Two genes specifying model mRNAs of minimal size and coding capacity, with or without the ShineDalgarno (SD) sequence, were assembled, cloned, and transcribed in high yields. These mRNAs, as well as synthetic polynucleotides, phage MS2 RNA, and a deoxyoctanucleotide complementary to the 3' end of 16S rRNA were used to study the mechanism of translation initiation in vitro. Escherichia coli 30S ribosomal subunits interact with all these nucleic acids, albeit with different affinities; the affinity for the mRNA with the SD sequence (Ka ==2 x 10 M-1) is more than an order of magnitude higher than that for the mRNA lacking this sequence. The initiation factors are equally required, regardless of the presence of the SD sequence, for 30S and 70S initiation complex formation and for mRNA translation, but the initiation factors do not affect the SD interaction or the binding of the mRNAs to the ribosomes. The SD interaction is also mechanistically irrelevant for 30S initiation complex formation and is not essential for translation in vitro or for the selection of the mRNA reading frame. It is suggested that the function of the SD interaction is to ensure a high concentration of the initiation triplet near the ribosomal peptidyl-tRNA binding site, whereas the selection of the translational start is achieved kinetically, under the influence of the initiation factors, during decoding of the iinitiator tRNA.Gene expression ultimately depends on how efficiently and accurately each mRNA translation initiation region is selected by the ribosomes, but we are often unable to rationalize why some genes are either poorly or highly expressed at the translational level (for reviews, see refs. 1-6).Comparison of several hundred translation initiation regions allowed the identification of a rather loose consensus sequence (1-3) and the conclusion that this region preferentially contains bases (i.e., adenine and uracil) that minimize secondary structure formation and includes an initiation triplet (AUG in >90% of the cases) (3). The initiation triplet is often preceded by a purine-rich Shine-Dalgarno (SD) sequence of variable length and potential complementarity to the 3' end of 16S rRNA (the anti-SD sequence). The SD sequence is separated from the initiation triplet by a spacer, most frequently 7-9 bases long (7,8). Convincing evidence for the participation of the SD sequence in initiation has been obtained (9, 10), but its precise function is unknown.To further the studies on the mechanism of translation and, more specifically, on mRNA-ribosome interaction at initiation, we designed, assembled, and expressed in vitro, a gene specifying a model mRNA (designated 002 mRNA) of minimal size and coding capacity containing the canonical translation initiation region of natural mRNAs. Deletion of a restriction fragment from this gene allowed us to compare the activity of two mRNAs identical to each other except for the presence of the SD sequence and to ask if the ribosomal binding of mRNA is different with natural and synthetic mRNA...
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...
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