Selection of the mRNA translation initiation region by Escherichia coli ribosomes.

Abstract

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...

Correlation of mRNA Expression and Protein Abundance Affected by Multiple Sequence Features Related to Translational Efficiency in Desulfovibrio vulgaris: A Quantitative Analysis

“…Therefore, all moderately or highly expressed genes should have a good combination of a good RBS and an efficient start codon. In contrast, some other findings suggested that the SD sequence has little influence on protein expression, as the binding of the 16S ribosomal subunit has been found not to be essential for translation initiation (Calogero et al 1988). In fact, there exist mRNAs without SD sequences (Boni et al 2001).…”

Ribosomal localization of the mRNA in the 30S initiation complex as revealed by UV crosslinking

“…very high Mg :÷ concentrations, absence of one or more of the initiation factors, use of non-natural forms of initiator tRNA). In this article we report on the topographical localization of the model 002 mRNA which has the consensus TIR sequence upstream of the AUG initiation triplet [7] crosslinked by UV-irradiation in a homogeneous 30S initiation complex (i.e. containing fMet-tRNA and initiation factors IFI, IF2 and IF3), …”

Modulation of the Rate of Peptidyl Transfer on the Ribosome by the Nature of Substrates

“…(12,26,27). The mRNAs used were 122-nucleotidelong derivatives of m022 mRNA (28) with the coding sequence 5Ј-AUG-NNN-UUC-3Ј, where NNN ϭ GCA, CGC, GAU, AAA, UUU, CCG, UCU, or GUG, coding for Ala, Arg, Asp, Lys, Phe, Pro, Ser, or Val, respectively. Initiation complexes were prepared by incubating 70 S ribosomes (1 M) with a 3-fold excess of mRNA; 1.5 M initiation factors 1, 2, and 3; 1.5 M [ 3 H]fMet-tRNA fMet ; and 1 mM GTP in buffer A for 30 min at 37°C.…”