Cleavage of the sarcin-ricin loop of 23S rRNA differentially affects EF-G and EF-Tu binding.
Ribotoxins are potent inhibitors of protein biosynthesis and inactivate ribosomes from a variety of organisms. The ribotoxin alpha-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 alpha-sarcin on defined steps of translation by the bacterial ribosome. alpha-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 alpha-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.
García-Ortega
Lucía
L
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.
Alvarez-García
Elisa
E
Gavilanes
José G
JG
Martínez-del-Pozo
Alvaro
A
Joseph
Simpson
S
D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D020653:Peptide Elongation Factor G / Q000737:chemistry / Q000378:metabolism*; D010444:Peptide Elongation Factor Tu / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D011500:Protein Synthesis Inhibitors / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism; D012276:Ricin / Q000378:metabolism
D005656:Fungal Proteins; D020653:Peptide Elongation Factor G; D011500:Protein Synthesis Inhibitors; D012338:RNA, Ribosomal, 23S; C014801:alpha-sarcin; D012276:Ricin; D004722:Endoribonucleases; D010444:Peptide Elongation Factor Tu