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Translation of the Sequence AGG-AGG Yields 50% Ribosomal Frameshift (Accuracy/Minor Trna/Rare Codon/Shine-Dalgarno Complementarity) R

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Translation of the Sequence AGG-AGG Yields 50% Ribosomal Frameshift (Accuracy/Minor Trna/Rare Codon/Shine-Dalgarno Complementarity) R Proc. Nat!. Acad. Sci. USA Vol. 85, pp. 7967-7971, November 1988 Biochemistry Translation of the sequence AGG-AGG yields 50% ribosomal frameshift (accuracy/minor tRNA/rare codon/Shine-Dalgarno complementarity) R. A. SPANJAARD AND J. VAN DuIN* Department of Biochemistry, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands Communicated by Marianne Grunberg-Manago, August 1, 1988 ABSTRACT We have inserted the sequence 5'-AAG- In spite of this degree of sophistication, the error-control GAGGU-3', which is complementary to the 3' terminus of mechanism is, by accident or by design, not at all foolproof. Escherichia coli 16S rRNA, in a reading frame and analyzed its We describe here an example of the breakdown of this effect on the accuracy and overall rate of translation in vivo. elaborate system in wild-type Escherichia coli cells. The Translation over the sequence yields a 50% ribosomal frame- presence of two consecutive AGG codons in a reading frame shift if the reading phase is A-AGG-AGG-U. The other two raises the ambiguity level by 3 orders of magnitude. Our possible frames do not give shifts. The introduction of a UAA results identify tRNAA`g shortage as the cause of frameshift- stop codon before (UAA-AGG-AGG-U) but not after (A-AGG- ing. Bacterial mechanisms to prevent such errors do not AGG-UAA) the AGG codons abolishes the frameshift. The appear to exist. change in the reading phase occurs exclusively' to the + 1 This is not the first time that an abnormally high error rate direction. Efficient frameshifting is also induced by the se- has been found in vivo at a single position. Recent studies quence A-AGA-AGA-U. The arginine codons AGG and AGA (reviewed in ref. 11) have shown that some messengers are read by minor tRNA. Suppression of frameshifting takes contain the information for the desired protein in two differ- place when a gene for minor tRNAArg is introduced on a ent reading frames and that, accordingly, specific phase multicopy plasmid. We suggest that frameshifting during changes must, and in fact do, occur. Notably, during trans- translation of the lation of the E. coli mRNA encoding peptide chain-release A-AGG-AGG-U sequence is due to the er- factor 2 (RF2), shifts of up to 50%o have been reported (12). roneous decoding of the tandem AGG codons and arises by Apparently, nature itself has found ways to bypass the rules depletion of tRNAAra. The complementarity of tandem AGG of triplet decoding. We will shortly discuss differences and codons to the 3' terminus of 16S rRNA is a coincidence and similarities'between shifts at naturally occurring sequences apparently not related to the shift. Replacing the AGG-AGG and the one we describe here. sequence by the optimal arginine codons CGU-CGU does not increase the overall rate of translation. MATERIALS AND METHODS The error accompanying the translation of mRNA into Bacterial Strains and Growth Conditions. In all experiments protein is on the order of5 x 1O- per codon (1). This degree the E. coli K-12 strain M5219 was used (13). This strain of accuracy does not simply reflect the specificity of codon- harbors a defective, nonexcisable A prophage carrying a anticodon interaction but is obtained through fidelity- mutant cI gene (cIts857) and the gene for the transcription amplification processes that involve a substantial mass of the antitermination factor N. Cultures were grown in L broth, ribosome (2-4). One such error-suppressing activity, for which contained (per liter) 10 g of tryptone, 5 g of'yeast which evidence has been presented, is a GTP-consuming extract (Difco), 8 g of NaCl, and 5 ml of 1 M Tris (pH 7.3). proofreading step at the level of aminoacyl-tRNA binding (5, Cells were grown at 280C to an OD650 of0.25 and then induced 6). The active role of the ribosome in setting the translational at 420C for 30 min. accuracy is confirmed by the existence of mutations in Plasmids. In clones 1, 2, and 3 the plasmids were derivatives ribosomal protein genes that profoundly affect the error rate. ofp13.3 (14). Clones 4-21 were derived from pPLc236 (13). In For instance, mutations in ribosomal protein S12 that confer all clones the MS2 cDNA present was the EcoRI fragment resistance to streptomycin result in a greatly increased containing the sequence 103-1628 (MS2 numbers) (15). fidelity (ref. 7 and references therein). Recombinant DNA Procedures. Standard cloning proce- A dures were used as described (16). Restriction enzymes, second potential source of translation errors is an bacteriophage T4 DNA ligase, and