The interaction between Escherichia coli translation-initiation factor IF-1 and ribosomes was studied in binding experiments by Airfuge centrifugation. IF-1 binds to the 30S, but not to the SOS, ribosomal subunit and its binding is strongly stimulated by IF-3 and IF-2, either alone or in combination. From the dependence of the Kd of the 30s-subunit -IF-1 complex on ionic strength, it can be concluded that IF-1 binds primarily via an ionic intcraction, most likely with the 16s rRNA, with the minimum number of ion pairs involved being 2.7-3.6. The 30S-subunit -IF-1 interaction is unaffected by temperature changes between 1 1 "C and 44°C and is thus accompanied by a negligible enthalpy change. It is concluded that the interaction is an entropy-driven process triggered mainly by the release of counter ions from the RNA phosphates.Titration of 30s-subunit -IF-1 complexes with 50s subunits causes the ejection of the factor indicating that IF-1 is released from the ribosomes during the subunit association step which marks the transition from a 30S-initiation-complex to a 70s initiation complex. Omission of IF-1 from reaction mixtures containing all purified components required for protein synthesis results in a much-reduced translational rate [8]. To define the mechanism by which IF-1 exercises its stimulation, structural, functional and genetic studies have been performed on this factor. By fluorescence stopped-flow kinetics, it was found that IF-1 produces a 2-fold or a 4-fold rate increase in 30s-initiationcomplex formation when IF-3 or IF-2-GTP or both are present [9]. By initial-rate kinetic analysis of 30s-initiationcomplex formation, it was found that IF-1 increases approximately 2.5-fold the limiting V,,, of this process. IF-1 was found not to affect the affinity of the ribosomes either for the initiator Met-tRNA or for the random poly(AUG) used as template. The kinetic effect of IF-I, on the other hand, was found to titrate roughly with the 30s ribosomal subunits suggesting that these represent the target of the IF-1 activity [lo].A thorough understanding of the functional role of IF-1 cannot be expected without a better knowledge of its way of interacting with ribosomes. Concerning this point, the data available so far are rather scarce and conflicting [ll]. In fact, the interaction between IF-I and ribosomes is very sensitive to the hydrostatic pressure generated by sucrose-gradient centrifugation, which makes this method unsuitable for obtaining quantitative results. To overcome the problem, lixation with glutaraldehyde has been used; this method is likely to produce artifacts, however, as suggested by the very low K, value estimated in these studies and by the fact that IF-1 was found to bind equally well to 30s and 50s subunits and 70s ribosomes [12]. The alternative approach of applying fluorescent spectroscopy to the study of the IF-1 -ribosome interaction unfortunately failed due to the inactivation of the factor brought about by the fluorescent labelling procedureIn the present study we have used cent...

Starting from a synthetic modular gene (infA) encoding Escherichia coli translation initiation factor IF1, we have constructed mutants in which amino acids are deleted from the carboxyl terminus or in which His29 or His34 are replaced by Tyr or Asp residues. The mutant proteins were overproduced, purified and tested in vitro for their properties in several partial reactions of the translation initiation pathway and for their capacity to stimulate MS2 RNA-dependent protein synthesis. The results allow for the conclusion that: (i) Arg69 is part of the 30S ribosomal subunit binding site of IF1 and its deletion results in the substantial loss of all IF1 function; (ii) neither one of its two histidines is essential for the binding of IF1 to the 30S ribosomal subunit, for the stimulation of fMet-tRNA binding to 30S or 70S ribosomal particles or for MS2 RNA-dependent protein synthesis; but (iii) His29 is involved in the 50S subunit-induced ejection of IF1 from the 30S ribosomal subunit.

Divisione di Oncologia Sperimentale E, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatcls I F 0 8605. This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with high similarity to the a-sarcin family of ribosome-inactivating proteins (RIPS). The cDNA encoding the mature protein was expressed in Escherichiu coli, and the recombinant protein, a 17-kDa polypeptide designated clavin was purified and characterized. Clavin shows typical type-1 RIP properties : specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cellfree and cellular systems. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration. Pharmacokinetics analysis in mice indicated an elimination half-life (tl,ZP) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and transient while no alteration of kidney function was observed. Clavin induced a late and very low antibody response in mice. The iii vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates.