Frameshifting in the expression of the Escherichia coli trpR gene

The trpR gene of Escherichia coli carries an open reading frame that encodes the trp repressor, 108 amino acids long. Here we show that translation of an additional (+1) reading frame of trpR occurs both in vivo and in vitro. This results in the synthesis of a stable +1 frame polypeptide. Using site-specific mutagenesis, immunological techniques and amino acid sequencing we have found that the N-terminus of the +1 frame product and that of the known 0 frame product are identical but that their C-termini differ. Our results are discussed in relation to the role of natural frameshifting as a regulatory mechanism of gene expression in general, and with respect to tryptophan biosynthesis in particular.     

Maintaining the ribosomal reading frame: the influence of the E site during translational regulation of release factor 2

Maintenance of the translation reading frame is one of the most remarkable achievements of the ribosome while decoding the information of an mRNA. Loss of the reading frame through spontaneous frameshifting occurs with a frequency of one in 30,000 amino acid incorporations. However, at many recoding sites, the mechanism that controls reading frame maintenance is switched off. One such example is the programmed +1 frameshift site of the prfB gene encoding the termination factor RF2, in which slippage into the forward frame by one nucleotide can attain an efficiency of approximately 100%, namely, four orders of magnitude higher than normally observed. Here, using the RF2 frameshift window, we demonstrate that premature release of the E site tRNA from the ribosome is coupled with high-level frameshifting. Consistently, in a minimal system, the presence of the E site tRNA prevents the +1 frameshift event, illustrating the importance of the E site for reading-frame maintenance.     

Pulling the ribosome out of frame by +1 at a programmed frameshift site by cognate binding of aminoacyl-tRNA.

Programmed translational frameshifts efficiently alter a translational reading frame by shifting the reading frame during translation. A +1 frameshift has two simultaneous requirements: a translational pause which occurs when either an inefficiently recognized sense or termination codon occupies the A site, and the presence of a special peptidyl-tRNA occupying the P site during the pause. The special nature of the peptidyl-tRNA reflects its ability to slip +1 on the mRNA or to facilitate binding of an incoming aminoacyl-tRNA out of frame in the A site. This second mechanism suggested that in some cases the first +1 frame tRNA could have an active role in frameshifting. We found that overproducing this tRNA can drive frameshifting, surprisingly regardless of whether frameshifting occurs by peptidyl-tRNA slippage or out-of-frame binding of aminoacyl-tRNA. This finding suggests that in both cases, the shift in reading frame occurs coincident with formation of a cognate codon-anticodon interaction in the shifted frame.     

A ribosomal frameshifting error during translation of the argl mRNA of Escherichla coli

Using fusions between the Escherichia coli genes argI and lacZ, it has been demonstrated that ribosomal frameshifting occurs at a frequency of between 3% and 16% within the argl mRNA, soon after the initiation codon. The frameshift involves a phenylalanyl-tRNA shifting into the + 1 frame at the sequence UUU-U/C. The shift does not occur if the in-frame phenylalanine codon UUU is replaced by UUC. The level of frameshifting is higher in dense cultures and is not dependent on phenylalanine starvation. In the wild-type argI gene this frameshifting event would be an error, leading to a truncated, non-functional protein. Therefore, it is unlike the numerous examples of required frameshifting events that have been described in other genes.     

A ribosomal frameshifting error during translation of the argl mRNA of Escherichla coli

Using fusions between the Escherichia coli genes argI and lacZ, it has been demonstrated that ribosomal frameshifting occurs at a frequency of between 3% and 16% within the argl mRNA, soon after the initiation codon. The frameshift involves a phenylalanyl-tRNA shifting into the + 1 frame at the sequence UUU-U/C. The shift does not occur if the in-frame phenylalanine codon UUU is replaced by UUC. The level of frameshifting is higher in dense cultures and is not dependent on phenylalanine starvation. In the wild-type argI gene this frameshifting event would be an error, leading to a truncated, non-functional protein. Therefore, it is unlike the numerous examples of required frameshifting events that have been described in other genes.     

Release factor 2 frameshifting sites in different bacteria

The mRNA encoding Escherichia coli polypeptide chain release factor 2 (RF2) has two partially overlapping reading frames. Synthesis of RF2 involves ribosomes shifting to the +1 reading frame at the end of the first open reading frame (ORF). Frameshifting serves an autoregulatory function. The RF2 gene sequences from the 86 additional bacterial species now available have been analyzed. Thirty percent of them have a single ORF and their expression does not require frameshifting. In the ~70% that utilize frameshifting, the sequence cassette responsible for frameshifting is highly conserved. In the E. coli RF2 gene, an internal Shine–Dalgarno (SD) sequence just before the shift site was shown earlier to be important for frameshifting. Mutagenic data presented here show that the spacer region between the SD sequence and the shift site influences frameshifting, and possible mechanisms are discussed. Internal translation initiation occurs at the shift site, but any functional role is obscure.     

