Translation of mRNAs with degenerate initiation triplet AUU displays high initiation factor 2 dependence and is subject to initiation factor 3 repression.

Abstract

The influence of the rare initiation triplet AUU on mRNA translation was investigated by comparing the activity of two pairs of model mRNAs that differ in the length of Shine-Dalgarno and spacer sequences. Irrespective of the initiation triplet (AUG or AUU), all mRNAs had similar template activity in vitro, but translation of AUU mRNAs depended more on initiation factor (IF) 2 and less on IF3 than that of AUG mRNAs. Increasing the IF3/ribosome ratio from 2 to 10 progressively inhibited the AUU mRNAs and abolished their capacity to compete for translating ribosomes with other mRNAs but did not affect activity of the AUG mRNAs. The effects induced by IF3 are from its different influence on onand off-rates of the transition 30S preinitiation complex -30S initiation complex; depending on the nature of the initiation triplet (AUG or AUU) of the mRNA, IF3 shifts the position of equilibrium toward binding or dissociation of fMet-tRNA, respectively. Stimulation of fMet-tRNA binding and dissociation yields superimposable IF3 titration curves that saturate at an IF3/30S ratio of -1, indicating that the data are from the interaction of one molecule of IF3 with the same 30S binding site. Both effects are either lost or strongly reduced with 30S mutants defective in IF3 binding. Translational repression of AUU mRNAs by IF3 is from the factor-dependent dissociation of fMet-tRNA from 30S subunits, which becomes relevant when excess IF3 interferes with the formation of 70S initiation complex, presumably by interacting with 50S subunit.

Altered discrimination of start codons and initiator tRNAs by mutant initiation factor 3

“…N-terminal sequencing to determine the influence of altered IF3 on initiation from internal sites in the p304 lacZ construct+ b-Galactosidase was purified from the R99L IF3 mutant containing the p304 lacZ plasmid+ The yield in picomoles for selected PTH amino acids analyzed for the first five cycles is shown on the upper panel and the lower panel depicts the mRNA sequence aligned with the experimentally determined amino acid sequence+ thus suggest that the impaired P site occupancy seen with these tRNAs is due, in part at least, to their rejection by IF3, which senses them as noninitiator tRNAs+ Discrimination against non-AUG codons in 30S initiation complexes appears to be due to IF3-mediated destabilization, rather than to any inherent instability of the mRNA-tRNA-30S complex (La Teana et al+, 1993)+ Both SD-anti-SD base pairing as well tRNA-mRNA interactions contribute to the stability of initiation complexes formed on mRNAs containing 59 untranslated leader regions+ Because no mRNA-rRNA base pairing occurs on leaderless mRNAs, such initiation complexes may not be able to withstand IF3-mediated destabilization+ A similar interpretation may explain the effect of IF3 on initiation from internal start codons: The mRNAs supporting internal initiation from non-AUG codons that we have examined lack recognizable SD sequences upstream of the (internal) start codon (Table 4)+ Initiation complexes formed on these mRNAs may be particularly susceptible to IF3-mediated destabilization because codon-anticodon mismatches are not offset by any compensating SD-anti-SD interaction+ IF3 is a two-domain protein and the structures of the separate domains have been solved by X-ray crystallography and NMR spectroscopy (Biou et al+, 1995;Garcia et al+, 1995aGarcia et al+, , 1995b)+ The C domain appears to contain most of the elements necessary for binding to 30S subunits and dissociation of 70S ribosomes (Garcia et al+, 1995b)+ Mutations affecting the fidelity of initiation have been isolated in both domains+ The R99 and R131 variants studied here, as well as the D106, E134, and P176 mutants (Sacerdot et al+, 1996Sussman et al+, 1996) are all in the C-terminal domain, whereas the Y75 (Sussman et al+, 1996) mutant lies in the N-terminal domain and the G71 mutant is at the beginning of the interdomain linker region (De Cock et al+, 1999)+ The mutant R99 and R131 E. coli factors have not been assayed for their ability to bind 30S subunits+ However, mutagenesis of R99, R133, and a number of neighboring residues in Bacillus stearothermophilus IF3 decreased factor binding to 30S subunits (Sette et al+, 1999)+ The E. coli P176 IF3 mutant had a decreased affinity for 30S subunits and the D103 mutant displayed a lack of binding specificity and bound to both ribosomal subunits )+ In contrast, the Y75 and G71 mutants in the N-terminal domain only affected the discriminatory properties of the factor and did not affect its affinity for 30S subunits (Sussman et al+, 1996;Sacerdot et al+, 1999)+ A model to explain these differential interactions of IF3 mutants with the ribosome, all of which compromise the fidelity of initiation, has been proposed by Sacerdot et al+ (1999)+ According to this model, the an...…”

Modulation of ribosomal recruitment to 5′‐terminal start codons by translation initiation factors IF2 and IF3

“…La Teana et al [24] have demonstrated that the discriminatory function of IF3 on mRNAs with start codons other than AUG can be compensated for by IF2. To further address the question whether the IF2^fMet-tRNA Met f interaction contributes to stabilization of 30S initiation complexes at 5P-terminal AUGs, we asked whether altering the ratio of the three initiation factors a¡ects the partitioning of 30S initiation events at AUG1 and AUGi on ompAv117 RNA.…”

Identification and Purification of Translation Initiation Factor 2 (IF2) from Thermus thermophilus

“…It distinguishes the initiator tRNA from elongator tRNAs (Hartz et al, 1989). Additionally, IF3 is probably responsible for direct control of the codon-anticodon recognition at initiation codons (Berkhout et al, 1986;La Teana et al, 1993).…”