Chemical evidence for the involvement of histidyl residues in the functioning of Escherichia coli elongation factor Tu

The 30S ribosomal subunits from Bacillus stearothermophilus were cross-linked under native conditions with the bifunctional reagent diepoxybutane. The dominant protein-protein cross-link in the 30S ribosomal subunit between proteins S13 and S19 [Brockmöller, J., & Kamp, R.M. (1986) Biol. Chem. Hoppe-Seyler 367, 925-935] was isolated on a preparative scale. The presence of a single cross-link site between cysteine-83 of protein S13 and histidine-68 of protein S19 was established by microsequence analysis of isolated cross-linked peptides. This cross-link site was further confirmed by different analytical methods including fast atom bombardment mass spectrometry of the cross-linked peptide. The cross-linking site is located in the highly conserved C-terminal regions of proteins S13 and S19. In addition, the complete amino acid sequence of protein S13 from B. stearothermophilus is determined. Sequence comparison with the homologous Escherichia coli protein S13 revealed 58% identical amino acid residues.

Section: Discussionmentioning

confidence: 99%

Cross-linked amino acids in the protein pair S13-S19 and sequence analysis of protein S13 of Bacillus stearothermophilus ribosomes

Brockmöller¹,

Rm²

1988

“…First, both DEPC and rose bengal have been shown to specifically modify histidyl residues in a number of soluble enzymes (Westhead, 1965;Hoffee et al, 1967;Brand et al, 1969;Holbrook & Ingram, 1973;Miles, 1977;Saluja & McFadden, 1980; Meyer & Cromartie, 1980). In addition, both reagents have been used to modify histidyl residues involved in the binding of EF-Tu to aminoacyl-tRNA and/or ribosomes (Jonak & Rychlik, 1980).…”

Section: Discussionmentioning

confidence: 99%

Mechanism of lactose transport in Escherichia coli membrane vesicles: evidence for the involvement of a histidine residue(s) in the response of the lac carrier to the proton electrochemical gradient

García¹,

Patel²,

Padan³

et al. 1982

that treatment with diethyl pyrocarbonate causes the lac transport system to exhibit biphasic kinetics. One component of the overall process exhibits kinetic parameters typical of active transport (i.e., low apparent Km), and the other has the characteristics of facilitated diffusion (i.e., high apparent Km). Since partial dissipation of the proton electrochemical gradient leads to similar biphasic kinetics [Robertson, D. E., Kaczo-

“…It is unknown whether this residue corresponds to His-66 in E. coli EF-Tu. Jonak & Rychlik (1980) have reported that the AA-tRNA binding activity of E. coli EF- Tu-GTP is also inactivated by photooxidation. Again it is not known whether the His residue destroyed is His-66.…”

Section: Discussionmentioning

confidence: 99%

Identification of a histidine residue near the aminoacyl transfer ribonucleic acid binding site of elongation factor Tu

Duffy¹,

Gerber²,

Johnson³

et al. 1981

The complex of elongation factor Tu with GTP (EF-Tu.GTP) reacts with N or epsilon -bromoacetyl-lys-tRNA ( or epsilon BrAcLys-tRNA) to form a functional covalently linked complex (XLTC). The site of cross-linking must be near the site on EF-Tu.GTP that binds the aminoacyl moiety of aminoacyl transfer ribonucleic acid (AA-tRNA). For identification of this site, a nanomole of purified XLTC prepared from or epsilon BrAc[(14)C]Lys-tRNA was digested first with RNase A and then with trypsin, and the peptides were resolved by high-performance liquid chromatography using a c8 reverse-phase column. A single peptide contained 80% of the label. The amino acid composition of this peptide was identical with that of residues 59-74 in EF-Tu. The NH2-terminal sequence of the peptide was determined to be Fly-Ile-Thr-Ile, which are residues 59-62 in EF-Tu. The modified amino acid was identified as pi - (carboxymethyl)histidine, which establishes that His-66 is at or near the AA-tRNA binding site on EF-Tu.GTP.