Interaction of Initiator Met-tRNAfMet (Escherichia coli) and Gly-tRNA1Gly (Staphylococcus epidermidis) with Bacterial Elongation Factor Tu: GTP Complex

Abstract

Jekowsky et al. reported recently that elongation factor Tu:GTP complex from Escherichia coli protected aminoacyl-tRNA from digestion by pancreatic RNase (I). On the basis of their finding, we have developed the "RNase-resistance assay" for determination of the dissociation constant of aminoacyl-tRNA from aminoacyl-tRNA:EF-Tu:GTP complex. By the use of this sensitive assay, the dissociation constants were estimated to be 3.6 x 10(-7) M for Ala-tRNA1Ala (Torulopsis utilis), 7.9 x 10(-8) M for Phe-tRNAPhe (Escherichia coli), 8.1 x 10(-7) M for initiator Met-tRNAfMet (Escherichia coli), and 5.4 x 10(-6) M for Gly-tRNA1Gly (Staphylococcus epidermidis) participating in cell wall biosynthesis. Moreover, using a relatively large amount of EF-Tu:GTP, we have been able to detect the ternary complexes of initiator Met-tRNAfMet and Gly-tRNA1Gly with EF-Tu:GTP even by the method of gel filtration.

Preliminary characterization by X-ray diffraction and Raman spectroscopy of a crystalline complex ofBacillus stearothermophilusinitiation factor 2 C-domain and fMet-tRNAfMet

“…Ribonuclease protection by IF2 proteins was assayed as described by Tanada et al (1981) and modi®ed by Louie & Jurnak (1985), using the same buffers and concentrations as described for the hydrolysisprotection experiments, with the exception that incubation took place on ice. The ribonuclease cleavage reaction was initiated by the addition of 10 mg ribonuclease A per reaction mixture.…”

Interaction of fMet-tRNAfMet and fMet-AMP with the C-terminal domain of Thermus thermophilus translation initiation factor 2

“…Protection of the 3 H end of f Met-tRNA f Met by IF2 and the C-terminal domain was also studied by the pancreatic RNase protection assay [19]. In the absence of IF2, f MettRNA f Met is rapidly degraded by the RNase A present (Fig.…”

Structural features in aminoacyl‐tRNAs required for recognition by elongation factor Tu

“…Comparable observations have been made with different aa-oligonucleotides using the nitrocellulose filter binding assay [27]. However, RNase protection experiments with various aminoacylated and misaminoacylated E. coli and yeast tRNA derivatives do not confirm this model [17,18].…”

“…The 3 '-end of aa-tRNA is sheltered in the ternary complex since the CCA end is protected from digestion by RNase A [ 15,17,18]. Various chemical and enzymatic modifications have been introduced into several nucleobase positions and into the 3 '-terminal ribose and their implications on ternary complex formation examined.…”

“…The Staphylococcus epidermis Gly-tRNAGLY involved in cell wall synthesis which also lacks the conserved T4C sequence also forms a complex with EF-Tu. GTP although with very low efficiency [17,18].…”