Dynamic and mechanistic studies of processes during bacterial ribosomal recycling / Yuanwei Chen.

Format:

Book

Manuscript

Thesis/Dissertation

Author/Creator:

Chen, Yuanwei, author.

Contributor:

Cooperman, Barry S., degree supervisor.

Dmochowski, Ivan J., degree committee member.

Baumgart, Tobias, degree committee member.

Kaji, Akira, degree committee member.

University of Pennsylvania. Department of Chemistry, degree granting institution.

Language:

English

Subjects (All):

Penn dissertations--Chemistry.

Chemistry--Penn dissertations.

Local Subjects:

Penn dissertations--Chemistry.

Chemistry--Penn dissertations.

Physical Description:

xv, 166 leaves : illustrations (some color) ; 29 cm

Production:

[Philadelphia, Pennsylvania] : University of Pennsylvania, 2015.

Summary:

In bacteria, nascent peptide is released from ribosome after the completion of protein synthesis, leaving a ribosome complex with deacylated transfer RNA (tRNA) and messenger RNA (mRNA) at the stop codon, referred as post-termination complex (PoTC). Ribosome recycling refers to the disassembly of PoTC by the action of ribosome release factor (RRF) and elongation factor G (EF-G) complexed with GTP. Nowadays it is widely accepted that recycling is an essential step in bacterial translation. Three processes, the release of mRNA and tRNA from ribosome and the splitting of ribosome into subunits, occur during ribosome recycling. Controversies remain on the mechanism of ribosome recycling, especially the sequence of the three processes. Two incompatible models with different sequence of processes have been proposed.

For better understanding on the mechanism of ribosome recycling, we implemented the stopped-flow fluorescence anisotropy and light scattering techniques to monitor the kinetics of three processes. Our results demonstrate a sequence in which the release of mRNA from ribosome is the first process induced by RRF/EF-G.GTP, followed by the release of tRNA from ribosome, after which the ribosome splits into subunits.

To reconcile our results with those obtained previously, we also studied the effects on the kinetics and sequence of the three processes in ribosome recycling of several factors, including the incorporation of an upstream Shine-Dalgarno (SD) sequence, the presence of initiation factor 3(IF3), the varying of magnesium ion the replacement of GTP with GDPNP or GDP, the identity of tRNA and the presence of three antibiotics. Our results demonstrate the incorporation of upstream SD sequence significantly reduces the rates of mRNA and tRNA release so that they become similar in magnitude to the rate of ribosome splitting. This suggests that the presence of internal SD sequence proximal to the stop codon may acts as a mechanism to modulate the rate of protein synthesis in bacteria. Although the other factors examined can affect the rates of the three processes, the sequence of these processes in ribosome recycling is unaffected. Our results also show that the release of mRNA and tRNA do not require GTP hydrolysis, and confirm that GTP hydrolysis is required for ribosome splitting.

Notes:

Ph. D. University of Pennsylvania 2015.

Department: Chemistry.

Supervisor: Barry S. Cooperman.

Includes bibliographical references.

OCLC:

950747172