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

Abstract

Bacillus stearothermophilus translation initiation factor 2 (IF2) specifically binds initiator fMet‐tRNAfMet and positions it into the ribosomal peptidyl site in the course of the initiation of protein biosynthesis. The isolated C‐terminal domain of IF2 is capable of binding fMet‐tRNAfMet, as shown by RNase A and hydrolysis protection experiments. In the presence of fMet‐tRNAfMet, the IF2 C‐domain yielded orthorhombic crystals of space group I222 (I212121) diffracting to 3.4 Å resolution. The existence of equimolar amounts of tRNA and protein in the crystals was proven by Raman spectroscopy. The observed unit cell suggests the presence of two IF2 C‐­domain–fMet‐tRNAfMet complexes per asymmetric unit of the crystal.

Interaction of fMet‐tRNAfMet with the C‐terminal domain of translational initiation factor IF2 from Bacillus stearothermophilus

“…Experiments of fMet‐tRNA fMet protection at 37°C yielded an apparent K d value of 1.8 μM for the complexes of fMet‐tRNA fMet with IF2, IF2 C and IF2 C‐2 [8,22]. Gel shift experiments described earlier, on the other hand, established an upper limit of 0.2 μM for the dissociation constant of the complex of fMet‐tRNA fMet and IF2 C at room temperature [5]. Considering the temperature dependence of K d observed here, these values agree reasonably well with our estimates at 30°C which gave K d values of 0.59 μM and 0.81 μM for IF2 C and IF2 C‐2, respectively.…”

“…Initiation of protein biosynthesis requires the correct positioning of charged initiator tRNA, fMet‐tRNA fMet , in the ribosomal P‐site of the mRNA‐programmed 70S ribosomes. This is accomplished by translation initiation factor IF2, which interacts with GTP and 50S ribosomal subunits through its central 40 kDa G domain and with fMet‐tRNA through its C‐terminal domain (IF2 C) [1–5]. Knowledge about the molecular nature of this interaction is particularly relevant in light of the vital importance of this interaction which represents a new target for a new class of specific antibacterial agents.…”

Development of a High-Throughput Scintillation Proximity Assay for the Identification of C-Domain Translational Initiation Factor 2 Inhibitors

“…Elucidation of the structure of IF2, free and complexed to fMet-tRNA, would enable the rational drug design of IF2 inhibitors that could represent a novel class of antibacterial agents. However, beside some preliminary characterization of IF2 by NMR 11,12 and X-ray 15 and the existence of crystal structures from structurally related proteins, 16 the full 3-D structure of IF2 is not available. Presently, the identification of specific IF2 inhibitors through target-directed high-throughput screening (HTS) of chemical libraries and natural products represents the only way to find hits to be developed into effective drugs.…”

Nano theoretical studies of fMet-tRNA structure in protein synthesis of prokaryotes and its comparison with the structure of fAla-tRNA

“…This tRNA does not recognize internal AUG codons. Initiation of protein biosynthesis requires the correct positioning of charged initiator tRNA, fMet-tRNA in the ribosomal P-site of the mRNA-programmed 70S ribosome's (Gualerzi and Pon, 1990;La Teana et al, 1996;Monajjemi et al, 2008;Spurio et al, 1993;Forster et al, 1999).…”