We have quantitatively analyzed the relationship between translational efficiency and the mRNA secondary structure in the initiation region. The stability of a defined hairpin structure containing a ribosome binding site was varied over 12 kcal/mol (1 cal = 4.184 J) by site-directed mutagenesis and the effects on protein yields were analyzed in vivo. The results reveal a strict correlation between translational efficiency and the stability of the helix. An increase in its AG0 of -1.4 kcal/mol (i.e., less than the difference between an A'U and a G-C pair) corresponds to the reduction by a factor of 10 in initiation rate. Accordingly, a single nucleotide substitution led to the decrease by a factor of 500 in expression because it turned a mismatch in the helix into a match. We find no evidence that exposure of only the Shine-Dalgarno region or the start codon preferentially favors recognition. Translational efficiency is strictly correlated with the fraction of mRNA molecules in which the ribosome binding site is unfolded, indicating that initiation is completely dependent on spontaneous unfolding of the entire initiation region. Ribosomes appear not to recognize nucleotides outside the ShineDalgarno region and the initiation codon.There is good evidence that mRNA secondary structure is a key factor in determining the efficiency of translational initiation in prokaryotes (1-3). The expression of several genes in Escherichia coli appeared inversely related to the stability of the secondary structure of their ribosome binding sites (4-9).Here, we present a quantitative analysis of the relationship between the stability of a local secondary structure and the level of gene expression in vivo. As a model system, we have chosen the coat protein gene of RNA bacteriophage MS2. (i) Analysis with structure-sensitive enzymes and chemicals as well as phylogenetic sequence comparison have provided evidence that its ribosome binding site adopts a defined hairpin structure (Fig. 1) (14).Western Blot Analysis. Cultures of the appropriate clones were grown at 280C to an OD650 of 0.2, followed by induction at 420C for 20 min. Samples of 1 ml were centrifuged and the pellets were boiled in SDS buffer (17). Serial dilutions were fractionated by SDS/PAGE (17) on a 15% gel and blotted onto nitrocellulose. Translation products were immunodetected by using antibodies raised against SDS-denatured MS2 coat protein and alkaline phosphatase-linked goat anti-rabbit antibodies (Sigma). RESULTSThe secondary structure of the initiation region of the MS2 coat protein gene is shown in Fig. 2 with the mutations introduced. All mutations leave the Shine-Dalgarno (SD) region (GGAG) and the amino acid sequence of the coat protein intact. In Fig. 3

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We have inserted the sequence 5'-AAG-GAGGU-3', which is complementary to the 3' terminus of Escherichia coli 16S rRNA, in a reading frame and analyzed its effect on the accuracy and overall rate of translation in vivo. Translation over the sequence yields a 50% ribosomal frameshift if the reading phase is A-AGG-AGG-U. The other two possible frames do not give shifts. The introduction of a UAA stop codon before (UAA-AGG-AGG-U) but not after (A-AGG-AGG-UAA) the AGG codons abolishes the frameshift. The change in the reading phase occurs exclusively' to the + 1 direction. Efficient frameshifting is also induced by the sequence A-AGA-AGA-U. The arginine codons AGG and AGA are read by minor tRNA. Suppression of frameshifting takes place when a gene for minor tRNAArg is introduced on a multicopy plasmid. We suggest that frameshifting during translation of the A-AGG-AGG-U sequence is due to the erroneous decoding of the tandem AGG codons and arises by depletion of tRNAAra. The complementarity of tandem AGG codons to the 3' terminus of 16S rRNA is a coincidence and apparently not related to the shift. Replacing the AGG-AGG sequence by the optimal arginine codons CGU-CGU does not increase the overall rate of translation.

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Arginine is coded for by CGN (N = G, A, U, C), AGA and AGG. In Escherichia coli there is little tRNA for AGA and AGG and the use of these codons is strongly avoided in virtually all genes. Recently, we demonstrated that the presence of tandem AGA or AGG codons in mRNA causes frameshifts with high frequency. Here, we show that phaseshifts can be suppressed when cells are transformed with the gene for tRNA(T4Arg) or E. coli tRNA(argU,Arg) demonstrating that such errors are the result of tRNA depletion. Bacteriophage T4 encoded tRNA(Arg) (anticodon UCU) corrects shifts at AGA-AGA but not at AGG-AGG, suggesting that this tRNA can only read AGA. Similarly, comparison of the translational efficiencies in an argU (Ts) mutant and in its isogenic wild type parent indicates that argU tRNA (anticodon UCU) reads AGA but not AGG. An argU (Ts) mutant barely reads through AGA-AGA at 42 degrees C but translation of AGG-AGG is hardly, if at all, affected. Overexpression of argU+ relaxes the codon specificity. The thermosensitive mutant in argU, previously called dnaY because it is defective in DNA replication, can be complemented for growth by the gene for tRNA(T4Arg). This implies that the sole function of the argU gene product is to sustain protein synthesis and that its role in replication is probably indirect.