Leftward ribosome frameshifting at a hungry codon.

Previous experiments have shown that limitation for certain aminoacyl-tRNA species results in phenotypic suppression of a subset of frameshift mutant alleles, including members in both the (+) and (-) incorrect reading frames. Here, we demonstrate that such phenotypic suppression can occur through a ribosome reading frame shift at a hungry AAG codon calling for lysyl-tRNA in short supply. Direct amino acid sequence analysis of the product and DNA sequence manipulation of the gene demonstrate that the ribosome frameshift occurs through a movement of one base to the left, so as to decode the triplet overlapping the hungry codon from the left or 5' side, followed by continued normal translation in the new, shifted reading frame.     

Expression levels influence ribosomal frameshifting at the tandem rare arginine codons AGG_AGG and AGA_AGA in Escherichia coli

The rare codons AGG and AGA comprise 2% and 4%, respectively, of the arginine codons of Escherichia coli K-12, and their cognate tRNAs are sparse. At tandem occurrences of either rare codon, the paucity of cognate aminoacyl tRNAs for the second codon of the pair facilitates peptidyl-tRNA shifting to the +1 frame. However, AGG_AGG and AGA_AGA are not underrepresented and occur 4 and 42 times, respectively, in E. coli genes. Searches for corresponding occurrences in other bacteria provide no strong support for the functional utilization of frameshifting at these sequences. All sequences tested in their native context showed 1.5 to 11% frameshifting when expressed from multicopy plasmids. A cassette with one of these sequences singly integrated into the chromosome in stringent cells gave 0.9% frameshifting in contrast to two- to four-times-higher values obtained from multicopy plasmids in stringent cells and eight-times-higher values in relaxed cells. Thus, +1 frameshifting efficiency at AGG_AGG and AGA_AGA is influenced by the mRNA expression level. These tandem rare codons do not occur in highly expressed mRNAs.     

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.     

Predicting ribosomal frameshifting efficiency

Many retroviruses use -1 ribosomal frameshifting as part of the mechanism in translational control of viral protein synthesis. Quantitative prediction of the efficiency of -1 frameshifting is crucial for understanding the viral gene expression. Here we investigate the free energy landscape for a minimal -1 programmed ribosomal frameshifting machinery, including the codon-anticodon base pairs at the slippery site, the downstream messenger RNA structure and the spacer between the slippery site and the downstream structure. The free energy landscape analysis leads to a quantitative relationship between the frameshifting efficiency and the tension force generated during the movement of codon-anticodon complexes, which may occur in the A/T to A/A accommodation process or the translocation process. The analysis shows no consistent correlation between frameshifting efficiency and global stability of the downstream mRNA structure.     

Imbalance of tRNAPro isoacceptors induces +1 frameshifting at near-cognate codons

Increased expression of the CCU/CCA/CCG-decoding tRNA^Pro₃ on a multicopy plasmid leads to suppression of several +1 frameshift mutations in Salmonella enterica serovar Typhimurium. Systematic analysis of the site of frameshifting indicates that excess tRNA^Pro₃ promotes near-cognate decoding at CCC codons. Re-phasing of the reading frame can be achieved by a subsequent slippage of the tRNA onto a cognate codon in the +1 reading frame. Frameshifting appears to be due to an imbalance of CCC-cognate and near-cognate tRNAs, as the effect of excess tRNA^Pro₃ on reading frame maintenance can be reversed by increasing simultaneously the concentration of the cognate tRNA^Pro2. Finally, the cmo⁵U modification present at position 34 of tRNA^Pro₃, which allows this tRNA to decode CCU in addition to CCG and CCA, also affects frameshifting, indicating that the ability of the near-cognate tRNA to decode a cognate codon efficiently in the alternative reading frame is important for re-phasing of the reading frame.     

Influence of the stacking potential of the base 3' of tandem shift codons on -1 ribosomal frameshifting used for gene expression

Translating ribosomes can shift reading frame at specific sites with high efficiency for gene expression purposes. The most common type of shift to the -1 frame involves a tandem realignment of two anticodons from pairing with mRNA sequence of the form X XXY YYZ to XXX YYY Z where the spaces indicate the reading frame. The predominant -1 shift site of this type in eubacteria is A AAA AAG. The present work shows that in Escherichia coli the identity of the 6 nt 3' of this sequence can be responsible for a 14-fold variation in frameshift frequency. The first 3' nucleotide has the primary effect, with, in order of decreasing efficiency, U > C > A > G. This effect is independent of other stimulators of frameshifting. It is detected with other X XXA AAG sequences, but not with several other heptameric -1 shift sites. Pairing of E. coli tRNALYS with AAG is especially weak at the third codon position. We propose that strong stacking of purines 3' of AAG stabilizes pairing of tRNALys, diminishing the chance of codon:anticodon dissociation that is a prerequisite for the realignment involved in frameshifting.     

