Document Type

Project

Publication Date

Spring 2013

Keywords

Ribosome, Protein L12, GTP hydrolysis

Abstract

The ribosome is a complex macromolecular machine that is responsible for the synthesis of proteins from a nucleic acid template. This process is largely regulated by various protein translation factors, many of which are GTPases. Ribosome-dependent GTPase activity has been observed to be coincident with the presence of ribosomal protein L12. Of current interest is to understand how L12 interacts with the GTPase factors on the 705 ribosome. A key to the investigation of these interactions is to produce ribosomes fully depleted of L12 for comparisons of factor activity and binding in the presence and absence of this protein. Here, we present a novel two-step depletion protocol that takes advantage of the JE28 ribosomes' engineered C-terminal (His)G-tag chromosomally encoded on protein L12. Fully depleted ribosomes were shown to be absent of L12 in Western blotting studies. Furthermore, these 705 ribosomes were shown not to stimulate ribosome-dependent GTP hydrolysis by translation factor EF-G in malachite green GTP hydrolysis assays. This population of ribosomes purified in the complete absence of protein L12 will make possible investigations of factor binding and ribosome-dependent GTP hydrolysis to further elucidate the role of L12 in translation.

Department

Chemistry

Subjects - Topical (LCSH)

Guanosine triphosphatase--Inhibitors; GTPase-activating protein; Ribosomes

Genre/Form

student projects; term papers

Type

Text

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

Rights Statement

http://rightsstatements.org/vocab/InC/1.0/

Language

English

Format

application/pdf

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Included in

Chemistry Commons

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