The Translational Fidelity Function of IF3 During Transition from the 30 S Initiation Complex to the 70 S Initiation Complex

Abstract

SUMMARYIF3 has a fidelity function in the initiation of translation, inducing the dissociation of fMettRNA fMet from 30S initiation complexes (30SIC) containing a non-canonical initiation triplet (e.g., AUU) in place of a canonical initiation triplet (e.g., AUG). IF2 plays a complementary role, selectively promoting initiator tRNA binding to the ribosome. Here we use parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light scattering, and changes in intensities of fluorophore-labeled IF2 and fMet-tRNA fMet to determine the effects on both 30SIC formation and 30SIC conversion to 70S initiation complexes (70SIC) of a) substituting AUG with AUU, and/or b) omitting IF3, and/or c) replacing GTP with the non-hydrolyzable analogue GDPCP. We demonstrate that the presence or absence of IF3 has at most minor effects on the rate of 30SIC formation using either AUG or AUU as the initiation codon, and conclude that the high affinity of IF2 for both 30S subunit and initiator tRNA overrides any perturbation of the codon-anticodon interaction resulting from AUU for AUG substitution. In contrast, replacement of AUG by AUU leads to a dramatic reduction in the rate of 70SIC formation from 30SIC upon addition of 50S subunits. Interpreting our results in the framework of a quantitative kinetic scheme leads to the conclusion that, within the overall process of 70SIC formation, the step most affected by substituting AUU for AUG involves the conversion of an initially labile 70S ribosome into a more stable complex. In the absence of IF3, the difference between AUG and AUU largely disappears, with each initiation codon affording rapid 70SIC formation, leading to the speculation that it is the rate of IF3 dissociation from the 70S ribosome during IC70S formation that is critical to its fidelity function.

Roles of helix H69 of 23S rRNA in translation initiation

“…IF3 is well known as an "antiassociation" factor that negatively regulates translation initiation to enhance start codon selection (26). In line with previous reports (10,15,16), omission of IF3 from 30SIC stimulated subunit joining in WT ribosomes, an effect most prominent in the presence of AUC ( Fig. 2B and Table 1).…”

“…IF3 has a well-established role in preventing spurious initiation (15,16,32,33), but the molecular basis of this activity has remained unclear. It has been proposed that mutually exclusive interactions between IF3 and H69 on the 30S subunit are key to the function of IF3 in initiation (2,8).…”

“…This is accompanied by movement of the initiator tRNA into the P/P site (14). IF3 inhibits subunit joining, an effect more pronounced in the presence of noncanonical codonanticodon base pairing (5,15). The mechanism by which IF3 prevents spurious initiation likely involves its interplay with fMet-tRNA fMet .…”

Role of helix 44 of 16S rRNA in the fidelity of translation initiation

“…Little binding was observed in the absence of 30S subunits under either set of conditions. The fact that a substantial proportion of complexes contain fMet-tRNA in the 30SIC(AUC) reactions suggests that the slow observed rate of 70SIC(AUC) formation stems primarily from a kinetic bottleneck during the 50S docking phase of initiation, in line with previous studies (Grigoriadou et al 2007b;Milon et al 2008). Consistent with this interpretation, when the stopped-flow experiment measuring 70SIC(AUC) formation was repeated using a 10-fold higher concentration of fMet-tRNA, virtually no increase in docking rate was observed (data not shown).…”

“…Analogous mutations in 16S rRNA have recently been identified, and those with the strongest phenotype cluster to the 790 loop and inhibit IF3 binding, further implicating IF3 in start codon selection (Qin et al 2007;Qin and Fredrick 2009). In vitro, IF3 negatively regulates both major steps of initiation, strongly inhibiting 70SIC formation in the presence of the near-cognate start codon AUU (Antoun et al 2006a,b;Grigoriadou et al 2007b;Milon et al 2008). While the importance of IF3 in start codon selection is undisputed, the molecular basis for its fidelity function remains unclear.…”