Structural and functional studies of retroviral RNA pseudoknots involved in ribosomal frameshifting: nucleotides at the junction of the two stems are important for efficient ribosomal frameshifting.

Ribosomal frameshifting, a translational mechanism used during retroviral replication, involves a directed change in reading frame at a specific site at a defined frequency. Such programmed frameshifting at the mouse mammary tumor virus (MMTV) gag-pro shift site requires two mRNA signals: a heptanucleotide shifty sequence and a pseudoknot structure positioned downstream. Using in vitro translation assays and enzymatic and chemical probes for RNA structure, we have defined features of the pseudoknot that promote efficient frameshifting. Heterologous RNA structures, e.g. a hairpin, a tRNA or a synthetic pseudoknot, substituted downstream of the shifty site fail to promote frameshifting, suggesting that specific features of the MMTV pseudoknot are important for function. Site-directed mutations of the MMTV pseudoknot indicate that the pseudoknot junction, including an unpaired adenine nucleotide between the two stems, provides a specific structural determinant for efficient frameshifting. Pseudoknots derived from other retroviruses (i.e. the feline immunodeficiency virus and the simian retrovirus type 1) also promote frameshifting at the MMTV gag-pro shift site, dependent on the same structure at the junction of the two stems.     

Stimulation of ribosomal frameshifting by antisense LNA

Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12–18 nt. Antisense oligonucleotides bearing locked nucleid acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element.     

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.     

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3-8 codon minigenes containing AGAAGA codons before the stop codon. Beta-galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, beta-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 microg/microl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of beta-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNA(Arg4). These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA.     

Stimulation of ribosomal frameshifting by RNA G-quadruplex structures

Guanine-rich sequences can fold into four-stranded structures of stacked guanine-tetrads, so-called G-quadruplexes (G4). These unique motifs have been extensively studied on the DNA level; however, exploration of the biological roles of G4s at the RNA level is just emerging. Here we show that G4 RNA when introduced within coding regions are capable of stimulating −1 ribosomal frameshifting (−1 FS) in vitro and in cultured cells. Systematic manipulation of the loop length between each G-tract revealed that the −1 FS efficiency positively correlates with G4 stability. Addition of a G4-stabilizing ligand, PhenDC3, resulted in higher −1 FS. Further, we demonstrated that the G4s can stimulate +1 FS and stop codon readthrough as well. These results suggest a potentially novel translational gene regulation mechanism mediated by G4 RNA.     

An "integrated model" of programmed ribosomal frameshifting

Many viral mRNAs, including those of HIV-1, can make translating ribosomes change reading frame. Altering the efficiencies of programmed ribosomal frameshift (PRF) inhibits viral propagation. As a new target for potential antiviral agents, it is therefore important to understand how PRF is controlled. Incorporation of the current models describing PRF into the context of the translation elongation cycle leads us to propose an 'integrated model' of PRF both as a guide towards further characterization of PRF at the molecular and biochemical levels, and for the identification of new targets for antiviral therapeutics.     

Translation initiation at alternate in-frame AUG codons in the rabies virus phosphoprotein mRNA is mediated by a ribosomal leaky scanning mechanism.

The phosphoprotein of rabies virus is a 297-amino-acid polypeptide encoded by the longest open reading frame of the P gene. Immunoprecipitation experiments using a monoclonal antiserum directed against the P protein detected the P protein and at least four additional shorter products in infected cells, cells transfected with a plasmid encoding the wild-type P protein, and purified virus (CVS strain). By means of deletion analyses, these proteins were shown to be translated from secondary downstream in-frame AUG initiation codons. Immunofluorescence experiments indicated that all these P products were found in the cytoplasm of transfected cells; however, the proteins initiated from the third, fourth, and fifth AUG codons were found mostly in the nucleus. Changes in the 5'-terminal region of the P mRNA (including site-specific mutations, deletions, and insertions) demonstrated that a leaky scanning mechanism is responsible for translation initiation of the P gene at several sites.     