Analysis of the roles of tRNA structure, ribosomal protein L9, and the bacteriophage T4 gene 60 bypassing signals during ribosome slippage on mRNA

A 50-nucleotide coding gap divides bacteriophage T4 gene 60 into two open reading frames. In response to cis-acting stimulatory signals encrypted in the mRNA, the anticodon of the ribosome-bound peptidyl tRNA dissociates from a GGA codon at the end of the first open reading frame and pairs with a GGA codon 47 nucleotides downstream just before the second open reading frame. Mutations affecting ribosomal protein L9 or tRNA(Gly)(2), the tRNA that decodes GGA, alter the efficiency of bypassing. To understand the mechanism of ribosome slippage, this work analyzes the influence of these bypassing signals and mutant translational components on -1 frameshifting at G GGA and hopping over a stop codon immediately flanked by two GGA glycine codons (stop-hopping). Mutant variants of tRNA(Gly)(2) that impair bypassing mediate stop-hopping with unexpected landing specificities, suggesting that these variants are defective in ribosomal P-site codon-anticodon pairing. In a direct competition between -1 frameshifting and stop-hopping, the absence of L9 promotes stop-hopping at the expense of -1 frameshifting without substantially impairing the ability of mutant tRNA(Gly)(2) variants to re-pair with the mRNA by sub-optimal pairing. These observations suggest that L9 defects may stimulate ribosome slippage by enhancing mRNA movement through the ribosome rather than by inducing an extended pause in translation or by destabilizing P-site pairing.Two of the bypassing signals, a cis-acting nascent peptide encoded by the first open reading frame and a stemloop signal located in the 5' portion of the coding gap, stimulate peptidyl-tRNA slippage independently of the rest of the gene 60 context. Evidence is presented suggesting that the nascent peptide signal may stimulate bypassing by destabilizing P-site pairing.     

Normal tRNAs promote ribosomal frameshifting.

The addition of Ser AGC AGU tRNA to an E. coli cell-free protein synthesizing system which contains the endogenous tRNA levels results in up to 100% of the ribosomes translating the MS2 coat gene shifting into the -1 reading frame. An analogous phenomenon is seen at a much lower level without the tRNA addition, where a shift into the +1 frame can also be detected. Thus translation with the endogenous tRNA levels yields proteins which have the amino terminus of the coat protein but which are substantially larger than the coat protein and comprise about 5% of the coat translation. Since the lysis gene overlaps the 3' end of the coat gene in the +1 frame, we conclude that the reading frame shift into the +1 frame yields a hybrid protein. Also, we present evidence that ribosomes translating the synthetase gene shift into the -1 frame near the distal end of the gene. This frameshifting is promoted by thrACU ACC tRNA. Specific competitor tRNAs for both Thr and Ser tRNA-promoted frameshifting have been characterized. The generality of this new mechanism for producing additional proteins is unclear, but it investigation should increase understanding of the coding mechanism and its origin.     

Generation of various amino acids mutants in the trpR gene of Escherichia coli by site-directed mutagenesis

Tryptophan repressor (trpR) gene lacks various amino acid codons. To establish these codons in the trpR gene, we created the mutants by site-directed mutagenesis in the trpR gene of pHK1 plasmid. The interested regions of trpR gene were amplified, cloned in pT7-5 plasmid and transformed in to the cells harboring pGP1-2 plasmid. These plasmid products were labeled with (35)S Met, and following sequencing we observed the presence of mutants for cysteine, glycine, serine and lysine in the trpR gene of E. coli. Therefore, using these approach mutants in various genes of E. coli could be established and used as a tool to study translational bypassing in trpR gene of E. coli.     

Antisense-induced ribosomal frameshifting

Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1–ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem–loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5′ of the shift site and to polyamine levels.     

