Dissertations, Theses, and Capstone Projects

Date of Degree

6-2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy

Program

Biochemistry

Advisor

Amedee des Georges

Committee Members

Oliver Clarke

Ranajeet Ghose

Diana Bratu

Subject Categories

Structural Biology

Keywords

43S, DHX29, ribosome

Abstract

The central dogma, proposed by Francis Crick in 1958, marks the direction of the flow of genetic information: from DNA to DNA (DNA replication), DNA to RNA (transcription), and RNA to protein (translation). Each of these is tightly regulated and important for cells and organisms to function correctly, grow, and reproduce.

In the eukaryotic system, the initiation stage of translation is the first, the rate-limiting, and the most regulated stage. It involves direct regulation of multiple eukaryotic initiation factors (eIFs), scanning factors such as DHX29, and other factors such as Poly-A binding protein (PABP). It starts with the small subunit of the 80S ribosomal subunit, the 40S ribosomal subunit. After the recycling of the 40S ribosomal subunit and initiation factors, the first step of eukaryotic translation initiation is the recruitment of the initiator tRNA to the 40S ribosomal subunit, which is bound with three initiation factors eIF1, eIF1A, and eIF3. The delivery of initiator tRNA starts with the formation of the Ternary Complex (TC), composed of the initiator tRNA, GTP, and eIF2, and forms the 43S Pre-Initiation Complex (PIC). The 43S PIC is then recruited to the m7G-cap of mRNA on its 5’-UTR, and the ribosomal association to the mRNA requires the eIF4F complex composed of eIF4A, eIF4E, and eIF4G. The ribosomal association of the mRNA marks the formation of the 48S Initiation Complex (IC). The translation initiation complex then scans the mRNA in the 5’-3’ direction until it recognizes the AUG start codon. Then the conformational change of the 48S IC triggers the release of eIF1, and the subsequent dissociation of other initiation factors that facilitate the joining of the 60S ribosomal subunit, which eIF5B mediates. The formation of the 80S ribosome marks the transition from the initiation stage to the elongation stage. Most such factors interact directly with the 40S ribosomal subunit, the central apparatus of translation. Since the early identification of initiation factors and the proposal of the scanning model of eukaryotic translation initiation in the 1970s, biochemical characterization and structural validation have significantly shed light on the mechanism of the recruitment of the initiator tRNA to the 40S ribosomal subunit and the mechanism of scanning; however, the exact structural role of individual initiation factor on this tightly-regulated pathway is yet to be explored. Such examples include the role of eIF1, eIF2, and eIF3 in promoting tRNA delivery, promoting mRNA association, and facilitating ribosomal scanning via the conformational change of the 40S ribosomal subunit. In addition, it remains elusive how the 40S ribosomal subunit scans structured mRNA, which is common in mammalian cells. Although DHX29 is characterized to be essential for this process, how it unwinds the mRNA secondary structures requires further investigation, especially considering that DHX29 is a non-processive helicase.

To answer the above questions, we used cryo-EM to visualize the reconstituted mammalian 43S PIC provided by our collaborators, Dr. Tatyana Pestova and Dr. Christopher Hellen. We collected four datasets in total, and extensive data processing improved the overall resolution of the mammalian 43S PIC to 3.1 Å. This is the first time for the 43S PIC to be captured in a closed state without the association of mRNA or other regulatory proteins. The cryo-EM structure shows the interaction network around tRNA in unprecedented detail and enables the direct visualization of the interaction between the 40S ribosomal subunit and eIF3’s octamer core. A focused classification with masks on the ternary complex and the eIF3’s octamer core successfully captured 43S PIC in 12 states, each containing different sets of initiation factors bound to the 40S ribosomal subunit. The comparison of the ribosome conformation between different states not only allows direct visualization of the allosteric regulation of the ribosomal mRNA channel by individual initiation factors but also implies a sequential model of the recruitment of the ternary complex, in which eIF1A recruits eIF1 that later recruits the β subunit of eIF2.

The ribosomal scanning on the structured mRNA additionally requires DExH-box RNA helicase DHX29. Although the cryo-EM structure of DHX29-bound 43S PIC was published back in 2015, the low resolution of 9 Å does not allow the accurate modeling of DHX29, and thus it was impossible to answer how DHX29 unwinds mRNA stem-loops during scanning. In a separate study, we used AI-empowered AlphaFold to predict the structure of human DHX29 and fit the prediction to a pre-determined cryo-EM structure of DHX29-bound 43S PIC, which allows us to build a precise and near-complete atomic model of human DHX29 bound to the 43S PIC. This helps us to decipher how DHX29 associates with the 40S ribosomal subunit, how the large insert of DHX29 inhibits its NTPase activity, and how it facilitates the unwinding of the mRNA stem loop by a 3’-5’ wrenching mechanism.

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