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Ribosome assembly factors Nsa2 and Rsa4 connect the ATPase Rea1 to the maturing catalytic center of the large subunit

Paternoga, Helge

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Abstract

Ribosome biogenesis is a highly complex process, which in eukaryotes depends on a myriad of assembly factors, including several energy-consuming enzymes. One of these is the ATPase Rea1, that is necessary for the formation of large ribosomal subunits. Rea1 is responsible for the removal of several assembly factors, including Rsa4, during a late step in 60S biogenesis. This release depends on a Rea1-generated pulling force, that is transmitted to Rsa4 and eventually results in its dissociation from pre-ribosomes. It is therefore of high interest to identify, which proteins or rRNA elements connect Rsa4 to the pre-ribosome, as these could transmit the Rea1 power stroke to the maturing 60S subunit and result in structural rearrangements at their binding site. This study builds on initial findings, that the 60S assembly factor Nsa2 is a putative interaction partner of Rsa4. Using genetic and biochemical approaches, I was able to verify the interaction and demonstrate, that it is essential for yeast growth. Furthermore, I was able to crystallize the Nsa2-Rsa4 hetero-dimer and the structure was solved in collaboration with the lab of Dr. Irmi Sinning (BZH, Heidelberg). A subsequent structurefunction analysis revealed the molecular details of the Nsa2-Rsa4 interaction and its impact on 60S biogenesis. Moreover, I was able to fit the Nsa2-Rsa4 crystal structure in an EM-volume of the Arx1 pre-ribosome, that places Nsa2 and Rsa4 at the nascent peptidyl transferase center (PTC). Here, Rsa4 is bound to the immature central protuberance and Nsa2 is oriented towards the nascent tRNA binding site. Using Nsa2 NMR structures, which were generated in collaboration with the lab of Dr. Elisar Barbar (Oregon State University), and crosslinking data from Dr. Sander Granneman (University of Edinburgh), I propose a model in which the globular C-domain of Nsa2 is located at the maturing peptidyl transferase center and the α-helical N-domain of Nsa2 reaches around immature rRNA helix 89 towards the P stalk region. Nsa2 and Rsa4 therefore connect Rea1 to the maturing PTC, which suggests an additional function of the Rea1-generated pulling force beyond the mere removal of assembly factors. The positioning and functional analysis of Nsa2 implies, that Rea1 exerts a mechanical force on immature helix 89, which is necessary for assembly of the catalytic center during 60S biogenesis.

Item Type: Dissertation
Supervisor: Hurt, Prof. Dr. Ed
Date of thesis defense: 21 June 2017
Date Deposited: 10 Jul 2017 08:24
Date: 2017
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 570 Life sciences
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