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Biophysical characterization of the Escherichia coli RNA degradosome


Type

Thesis

Change log

Authors

Yang, Yuchen 

Abstract

In Escherichia coli, post-transcriptional regulation is a tightly controlled process facilitated by a multi-enzyme complex, the RNA degradosome. The core components of the RNA degradosome consist of the endoribonuclease RNase E, the DEAD-box helicase RhlB, the glycolytic enzyme enolase, and the exoribonuclease PNPase. The main scaffold component of the RNA degradosome, RNase E, contains an amphipathic alpha helix that recruits the degradosome to the cytoplasmic membrane. Investigations on the membrane binding properties of the RNase E as well as the RNA unwinding properties of RhlB were performed in this study.

Total internal reflection fluorescent (TIRF) microscopy of the RNA degradosome fluorescently labeled using a HaloTag showed discrete particles of the enzyme complex on a supported lipid bilayer on glass. To enable in situ studies of the degradosome, the HaloTag was encoded at the end of the rne gene within the E. coli chromosome using CRISPR/Cas. In order to extract the labeled degradosome from its native lipid environment for in vitro studies, efforts were made to generate nanodisc supports using amphipathic copolymers. Furthermore, various mutants in the membrane-tethering segment of RNase E were created in order to stabilize the complex by crosslinking onto the lipid membrane. Using biophysical methods, the association and dissociation constants and the thermodynamic parameters of the interactions of the degradosome with the lipid membrane were measured.

To investigate in detail on the helicase activity of the RNA degradosome, optical tweezers experiments were designed using the malE REP stabilizer as the substrate. A RNA/DNA hybrid was prepared to generate handles that facilitate incorporation into the device and labeling of the DNA components with biotin and digoxigenin was performed. Rigorous quality control tests demonstrated successful annealing of the RNA/DNA hybrid, paving the way for single-molecule studies of degradosome activity.

Description

Date

2022-05-11

Advisors

Luisi, Ben

Keywords

biophysics, E. coli, ITC, lipids, RNA, RNA degradosome, RNase E, single-molecule, SPR

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge