Translational regulation of infC operon in Bacillus stearothermophilus.

A Bacillus stearothermophilus in vitro translational system has been developed to study the expression of the three cistrons (infC, rpml, and rplT) constituting the infC operon of this bacterium. When directed by homologous in vitro transcribed infC tricistronic mRNA, this system, which consists of partially purified and purified components of the B. stearothermophilus translational apparatus, synthesizes with high efficiency and specificity the three gene products (IF3, L35, and L20) in a ratio similar to that found in vivo (i.e., about 1:6:6). The three cistrons are translationally coupled and expressed in a specific temporal order: a low level of IF3 synthesis stimulates the expression of L35 which, in turn, greatly stimulates the synthesis of L20 and IF3. Protein L20 and an excess of IF3 were found to act as translational feedback inhibitors of the entire operon. The synthesis of IF3 displayed a strong dependence on IF2. This dependence as well as the repressibility by excess IF3 were found to be due to the presence of the rare AUU initiation triplet at the beginning of infC

Translation initiation factor IF3: two domains, five functions, one mechanism?

Initiation factor IF3 contains two domains separated by a flexible linker. While the isolated N-domain displayed neither affinity for ribosomes nor a detectable function, the isolated C-domain, added in amounts compensating for its reduced affinity for 30S subunits, performed all activities of intact IF3, namely: (i) dissociation of 70S ribosomes; (ii) shift of 30S-bound mRNA from ‘stand-by’ to ‘P-decoding’ site; (iii) dissociation of 30S–poly(U)–NacPhe-tRNA pseudo- initiation complexes; (iv) dissociation of fMet-tRNA from initiation complexes containing mRNA with the non-canonical initiation triplet AUU (AUUmRNA); (v) stimulation of mRNA translation regardless of its start codon and inhibition of AUUmRNA translation at high IF3C/ribosome ratios. These results indicate that while IF3 performs all its functions through a C-domain–30S interaction, the N-domain function is to provide additional binding energy so that its fluctuating interaction with the 30S subunit can modulate the thermodynamic stability of the 30S–IF3 complex and IF3 recycling. The localization of IF3C far away from the decoding site and anticodon stem–loop of P-site-bound tRNA indicates that the IF3 fidelity function does not entail its direct contact with these structures