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PubMed2001

Frameshift signal transplantation and the unambiguous analysis of mutations in the yeast retrotransposon Ty1 Gag-Pol overlap region.

The yeast retrotransposon Ty1 encodes a 7-nucleotide RNA sequence that directs a programmed, +1 ribosomal frameshifting event required for Gag-Pol translation and retrotransposition. We report mutations that block frameshifting, which can be suppressed in cis by "transplanting" the frameshift signal to a position upstream of its native location. These "frameshift transplant" mutants transpose with only a modest decrease in efficiency, suggesting that the location of the frameshift signal in a functional Ty1 element may vary. The genomic architecture of Ty1 is such that Gag, Ty1 PR (PR), and the Gag-derived p4 peptide share a common sequence. The functional independence of the movement of the frameshift signal to a new location within the Ty1 element is used to unambiguously attribute the effect of mutations deleterious to transposition in this region of overlapping coding sequences to effects on the Ty1 (PR). This work defines the amino terminus of the Ty1 PR and introduces a new technique for studying viral genome organization.

Amino Acid Sequence↗
PubMed2004

The human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy.

Human immunodeficiency virus type 1 (HIV-1) utilizes a distinctive form of gene regulation as part of its life cycle, termed programmed -1 ribosomal frameshifting, to produce the required ratio of the Gag and Gag-Pol polyproteins. We carried out a sequence comparison of 1,000 HIV-1 sequences at the slippery site (UUUUUUA) and found that the site is invariant, which is somewhat surprising for a virus known for its variability. This prompted us to prepare a series of mutations to examine their effect upon frameshifting and viral infectivity. Among the series of mutations were changes of the HIV-1 slippery site to those effectively utilized by other viruses, because such mutations would be anticipated to have a relatively mild effect upon frameshifting. The results demonstrate that any change to the slippery site reduced frameshifting levels and also dramatically inhibited infectivity. Because ribosomal frameshifting is essential for HIV-1 replication and it is surprisingly resistant to mutation, modulation of HIV-1 frameshifting efficiency potentially represents an important target for the development of novel antiviral therapeutics.

Anti-HIV Agents↗
PubMed1994

Frameshift mutations in the v-src gene of avian sarcoma virus act in cis to specifically reduce v-src mRNA levels.

A portion of the avian sarcoma virus (ASV) primary RNA transcripts is alternatively spliced in chicken embryo fibroblast cells to two different messages, the src and env mRNAs. Frameshift mutations of the viral genome causing premature translation termination within the src gene result in a decreased steady-state level of the src mRNA. In marked contrast, frameshift mutations at various positions of the env gene do not decrease the level of the env mRNA. We show that the src gene product is not required in trans for splicing and accumulation of src mRNA. Conversely, the truncated Src proteins do not act negatively in trans to decrease specifically the levels of src mRNA. Taken together, these results indicate that the frameshift mutations act in cis to reduce src mRNA levels. A double mutant with a lesion in the src initiator AUG and a frameshift within the src gene demonstrated wild-type RNA levels, indicating that the src mRNA must be recognized as a translatable mRNA for the effect on src mRNA levels to occur. Our results indicate that the reduced levels do not result from decreased cytoplasmic stability of the mature src mRNA. We also show that the src gene frameshift mutations affect src mRNA levels when expressed from intronless src cDNA clones. We conclude that the reduction of src mRNA levels triggered by the presence of frameshift mutations within the src gene occurs while it is associated with the nucleus. Our data also strongly suggest that this occurs at a step of RNA processing or transport independent of RNA splicing.

Animals↗
PubMed2000

Kinetics of ribosomal pausing during programmed -1 translational frameshifting.

