Poster abstracts

Poster number 50 submitted by Sepideh Fakhretaha Aval

Role of the sarcin-ricin loop (SRL) of 23S rRNA in assembly of the 50S ribosomal subunit

Sepideh Fakhretaha Aval (Ohio state Biochemistry program, The Ohio State University), Kurt Fredrick (Department of Microbiology, The Ohio State University)

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 and 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. Lancaster et al. (2008) showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype in E. coli. These ΔSRL ribosomes were purified from cells and 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. These reconstituted ΔSRL particles exhibit full activity and similar reconstitution rates to WT, in contrast to the mutant ΔSRL subunits from which the rRNA came. SILAC analysis has also revealed differences in ribosomal protein levels of reconstituted particle compared to in vivo isolated subunits. These data suggest that the assembly defect conferred by ΔSRL is specific to 50S biogenesis in the cell. During the assembly of large subunit, the SRL folds early on as part of block 1. We propose that one or more trGTPases then transiently bind via the SRL and facilitate subsequent folding events.

References:
Lancaster L, Lambert NJ, Maklan EJ, Horan LH, Noller HF. The sarcin-ricin loop of 23S rRNA is essential for assembly of the functional core of the 50S ribosomal subunit. RNA. 2008;14(10):1999-2012. doi:10.1261/rna.1202108

Davis JH, Williamson JR. Structure and dynamics of bacterial ribosome biogenesis. Philos Trans R Soc Lond B Biol Sci. 2017;372(1716):20160181. doi:10.1098/rstb.2016.0181

Keywords: Ribosome assembly, Sarcin-ricin loop, 23S rRNA