Structural and Mechanistic Studies on Chloroplast Translational Initiation Factor 3 fromEuglena gracilis†

Biochemistry ◽  
1997 ◽  
Vol 36 (48) ◽  
pp. 14827-14835 ◽  
Author(s):  
Nan-Jun Yu ◽  
Linda L. Spremulli
Keyword(s):  
Initiation Factor ◽  
Mechanistic Studies ◽  
Translational Initiation ◽  
Translational Initiation Factor

Structure of the β subunit of translational initiation factor elF-2

Cell ◽  
1988 ◽  
Vol 54 (5) ◽  
pp. 633-639 ◽  
Author(s):  
Vinay K. Pathak ◽  
Peter J. Nielsen ◽  
Hans Trachsel ◽  
John W.B. Hershey
Keyword(s):  
Initiation Factor ◽  
Β Subunit ◽  
Translational Initiation ◽  
Translational Initiation Factor

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

2002 ◽  
Vol 7 (6) ◽  
pp. 541-546 ◽  
Author(s):  
Sonia Delle Fratte ◽  
Chiara Piubelli ◽  
Enrico Domenici
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Ribosomal Subunit ◽  
New Drugs ◽  
Initiation Factor ◽  
Translational Initiation ◽  
Scintillation Proximity Assay ◽  
Initiation Factor 2 ◽  
Subsequent Capture ◽  
Translational Initiation Factor

Translational initiation factor 2 (IF2) is the largest of the 3 factors required for translation initiation in prokaryotes and has been shown to be essential in Escherichia coli. It stimulates the binding of fMet-tRNAfMet to the 30S ribosomal subunit in the presence of GTP. The selectivity is achieved through specific recognition of the tRNAfMet blocked α-amino group. IF2 is composed of 3 structural domains: N-domain, whose function is not known; G-domain, which contains the GTP/GDP binding site and the GTPase catalytic center; and C-domain, which recognizes and binds fMet-tRNAfMet. Its activity is strictly bacteria specific and highly conserved among prokaryotes. So far, antibiotics targeting IF2 function are not known, and this makes it an ideal target for new drugs with mechanisms of resistance not yet developed. A few assays have been developed in the past, which allow the detection of IF2 activity either directly or indirectly. In both instances, the assays are based on radioactive detection and do not allow for high throughput because of the need for separation or solvent extraction steps. The authors describe a novel biochemical assay for IF2 that exploits the molecular recognition of fMet-tRNAfMet by the C-domain. The assay is based on the incubation of biotinyl-IF2 with fMet-tRNAfMet and the subsequent capture of the radiolabeled complex by streptavidin-coated beads, exploiting the scintillation proximity assay (SPA) technology. The assay has been designed in an automatable, homogeneous, miniaturized fashion suitable for high-throughput screening and is rapid, sensitive, and robust to dimethyl sulfoxide (DMSO) up to 10% v/v. The assay, used to screen a limited chemical collection of about 5000 compounds and a subset of compounds originated by a 2-D substructural search, has shown to be able to detect potential IF2 inhibitors.

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