

Cellular environment

Index Term-Uncontrolled  

Structural biology

Added Entry-Corporate Name  

The Scripps Research Institute Structural Biology/Biophysics

Host Item Entry  

Dissertations Abstracts International. 85-12B.

Electronic Location and Access  

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Control Number  

joongbu:655225