In the Saccharomyces cerevisiae double-stranded RNA virus, programmed -1 ribosomal frameshifting is responsible for translation of the second open reading frame of the essential viral RNA. A typical slippery site and downstream pseudoknot are necessary for this frameshifting event, and previous work has demonstrated that ribosomes pause over the slippery site. The translational intermediate associated with a ribosome paused at this position is detected, and, using in vitro translation and quantitative heelprinting, the rates of synthesis, the ribosomal pause time, the proportion of ribosomes paused at the slippery site, and the fraction of paused ribosomes that frameshift are estimated. About 10% of ribosomes pause at the slippery site in vitro, and some 60% of these continue in the -1 frame. Ribosomes that continue in the -1 frame pause about 10 times longer than it takes to complete a peptide bond in vitro. Altering the rate of translational initiation alters the rate of frameshifting in vivo. Our in vitro and in vivo experiments can best be interpreted to mean that there are three methods by which ribosomes pass the frameshift site, only one of which results in frameshifting.

Base Sequence↗
PubMed2001

Amino acid translation program for full-length cDNA sequences with frameshift errors.

Here we present an amino acid translation program designed to suggest the position of experimental frameshift errors and predict amino acid sequences for full-length cDNA sequences having phred scores. Our program generates artificial insertions into artificial deletions from low-accuracy positions of the original sequence, thereby generating many candidate sequences. The validity of the most probable sequence (the likelihood that it represents the actual protein) is evaluated by using a score (V(a)) that is calculated in light of the Kozak consensus, preferred codon usage, and position of the initiation codon. To evaluate the software, we have used a database in which, out of 612 cDNA sequences, 524 (86%) carried 773 frameshift errors in the coding sequence. Our software detected and corrected 48% of the total frameshift errors in 62% of the total cDNA sequences with frameshift errors. The false positive rate of frameshift correction was 9%, and 91% of the suggested frameshifts were true.

Base Composition↗
PubMed2006

Evolutionary specialization of recoding: frameshifting in the expression of S. cerevisiae antizyme mRNA is via an atypical antizyme shift site but is still +1.

An autoregulatory translational shift to the +1 frame is required for the expression of ornithine decarboxylase antizyme from fungi to mammals. In most eukaryotes, including all vertebrates and a majority of the studied fungi/yeast, the site on antizyme mRNA where the shift occurs is UCC-UGA. The mechanism of the frameshift on this sequence likely involves nearly universal aspects of the eukaryotic translational machinery. Nevertheless, a mammalian antizyme frameshift cassette yields predominantly -2 frameshift in Saccharomyces cerevisiae, instead of the +1 in mammals. The recently identified endogenous S. cerevisiae antizyme mRNA has an atypical shift site: UGC-GCG-UGA. It is shown here that endogenous S. cerevisiae antizyme frameshifting is +1 rather than -2. We discuss how antizyme frameshifting in budding yeasts exploits peculiarities of their tRNA balance, and relate this to prior studies on Ty frameshifting.

Animals↗
PubMed2004

Detection of frameshift mutations of RIZ in gastric cancers with microsatellite instability.

AIM: To study the frameshift mutations of the retinoblastoma protein-interacting zinc finger gene RIZ in gastric cancer with microsatellite instability, and to identify two coding polyadenosine tracts of RIZ. METHODS: Frameshift mutations at (A)8 and (A)9 tracts of RIZ were detected in 70 human gastric cancer (HGC) specimens by DHPLC and DNA sequencing. Microsatellite instability (MSI) status was assessed by two mononucleotide markers, BAT26 and BAT25, by means of denaturing high-performance liquid chromatography (DHPLC). RESULTS: In 70 HGC samples, 8 (11.4%) were found positive for instabilities at BAT26 and BAT25. In 7 of the 8 cases with instabilities at both BAT26 and BAT25 (MSI-H), 1 was unstable at BAT26 but stable at BAT25. Frameshift mutations were identified in 4 (57.1%) of the 7 samples with MSI-H in the (A)9 tract of RIZ without mutations in the (A)8 tract. In contrast, frameshift mutations were found in neither of the polyadenosine tracts in 63 samples of MSI-L or MSI stable tumors. Pro704 LOH detection in 4 cases with frameshift mutations did not find LOH in these cases. CONCLUSION: Frameshift mutations of RIZ may play an important role in gastric cancers with MSI.

