Cleavage of the sarcin-ricin loop of 23S rRNA differentially affects EF-G and EF-Tu binding

Abstract

Ribotoxins are potent inhibitors of protein biosynthesis and inactivate ribosomes from a variety of organisms. The ribotoxin α-sarcin cleaves the large 23S ribosomal RNA (rRNA) at the universally conserved sarcin–ricin loop (SRL) leading to complete inactivation of the ribosome and cellular death. The SRL interacts with translation factors that hydrolyze GTP, and it is important for their binding to the ribosome, but its precise role is not yet understood. We studied the effect of α-sarcin on defined steps of translation by the bacterial ribosome. α-Sarcin-treated ribosomes showed no defects in mRNA and tRNA binding, peptide-bond formation and sparsomycin-dependent translocation. Cleavage of SRL slightly affected binding of elongation factor Tu ternary complex (EF-Tu•GTP•tRNA) to the ribosome. In contrast, the activity of elongation factor G (EF-G) was strongly impaired in α-sarcin-treated ribosomes. Importantly, cleavage of SRL inhibited EF-G binding, and consequently GTP hydrolysis and mRNA–tRNA translocation. These results suggest that the SRL is more critical in EF-G than ternary complex binding to the ribosome implicating different requirements in this region of the ribosome during protein elongation.

Structure and assembly model for the Trypanosoma cruzi 60S ribosomal subunit

“…3A). This places KSD in a pivotal position interacting with ribosomal functional centers (SI Appendix, Table S6) (20,21). ES42L covers the hub region encompassing the cleavage sites of srRNAs2-4 ( Fig.…”

Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

“…These alterations in the SRL result in a complete block of translation, which is thought to be the consequence of an impaired activation/binding of trGTPases (17). Recent crystal structures of EF-Tu (12), or EF-G (9,(18)(19)(20)(21) bound to the ribosome stimulated considerations about the mechanism of ribosome-triggered GTP hydrolysis.…”

Structural Implications of Universal Complementarities in Translation—High Accuracy at the Decoding Site

“…Since it has been known that spectinomycin inhibits EF-G dependent translocation [22] [23], the conformational change of the above region of 16S rRNA must have something to do with the mechanism of translocation. Although the binding site of both EF-G·GTP complex and aa-tRNA•EF-Tu•GTP TC on the 30S subunit ribosome could be similar, a recent result of α-sarcin-treated ribosomes has clarified that cleavage of SRL inhibited EF-G binding, GTP hydrolysis, and mRNA-tRNA translocation, while it slightly affected binding of the TC to the ribosome [24]. A big difference in binding of the GTP complexes between EF-G and EF-Tu•tRNA from the viewpoint of the present work is the absence of helix formation of the region 1103 -1107 of 16S rRNA with the region of EF-G corresponding to the T-loop of tRNAs.…”

“…A big difference in binding of the GTP complexes between EF-G and EF-Tu•tRNA from the viewpoint of the present work is the absence of helix formation of the region 1103 -1107 of 16S rRNA with the region of EF-G corresponding to the T-loop of tRNAs. Accordingly, both spectinomycin binding to the nucleotides, G1064, C1066 and C1192 of 16S rRNA [21] and splitting of the nucleotide bond between A2660 and G2661 in SRL [24] obstructs the transition from the left-most conformations to the second from the right-most conformation in Figure 1. This transition seems to produce movement of the 30S subunit relative to the 50S subunit [8]- [25].…”