The Mof2/Sui1 Protein Is a General Monitor of Translational Accuracy

Although it is essential for protein synthesis to be highly accurate, a number of cases of directed ribosomal frameshifting have been reported in RNA viruses, as well as in procaryotic and eucaryotic genes. Changes in the efficiency of ribosomal frameshifting can have major effects on the ability of cells to propagate viruses which use this mechanism. Furthermore, studies of this process can illuminate the mechanisms involved in the maintenance of the normal translation reading frame. The yeast Saccharomyces cerevisiae killer virus system uses programmed −1 ribosomal frameshifting to synthesize its gene products. Strains harboring the mof2-1 allele demonstrated a fivefold increase in frameshifting and prevented killer virus propagation. In this report, we present the results of the cloning and characterization of the wild-type MOF2 gene. mof2-1 is a novel allele of SUI1, a gene previously shown to play a role in translation initiation start site selection. Strains harboring the mof2-1 allele demonstrated a mutant start site selection phenotype and increased efficiency of programmed −1 ribosomal frameshifting and conferred paromomycin sensitivity. The increased frameshifting observed in vivo was reproduced in extracts prepared from mof2-1 cells. Addition of purified wild-type Mof2p/Sui1p reduced frameshifting efficiencies to wild-type levels. Expression of the human SUI1 homolog in yeast corrects all of the mof2-1 phenotypes, demonstrating that the function of this protein is conserved throughout evolution. Taken together, these results suggest that Mof2p/Sui1p functions as a general modulator of accuracy at both the initiation and elongation phases of translation.     

Analysis of effects of tRNA:message stability on frameshift frequency at the Escherichia coli RF2 programmed frameshift site.

The codon that is in-frame prior to +1 frameshifting at the E.coli prfB (RF2 gene) frameshift site is randomized to create thirty-two variants. These alleles vary 1000-fold in frameshift-dependent expression in fusions to lacZ. Frameshifting is more frequent at sites where the in-frame codon ends in uridine, as if third position wobble pairs to message uridine facilitate slippage into the +1 frame. Consistent with other studies of programmed frameshift sites, efficient frameshifting depends on stable message:tRNA base pairs after rephasing. For complexes with mispairs, frameshift frequency depends on the nature, number, and position of mispairs. Central purine:purine mispairs are especially inhibitory. Relative stabilities of +1 rephased complexes are estimated from published data on the stabilities of tRNA:tRNA complexes. Stability correlates with frameshifting over its entire range, which suggests that stability is an important determinant of the probability of translation of the rephased complex.     

The interplay of mRNA stimulatory signals required for AUU-mediated initiation and programmed -1 ribosomal frameshifting in decoding of transposable element IS911

The IS911 bacterial transposable element uses -1 programmed translational frameshifting to generate the protein required for its mobility: translation initiated in one gene (orfA) shifts to the -1 frame and continues in a second overlapping gene (orfB), thus generating the OrfAB transposase. The A-AAA-AAG frameshift site of IS911 is flanked by two stimulatory elements, an upstream Shine-Dalgarno sequence and a downstream stem-loop. We show here that, while they can act independently, these stimulators have a synergistic effect when combined. Mutagenic analyses revealed features of the complex stem-loop that make it a low-efficiency stimulator. They also revealed the dual role of the upstream Shine-Dalgarno sequence as (i) a stimulator of frameshifting, by itself more potent than the stem-loop, and (ii) a mandatory determinant of initiation of OrfB protein synthesis on an AUU codon directly preceding the A6G motif. Both roles rely on transient base pairing of the Shine-Dalgarno sequence with the 3' end of 16S rRNA. Because of its effect on frameshifting, the Shine-Dalgarno sequence is an important determinant of the level of transposase in IS911-containing cells, and hence of the frequency of transposition.     

Ribosome can resume the translation in both +1 or -1 frames after encountering an AGA cluster in Escherichia coli

In Escherichia coli the rare codons AGG, AGA and CGA are reported to have a detrimental effect on protein synthesis, especially during the expression of heterologous proteins. In the present work, we have studied the impact of successive clusters of these rare codons on the accuracy of mRNA translation in E. coli. For this purpose, we have analyzed the expression of an mRNA which contains in its 3' region a triplet and a tandem of AGA codons. This mRNA is derived from the human hepatitis B virus (HBV) preC gene. Both in eukaryotic cells and in eukaryotic cell-free translation system, this mRNA, directs the synthesis of a single 25 kDa protein. However, in a conventional E. coli strain BL 21 (DE3), transformed with a plasmid expressing this protein the synthesis of four polypeptides ranging from 30 to 21.5 kDa can be observed. Using different approaches, notably expression of i) precore mutated proteins or ii) chimeric proteins containing HA- and Myc-tags downstream of the AGA clusters (respectively in the -1 or +1 frame), we have found that when the ribosome encounters the AGA clusters, it can then resume the translation in both +1 and -1 frames. This result is in agreement with the model proposed recently by Baranov et al. (Baranov, P.V., Gesteland, R.F., Atkins, J.F., 2004. P-site tRNA is a crucial initiator of ribosomal frameshifting. RNA 10, 221-230), thus confirming that AGA/AGG codons can serve as sites for -1 frameshifting events. Only +1 frameshifting was suggested previously to occur at the AGA/AGG clusters.     

Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide minimal site

Ribosomal frameshifting regulates expression of the TYB gene of yeast Ty retrotransposons. We previously demonstrated that a 14 nucleotide sequence conserved between two families of Ty elements was necessary and sufficient to support ribosomal frameshifting. This work demonstrates that only 7 of these 14 nucleotides are needed for normal levels of frameshifting. Any change to the sequence CUU-AGG-C drastically reduces frameshifting; this suggests that two specific tRNAs, tRNA(UAGLeu) and tRNA(CCUArg), are involved in the event. Our tRNA overproduction data suggest that a leucyl-tRNA, probably tRNA(UAGLeu), an unusual leucine isoacceptor that recognizes all six leucine codons, slips from CUU-Leu onto UUA-Leu (in the +1 reading frame) during a translational pause at the AGG-Arg codon induced by the low availability of tRNA(CCUArg), encoded by a single-copy essential gene. Frameshifting is also directional and reading frame specific. Interestingly, frameshifting is inhibited when the "slip" CUU codon is located three codons downstream, but not four or more codons downstream, of the translational initiation codon.     

An mRNA sequence derived from a programmed frameshifting signal decreases codon discrimination during translation initiation

Sequences and structures in the mRNA can alter the accuracy of translation. In some cases, mRNA secondary structures like hairpin loops or pseudoknots can cause frequent errors of translational reading frame (programmed frameshifting) or misreading of termination codons as sense (nonsense readthrough). In other cases, the primary mRNA sequence stimulates the error probably by interacting with an element of the ribosome to interfere with error correction. One such primary mRNA sequence, the Ty3 stimulator, increases programmed +1 frameshifting 7.5 times in the yeast Saccharomyces cerevisiae. Here we show that this stimulator also increases the usage of non-AUG initiation codons in the bacterium Escherichia coli but not in S. cerevisiae. These data suggest that in E. coli, though not in yeast, an element of the ribosome's elongation accuracy mechanism ensures initiation accuracy.     

Enhancement of ribosomal frameshifting by oligonucleotides targeted to the HIV gag-pol region.

The pol gene of all retroviruses is expressed as a gag-pol fusion protein which is proteolytically processed to produce all viral enzymes. In the human immunodeficiency virus (HIV), the gag and pol genes overlap by 241 nucleotides with pol in the -1 phase with respect to gag. The gag-pol fusion is produced via a -1 ribosomal frameshifting event that brings the overlapping, out-of-phase gag and pol genes into translational phase. Frameshifting occurs at a so called 'shift site' 8-10 nucleotides upstream of a hairpin loop which may play a role in the regulation of frameshifting. We have fused this region of HIV-1 to the 5' end of the firefly luciferase reporter gene in order to quantitatively measure ribosomal frameshifting both in cells and by in vitro translation. A series of 2'-O-methyl oligonucleotides was designed to specifically bind the sequences which flank the gag-pol hairpin. Ribosomal frameshifting is enhanced up to 6 fold by those oligonucleotides which bind the area just 3 to the stem. Oligonucleotides which bind 5' to the stem have no effect on frameshift efficiency. In addition, we have constructed a series of fusion genes which mimic the effect of the bound oligonucleotides with intramolecular hairpins. The results suggest that increasing RNA secondary structure downstream of the shift site increases the frequency of ribosomal frameshifting, and that this effect can be mimicked by antisense oligonucleotides.     

Frameshifting by eukaryotic ribosomes during expression of Escherichia coli release factor 2.

Normal translation of the gene for E. coli release factor 2 (RF-2) is characterized by a +1 frameshift event that occurs with 30-50% efficiency. Frameshifting on synthetic RF-2 mRNA by eukaryotic ribosomes has also been observed, even though they lack the capability to interact with the frameshift signal in the same manner as prokaryotic ribosomes. We have mutagenized the sequence of the RF-2 gene to eliminate the need for a frameshift, thereby allowing frameshifting efficiency to be measured by direct comparison of RF-2 production from the mutant with production from the wild-type. Measurements using this approach confirm that frameshifting by rabbit reticulocyte lysate ribosomes occurs at the frameshift region, but with a limited efficiency of approximately 0.4%.