DNA-Binding Proteins↗
PubMedDate not supplied

Microsatellite instability(MSI) in non-small cell lung cancer(NSCLC) is highly associated with transforming growth factor-beta type II receptor(TGF-beta RII) frameshift mutation.

BACKGROUND: TGF-beta type II receptor (TGF-beta RII) mutations associated with microsatellite instability(MSI) are characteristically frameshift mutations within a 10 bp poly-A tract. These frameshift mutations have been reported to be common in colorectal and gastric cancers with MSI, though, rarely reported in non-small cell lung cancer (NSCLC). MATERIALS AND METHOD: In this study, we analysed MSI and TGF-beta RII frameshift mutations in 7 NSCLC cell lines and 21 surgically resected NSCLC tissues. Determination of MSI in NSCLC was performed using primer sets for BAT-25, BAT-26 and BAT-40. In order to examine the presence of the frameshift mutations of TGF-beta RII in samples with MSI, sequencing for TGF-beta RII poly-A tract was performed. RESULTS: MSI was observed in 5 out of 7 NSCLC cell lines and 3 out of 21 NSCLC tissues. Six out of 8 samples with MSI(75%) showed frameshift mutations in TGF-beta RII poly-A tract. CONCLUSION: These results suggest that MSI is highly associated with TGF-beta RII frameshift mutations in NSCLC and further support the hypothesis that TGF-beta RII plays an important role in NSCLC carcinogenesis.

Carcinoma, Non-Small-Cell Lung↗
PubMed2001

[Suppression of frameshift mutation as a result of partial inactivation of translation termination factors in Saccharomyces cerevisiae yeast].

Special search for frameshift mutations, which are suppressed by the cytoplasmic [PSI] factor and by omnipotent nonsense suppressors (recessive mutations in the SUP35 and SUP45 genes), partially inactivating a translation termination complex, was initiated in the LYS2 gene in the yeast Saccharomyces cerevisiae. Mutations were obtained after exposure to UV light and treatment with a mixture consisting of 1.6- and 1.8-dinitropyrene (DNP). This mixture was shown to induce mutations of the frameshift type with a high frequency. The majority of these mutations were insertions of one A or T, which is in good agreement with the data obtained in studies of DNP-induced mutagenesis in other eukaryotes. Frameshift suppression in yeast was first shown on the example of the mutation obtained in this work (lys2-90), which carried the insertion of an extra T in the sequence of five T. This frameshift suppression was shown to occur in the presence of the [PSI] factor (i.e., due to the prion form of the translation release factor eRF3) and as a result of mutations in genes SUP35 or SUP45, which partially inactivate translation termination factors eRF3 and eRF1, respectively. Alternative mechanisms of programmed translational frameshifting in the course of translation and the possibility of enhancing the effectiveness of such frameshifting in the presence of the [PSI] factor are considered.

Base Sequence↗
PubMed1997

Translational misreading: mutations in translation elongation factor 1alpha differentially affect programmed ribosomal frameshifting and drug sensitivity.

The translation elongation feactor 1alpha (EF-1alpha) catalyzes the critical step of delivering aminoacyl-tRNAs to the elongating ribosome. A series of Saccharomyces cerevisiae strains containing mutant alleles of the TEF2 gene encoding EF-1alpha have phenotypes consistent with effects on cellular processes related to translation. These include (1) conditional growth defects, (2) antibiotic sensitivity or resistance, (3) altered +1 or -1 ribosomal frameshifting efficiencies, and (4) altered maintenance of the killer phenotype. Although all the mutant alleles were isolated as dominant +1 frameshift suppressors, the effects of these mutations on the cell are quite different when present as the only form of EF-1alpha. Allele-specific effects are observed with regard to their ability to alter the efficiency of programmed +1 frameshifting as opposed to programmed -1 ribosomal frameshifting. The significantly altered efficiency of -1 frameshifting in strains containing the TEF2-4 and TEF2-9 mutant alleles further correlates with a reduced ability to maintain the killer phenotype and the M1 satellite virus of L-A, an in vivo assay of translational fidelity. In light of the proposed models regarding the different A- and P-site occupancy states required for +1 or -1 ribosomal frameshifting, these results aid analysis of interactions between EF-1alpha and the translational apparatus.

