Mutations that alter initiation codon discrimination by Escherichia Coli initiation factor IF3

In vivo involvement of mutated initiation factor IF1 in gene expression control at the translational level

“…No significant effect on gene expression was observed for any of the five other IF1 mutants, also having Arg to Leu alterations [22], compared to wild-type strain (data not shown). Also for these mutants, all analyzed near-cognate initiation codons were less efficient than the canonical AUG initiation codon, thus resembling the situation for the wildtype strain [28,29].…”

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...…”

The N-terminal extension of Escherichia coli ribosomal protein L20 is important for ribosome assembly, but dispensable for translational feedback control

“…The N-terminal extension of ribosomal protein L20 www.rnajournal.org replace the corresponding SstII/NheI fragment in plasmid pMAK5-57 (Sacerdot et al 1999), a derivative of plasmid pMAK705 (Hamilton et al 1989) that contains the 3Ј region of thrS, infC, rpmI, rplT, and the 5Ј region of pheS. The resulting plasmid, named pNIBLE, carries a temperature-sensitive pSC101 origin of replication, the choramphenicol and the kanamycin resistance markers, the 3Ј region of thrS, infC, rpmI, the disrupted rplT gene, and the 5Ј region of pheS.…”