E. coli DNA polymerase unbalanced supply of aminoacyl-tRNA. Since the accuracy I (Klenow) were obtained from Pharmacia. Unphosphoryl- of selection is basically proportional to the ratio of correct to ated, annealed oligonucleotides were inserted into linearized incorrect substrates available, it follows that limitations in vector DNA at a 250-fold molar excess. The nucleotide one or more amino acids could drastically increase the error sequence of cloned inserts was determined in the phage M13 at those codons for which aminoacyl-tRNA is in short supply. system developed by Messing and Vieira (17). Only in one Such an increase has indeed been found in vitro and in relA instance (clone 7) was the sequence different than projected. mutants, but in wild-type bacteria this error is suppressed by Radiolabeling of Proteins. 'Cells were grown in M9CA the induction of guanosine 3'-diphosphate 5'-diphosphate medium (16). After induction at 420C, 30 ,uCi (11.1 x 105 (ppGpp) (8-10). ppGpp is produced on the ribosome by the becquerels) of [3H]valine (New England Nuclear) was added relA gene product when, due to aminoacyl-tRNA shortage, and growth continued for 30 min at 420C. The pellet of 1 ml uncharged tRNA binds to the ribosomal aminoacyl-tRNA (A) of cells was dissolved in 25 tkl of 2 x Laemmli sample buffer site. (18) and analyzed as indicated in the text. The publication costs of this article were defrayed in part by page charge Abbreviations: ppGpp, guanosine 3'-diphosphate 5'-diphosphate; A payment. This article must therefore be hereby marked "advertisement" site, aminoacyl-tRNA site; RF2, peptide chain-release factor 2. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7967 Downloaded by guest on September 29, 2021 7968 Biochemistry: Spanjaard and van Duin Proc. Natl. Acad. Sci. USA 85 (1988) RESULTS C 1 #2z 3 The Sequence A-AGG-AGG-U Induces a + 1 Frameshift. our to express the rat interferon a1 gene in E. During efforts - coat-I FN coli by translational reinitiation, we constructed the clone fusion depicted in Fig. 1. Our strategy was that the efficiently translated coat-protein gene of RNA phage MS2 will direct a large proportion of the cell's ribosomes to the stop codon present 5 bases downstream of the start codon of the inter- - coot feron gene. There is a variable probability that terminating f ragment ribosomes will reinitiate at a nearby start codon (refs. 19-22 and references therein). In an attempt to boost the reinitiation efficiency, an 8-base-long Shine-Dalgarno region (AAGGA- GGU) was placed before the interferon start. Immunoblots of FIG. 2. Immunoblot analysis showing the presence of a coat- cell extracts of this clone showed that beside mature inter- interferon fusion protein in clone 1. Stronger staining of the upper feron, a coat-interferon fusion protein was produced that background bands in the control (C) and clone 1 lanes reflects higher could only have arisen by a + 1 frameshift. Fig. 2 shows an cell extract loading. The relevant sequences of clones 1-3 are shown immunoblot of this clone (no. 1) developed with antibodies in Fig. 1. In lane C the coat gene fragment is cloned in the opposite against the MS2 coat protein. The upper indicated band orientation. An antiserum against MS2 coat protein was used and the corresponds to the fusion product, and the lower band corre- procedure described by Towbin et al. (23) was followed to develop sponds to the coat-protein fragment that is produced by the blot. termination just behind the interferon start. stop codon (Fig. 3 and Table 1). Immunoblot analysis of this To roughly identify the region where the shift had oc- clone two bands (Fig. 4). The upper one was identified curred, we placed a UAA stop codon before the SD region gave 147-residue coat because it had the correct (clone 2); as shown in Fig. 2, this change abolished the as the protein, frameshift product. In clone 3, the guanine residues in the SD mobility and was absent in the control (lane C). The lower band needed identification as the product originating from region were replaced by pyrimidines (AAGGAGGU AACUAUCU). This change also prevented appearance of out-of-phase termination at the overlined ochre (UAA) codon the frameshift product. We consider these results as good (Table 1). It was not a proteolytic degradation product of the indications that the frameshift originates in the AAGGAGGU 147-amino acid protein, because when we substituted the region. arginine codons AGG-AGG for their synonyms CGU-CGU Since the construct shown in Fig. 1 is not convenient to the lower band disappeared (Fig. 4, clone 6). In addition, assay large numbers of sequences for their capacity to change when the stop codon in the + 1 frame was eliminated as in the ribosomal reading frame, we set up a simpler system clones 5 and 7, the lower band disappeared (Table 1). The (outlined in Fig.
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