Alternative occupancy of a dual ribosomal binding site by mRNA affected by translation initiation factors

Abstract

The interaction between Escherichia coli 30s ribosomal subunits and mRNAs, and the effect of the initiation factors on this process, have been studied using MS2 RNA, polyribonucleotides and model mRNAs encoded by synthetic genes. The interactions were analyzed by gel filtration, by sucrose gradient centrifugation and by competition for ribosome binding between the various mRNAs and a Shine-Dalgarno deoxyoctanucleotide. It was found that the initiation factors do not significantly affect the Shine-Dalgarno interaction nor the apparent K, values of the 30s-subunit -mRNA binary complexes, but influence the positioning of the mRNAs on the 30s subunit with respect to the Shine-Dalgarno octanucleotide. The results suggest that, in the absence of initiation factors, the mRNA occupies a ribosomal "stand-by" site which is close to or includes the region where the ShineDalgarno interaction takes place; in the presence of the factors, the mRNA is shifted away from the stand-by site, towards another ribosomal site with similar affinity for the mRNA. This shift does not require the presence of fMet-tRNA and, depending upon the type of mRNA, is mediated by IF-2 and/or IF-3.A key step in the initiation of protein biosynthesis is the recognition of the translation initiation region of mRNAs by ribosomes. In prokaryotes, this mRNA region is characterized by the presence of an initiation triplet (AUG in most cases) and, very often, by the purine-rich Shine-Dalgarno sequence. The latter is of variable length and potential complementarity to the 3' end of the 16s rRNA and is separated from the initiation triplet by a spacer, also of variable length [l -51. Even though the Shine-Dalgarno sequence was found to be dispensable in vivo [6] and mechanistically irrelevant for the initiation process and for the selection of the mRNA reading frame in vitro [7], it has been demonstrated that protein synthesis in vivo makes use of this interaction and is strongly enhanced by its presence [8,9]. It has been suggested that the function of the Shine-Dalgarno sequences is to provide highaffinity sites of the mRNA for ribosomes in order to ensure high concentrations of potential initiation triplets near the ribosomal P-site decoding region [7] where the initiation factors (IFs) influence the kinetics of codon-anticodon interaction [4, 51. A function commonly attributed to the IFs, IF-3 in particular, is to promote the binding of the mRNA to the 30s ribosomal subunits, thus allowing the formation of a 30s -mRNA binary complex [lo, 3 11. To reconcile conceptually the requirement for the IFs in translation with the existence of the Shine-Dalgarno interaction, which should be sufficient by itself to ensure an efficient mRNA-ribosome interaction, it has been proposed that IF-3 is especially needed to confer stability on those mRNA-ribosome complexes involving very weak or no Shine-Dalgarno base-pairing [lo, 12, 131; alternatively, it has been suggested that IF-3 is required to free the anti-Shine-Dalgarno region of 16s rRNA from an intramolecular...

A new model for the three-dimensional folding of Escherichia coli 16 s ribosomal RNA. III † . The topography of the functional centre 1 †Paper II in this series is an accompanying paper, Mueller & Brimacombe (1997b). 1Edited by D. E. Draper

“…The thickness of the latter contact is seen to vary between different EM reconstructions (cf. Such a mechanism would ensure that the incoming mRNA is precisely positioned as it approaches the decoding area, and is consistent with the concept of a``stand-by'' site for the mRNA (Canonaco et al, 1989;La Teana et al, 1995), whereby the mRNA becomes partially relocated under the in¯uence of the initiation factors. Thus, after formation of the Shine-Dalgarno interaction, the downstream region of the mRNA would wrap around the 30 S subunit so as to ®nd its contacts with the 16 S rRNA (Figure 10(b) and (c)), and the right-hand head-body junction would then close, clamping the mRNA into position.…”

Ribosome‐messenger recognition in the absence of the Shine‐Dalgarno interactions

“…As was shown earlier, IF3 does not influence the affinity of the 30s subunit for mRNAs with or without SD sequence [24,25], its role in the initiation process (besides subunit anti-association function) concerns mainly the selection of the initiator tRNA [ 191. IF3 can be regarded as kinetic effector and fidelity factor of 30s initiation complex formation [26].…”

“…In this article we present evidence for that E. coli 30s ribosomal subunits are able to recognize the translational starts on (-SD)mRNAs bearing 5' untranslated regions of plant viral RNAs or derivatives thereof, and to form stable ternary initiation complexes only in the presence of ribosomal protein Sl and IF3. As was shown earlier, IF3 does not influence the affinity of the 30s subunit for mRNAs with or without SD sequence [24,25], its role in the initiation process (besides subunit anti-association function) concerns mainly the selection of the initiator tRNA [ 191. IF3 can be regarded as kinetic effector and fidelity factor of 30s initiation complex formation [26].…”

Identification of Mammalian Mitochondrial Translational Initiation Factor 3 and Examination of Its Role in Initiation Complex Formation with Natural mRNAs

“…IF3 binds to the 30 S subunit and inhibits its association with the 50 S subunit, thus ensuring a supply of 30 S subunits for initiation (13,14). IF3 also promotes an adjustment of the position of the mRNA on the 30 S subunit facilitating codon-anticodon interactions between the AUG codon and fMet-tRNA in the P site (15)(16)(17)(18). IF3 acts to switch the decoding preference of the small ribosomal subunit from elongator tRNAs to the initiator tRNA in the P site, thus playing a proofreading role in initiation (19 -21).…”