Poster abstracts
Poster number 50 submitted by Sepideh Fakhretaha Aval
Role of the sarcin-ricin loop of 23S rRNA in biogenesis of the 50S ribosomal subunit
Sepideh Fakhretaha Aval (Ohio state Biochemistry program, The Ohio State University, Columbus, OH 43210, USA.), Amal Seffouh (Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, Canada), Kyung-Mee Moon (Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.), Leonard J. Foster (Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.), Joaquin Ortega (Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 0C7, Canada.), Kurt Fredrick (Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA.)
Abstract:
The sarcin-ricin loop (SRL) is one of the most conserved segments of the large subunit ribosomal RNA (rRNA). Translational GTPases (trGTPases), such as EF-G, EF-Tu, and IF2, interact with the SRL, and this interaction is essential for GTP hydrolysis and factor function. Cleavage and modification of the SRL by cytotoxins α-sarcin and ricin disrupt GTPase-ribosome interaction, leading to reduced protein synthesis and cell death. However, the full role of the SRL remains unclear. Previous studies showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype in E. coli. When ΔSRL particles were purified from cells, they were found to be not only inactive in protein synthesis but also incompletely assembled. In particular, block 4 of the subunit, which includes the peptidyl transferase center, remained unfolded. Here, we explore the basis of this assembly defect. We show that 23S rRNA extracted from ΔSRL subunits can be efficiently reconstituted into 50S subunits, and these reconstituted ΔSRL particles exhibit full peptidyl transferase activity. We also further characterize ΔSRL particles purified from cells, using cryo-EM and proteomic methods. These particles lack density for rRNA and r-proteins of block 4, consistent with earlier chemical probing data. Incubation of these particles with excess total r-protein of the large subunit (TP50) fails to restore substantial peptidyl transferase activity. Interestingly, proteomic analysis of control and mutant particles shows an overrepresentation of multiple assembly factors in the ΔSRL case. We propose that one or more GTPases normally act to release assembly factors, and this function is blocked in the absence of the SRL.
Keywords: Ribosome assembly, Sarcin-ricin loop, Ribosomal RNA