Tyrosine Residues in the C‐Terminal Domain of the Elongation Factor G Are Essential for Its Interaction with the Ribosome

Abstract

Chemical modification of the elongation factor G (EF‐G) with tetranitromethane and iodine has been studied. It has been shown by spectrophotometric titration that EF‐G contains two exposed tyrosine residues, one of which has an unusually low pK value for a phenol hydroxyl group at pH 8.5. Modification of one tyrosine residue with either tetranitromethane or iodine results in a 70‐80% loss of EF‐G activity in all ribosome‐dependent reactions. Modification of three or four residues inhibits 90‐100 % of activity. Binding of EF‐G with the 70‐S ribosome and 50‐S subunit is equally effective for protection of tyrosine residues against modification. The rate of EF‐G modification with tetranitromethane is considerably higher in the presence of guanyl nucleotides than for free EF‐G. The modified residues are located in the C‐terminal domain of EF‐G and are presumably contained in one of the sites of EF‐G interaction with the ribosome.

Interaction of the N‐Terminal and C‐Terminal Domains of Elongation Factor G on Formation of Complexes with Guanyl Nucleotides

“…Nonetheless, the existence of local conformational changes in the structure of EF-G on the formation of binary complexes with guanyl nucleotides has been shown by the method of hydrophobic [2] and spin-label probes [3] as well as by investigations of the rate of the exposed -SH group modification [ll,12]. If the exposed -SH group is effectively protected from modification by guanyl nucleotides [ l l , 121, then, as has been recently shown by us [13], the rate of modification of tyrosine residues in EF-G in the presence of GTP or GDP is essentially higher than in the free protein.…”

“…Nonetheless, the existence of local conformational changes in the structure of EF-G on the formation of binary complexes with guanyl nucleotides has been shown by the method of hydrophobic [2] and spin-label probes [3] as well as by investigations of the rate of the exposed -SH group modification [ll,12]. If the exposed -SH group is effectively protected from modification by guanyl nucleotides [ l l , 121, then, as has been recently shown by us [13], the rate of modification of tyrosine residues in EF-G in the presence of GTP or GDP is essentially higher than in the free protein.It ha5 been reported [lo] that EF-G has a domain structure and'under the action of proteases is cleaved into a limited number of fragments resistant to further proteolysis [14,15]. The resistance of the fragments formed to proteolysis is due to the maintenance of a compact structure by them.…”

“…molecule as independent structural units [lo]. The exposed -SH group [16,17] and the GTP-binding site [18] ate located within the N-terminal domain, while the tyrosine residues whose rate of modification is affected by guanyl nucleotides are in the C-terminal domain [13]. From these data it follows that on the formation of binary complexes with guanyl nucleotides the noted local conformational changes in the EF-G molecule involve both the N-terminal and C-terminal parts of the protein molecule.…”