P-site tRNA is a crucial initiator of ribosomal frameshifting

The expression of some genes requires a high proportion of ribosomes to shift at a specific site into one of the two alternative frames. This utilized frameshifting provides a unique tool for studying reading frame control. Peptidyl-tRNA slippage has been invoked to explain many cases of programmed frameshifting. The present work extends this to other cases. When the A-site is unoccupied, the P-site tRNA can be repositioned forward with respect to mRNA (although repositioning in the minus direction is also possible). A kinetic model is presented for the influence of both, the cognate tRNAs competing for overlapping codons in A-site, and the stabilities of P-site tRNA:mRNA complexes in the initial and new frames. When the A-site is occupied, the P-site tRNA can be repositioned backward. Whether frameshifting will happen depends on the ability of the A-site tRNA to subsequently be repositioned to maintain physical proximity of the tRNAs. This model offers an alternative explanation to previously published mechanisms of programmed frameshifting, such as out-of-frame tRNA binding, and a different perspective on simultaneous tandem tRNA slippage.     

Regulated ribosomal frameshifting by an RNA-protein interaction.

Ribosomal frameshifting is a translational mechanism used as an essential step in the replication cycle of retroviruses. Programmed frameshifting in retroviral translation involves two sequence elements: A heptanucleotide slippery sequence which induces a low basal level of frameshifting and a downstream RNA structure as an enhancer of the process. The precise mechanism of function of these downstream elements is still unclear, but their effect does not solely depend on their stability. Likewise, the possibility that frameshifting could be controlled by specific proteins that bind to these elements and enable or modulate their effects has yet not been substantiated. The RNA hairpin of the HIV-1 gag-pol frameshift cassette was replaced by the iron-responsive element (IRE) from ferritin mRNA, a stem-loop structure that binds iron regulatory proteins (IRPs) in dependence of the iron status of the cell. When a lacZ/luciferase reporter construct was expressed in transfected BHK-21 cells, the IRE or a point-mutated version that is unable to bind IRPs were found to functionally substitute for the HIV-1 hairpin. When cells were treated with the iron chelator desferrioxamine to stimulate IRP binding to the wild-type IRE, frameshift activity was specifically and strongly augmented by protein binding Our data establish that frameshifting can be regulated in a reversible fashion by mRNA-binding proteins.     

Quantitative analysis of in vivo ribosomal events at UGA and UAG stop codons.

An in vivo translation assay system has been designed to measure, in one and the same assay, the three alternatives for a ribosome poised at a stop codon (termination, read-through and frameshift). A quantitative analysis of the competition has been done in the presence and absence of release factor (RF) mutants, nonsense suppressors and an upstream Shine-Dalgarno-like sequence. The ribosomal +1 frameshift product is measurable when the stop codon is decoded by wild-type or mutant RF (prf A1 or prf B2) and also in the presence of competing suppressor tRNAs. Frameshift frequency appears to be influenced by RF activity. The amount of frameshift product decreases in the presence of competing suppressor tRNAs, however, this decrease is not in proportion to the corresponding increase in the suppression product. Instead, there is an increase in the total amount of protein expressed from the gene, perhaps due to the purging of queued ribosomes. Mutated RFs reduce the total output of the reporter gene by reducing the amount of all three protein products. The nascent peptide has earlier been shown to influence the translation termination process by interacting with the RFs. At 42 degrees C in a temperature-sensitive RF mutant strain, protein measurements indicate that the nascent peptide seems to influence the binding efficiencies of the RFs.     

High-level ribosomal frameshifting directs the synthesis of IS150 gene products.

IS150 contains two tandem, out-of-phase, overlapping genes, ins150A and ins150B, which are controlled by the same promoter. These genes encode proteins of 19 and 31 kD, respectively. A third protein of 49 kD is a transframe gene product consisting of domains encoded by both genes. Specific -1 ribosomal frameshifting is responsible for the synthesis of the large protein. Expression of ins150B also involves frameshifting. The IS150 frameshifting signals operate with a remarkably high efficiency, causing about one third of the ribosomes to switch frame. All of the signals required for this process are encoded in a 83-bp segment of the element. The heptanucleotide A AAA AAG and a potential stem-loop-forming sequence mark the frameshifting site. Similar sequence elements are found in -1 frameshifting regions of bacterial and retroviral genes. A mutation within the stem-loop sequence reduces the rate of frameshifting by about 80%. Artificial transposons carrying this mutation transpose at a normal frequency, but form cointegrates at a approximately 100-fold reduced rate.