DNA-Binding Proteins↗
PubMed1983

Mutator versus antimutator activity of a T4 DNA polymerase mutant distinguishes two different frameshifting mechanisms.

Classical "antimutator" DNA polymerases of bacteriophage T4 were examined for their effects upon frameshift mutation rates at a number of positions within rII cistrons. Their antimutagenic activities reduced frameshift frequencies at a number of sites, but at other sites the opposite occurred: the mutant polymerases exhibited clear mutator activities. This dichotomy reveals the operation of two distinct mechanisms of frameshift mutagenesis that are correlated with the DNA sequences at the frameshift sites. Frameshift mutants subject to the antimutator effects of the mutant polymerase lie in A: T-run DNA sequences, where mutations presumably arise by means of the interstrand DNA misalignments postulated by classical theory. The frameshift mutants produced by the mutator activity of these same polymerases lie in quasipalindromic DNA sequences, where mutations are postulated to arise by aberrant metabolism of DNA secondary structures such as hairpins.

Alleles↗
PubMed1988

On the mechanism of ribosomal frameshifting at hungry codons.

In a few, rather rare cases, frameshift mutant alleles are phenotypically suppressed during limitation for particular aminoacyl-tRNA species. The simplest interpretation is compensatory ribosome frameshifting at a "hungry" codon in the vicinity of the suppressed frameshift mutation. We have now tested this interpretation directly by obtaining amino acid sequence data on such a phenotypically suppressed protein. We used a plasmid-borne lacZ gene, engineered to be in the (+) reading frame. Its background leakiness is increased by two orders of magnitude during lysyl-tRNA limitation. The enzyme made under this condition has the amino acid sequence expected from the DNA sequence up to the first lysine codon, then shifts in the (-) direction to recreate the correct lacZ reading frame. The lysine is replaced by serine, presumably due to cognate reading of an overlapping AGC codon displaced by one base to the 3' side of the AAG codon. When the 3' overlapping codon is AGA or AGG, there is no ribosome frameshifting; when it is AGU (read by the same serine tRNA) there is frameshifting, although less efficiently than in the case of AGC. The mechanism of cognate overlapping reading contradicts more elaborate models that two of the authors have suggested previously. However, the possibility remains that there is more than one mechanism of ribosome frameshifting at hungry codons.

Amino Acid Sequence↗
PubMed1985

Frameshift mutagenesis by ultraviolet and gamma radiations in Salmonella: SOS inducibility and effects of DNA polymerase I.

Ultraviolet (UV) and gamma-induced mutagenesis have been studied using a doubly auxotrophic strain of Salmonella typhimurium carrying the amber leuA150 mutation (which reverts by base-pair substitution) and the frameshift hisC3076 marker (which reverts by compensating frameshifts). In the initially constructed LT2 background, both markers were poorly revertible by UV and essentially non-revertible by gamma-radiation. A derivative of this strain carrying the mutation-enhancing plasmid pKM101 was however readily reverted by both UV and gamma, with either Leu+ (base substitution) or His+ (frameshift) revertants being observed on appropriate selective media. Photoreactivation experiments suggested that the lesions leading to formation of the two types of mutagenic event were similar if not identical. Support for this suggestion was obtained when it was found that yields of both types of UV-induced revertant were significantly increased in an excision-deficient background, while no revertants of either type were found in a recA background. Yields of gamma-induced revertants were not greatly altered in a uvrB background, but were also reduced to zero (for both markers) in the recA background. These results are consistent with what has previously been well-documented for UV and gamma-induced base-pair substitution mutagenesis, and serve to emphasize the similarities between base-pair substitution mutagenesis and frameshift mutagenesis by these agents. There are differences, however, since although UV-induced reversion of the leuA150 marker was little affected and gamma-induced reversion of leuA150 was somewhat reduced in the presence of a polA mutation (polA3), the yields of His+ frameshift revertants were significantly increased in the polA3 background following treatment with either UV or gamma. Thus while inducible DNA repair (SOS repair) appears to be involved in generating both types of mutational event following either UV- or gamma-irradiation, at some stage in the processing of premutational lesions the level (or type) of DNA polymerase I activity in the cell seems to have an important role in determining whether or not frameshifts or base-pair substitutions will be produced at a particular frequency.

