Chemical crosslinking of elongation factor G to the 23S RNA in 70S ribosomes from Escherichia coli

Abstract

Elongation factor G was crosslinked to the 23S RNA of 70S Escherichia coli ribosomes with the bifunctional, cleavable reagent diepoxybutane (DEB). The EF-G-23S RNA complex was isolated and digested with ribonuclease A. After digestion, an RNA fragment, protected by EF-G was cleaved from the complex and isolated. The nucleotide sequence of this RNA fragment was determined by partial ribonuclease digestion. It proved to be 27 nucleotides long and it could be identified with residues 1055 to 1081 of the nucleotide sequence of E. coli 23S RNA. In the presence of thiostrepton, which prevents binding of EF-G to the ribosome, there was a dramatic decrease in the yield of this complex.