DNA Polymerase I↗
PubMed1988

Frameshift mutagenesis in Escherichia coli by reversible DNA intercalators: sequence specificity.

The mutagenic potency of the simple reversible intercalators isopropyl-OPC (iPr-OPC) and 9-aminoacridine (9-AA) is assessed in E. coli using reversion assays based on plasmids derived from pBR322 carrying various frameshift mutations within the tetracycline resistance gene in repetitive sequences: +/- 2 frameshift mutations within alternating GC sequences; +/- 1 frameshift mutation at runs of guanines. The results obtained show that iPr-OPC and 9-AA have a sequence specificity for mutagenesis: they revert +1 and -1 frameshift mutations within runs of monotonous G:C base pairs. The precise determination of the size of a small restriction fragment which contains the mutation allowed us to demonstrate that reversion occurred by -1 deletions for the +1 frameshift mutations and by +1 additions for the -1 frameshift mutations. The possible relations of this specific reversion with the base sequence specificity of the mutagenesis are briefly discussed.

Alkaloids↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

The gamma subunit of DNA polymerase III holoenzyme of Escherichia coli is produced by ribosomal frameshifting.

The tau and gamma subunits of DNA polymerase III holoenzyme are both products of the dnaX gene. Since tau and gamma are required as stoichiometric components of the replicative complex, a mechanism must exist for the cell to coordinate their synthesis and ensure that both subunits are present in an adequate quantity and ratio for assembly. We have proposed that gamma is produced by a translational frameshift event. In this report, we describe the use of dnaX-lacZ fusions in all three reading frames to demonstrate that gamma, the shorter product of dnaX, is generated by ribosomal frameshifting to the -1 reading frame of the mRNA within an oligo(A) sequence that is followed by a sequence predicted to form a stable secondary structure. Immediately after frameshifting a stop codon is encountered, leading to translational termination. Mutagenesis of the oligo(A) sequence abolishes frameshifting, and partial disruption of the predicted distal secondary structure severely impairs the efficiency. Comparison of the expression of lacZ fused to dnaX distal to the site of frameshifting in the -1 and 0 reading frames indicates that the efficiency of frameshifting is approximately 40%.

Amino Acid Sequence↗
PubMed1999

Specific mutations in a viral RNA pseudoknot drastically change ribosomal frameshifting efficiency.

Many viruses regulate protein synthesis by -1 ribosomal frameshifting using an RNA pseudoknot. Frameshifting is vital for viral reproduction. Using the information gained from the recent high-resolution crystal structure of the beet western yellow virus pseudoknot, a systematic mutational analysis has been carried out in vitro and in vivo. We find that specific nucleotide tertiary interactions at the junction between the two stems of the pseudoknot are crucial. A triplex is found between stem 1 and loop 2, and triplex interactions are required for frameshifting function. For some mutations, loss of one hydrogen bond is sufficient to abolish frameshifting. Furthermore, mutations near the 5' end of the pseudoknot can increase frameshifting by nearly 300%, possibly by modifying ribosomal contacts. It is likely that the selection of suitable mutations can thus allow viruses to adjust frameshifting efficiencies and thereby regulate protein synthesis in response to environmental change.

Cells, Cultured↗
PubMed1991

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.

Amino Acid Sequence↗