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At least 235 records · Page 13Linked to original sources
PubMed1993

A rare tRNA-Arg(CCU) that regulates Ty1 element ribosomal frameshifting is essential for Ty1 retrotransposition in Saccharomyces cerevisiae.

Translation of the yeast retrotransposon Ty1 TYA1(gag)-TYB1(pol) gene occurs by a +1 ribosomal frameshifting event at the sequence CUU AGG C. Because overexpression of a low abundance tRNA-Arg(CCU) encoded by the HSX1 gene resulted in a reduction in Ty1 frameshifting, it was suggested that a translational pause at the AGG-Arg codon is required for optimum frameshifting. The present work shows that the absence of tRNA-Arg(CCU) affects Ty1 transposition, translational frameshifting, and accumulation of mature TYB1 proteins. Transposition of genetically tagged Ty1 elements decreases at least 50-fold and translational frameshifting increases 3-17-fold in cells lacking tRNA-Arg(CCU). Accumulation of Ty1-integrase and Ty1-reverse transcriptase/ribonuclease H is defective in an hsx1 mutant. The defect in Ty1 transposition is complemented by the wild-type HSX1 gene or a mutant tRNA-Arg(UCU) gene containing a C for T substitution in the first position of the anticodon. Overexpression of TYA1 stimulates Ty1 transposition 50-fold above wild-type levels when the level of transposition is compared in isogenic hsx1 and HSX1 strains. Thus, the HSX1 gene determines the ratio of the TYA1 to TYA1-TYB1 precursors required for protein processing or stability, and keeps expression of TYB1 a rate-limiting step in the retrotransposition cycle.

Base Sequence↗
PubMed1998

Spectra of spontaneous frameshift mutations at the hisD3052 allele of Salmonella typhimurium in four DNA repair backgrounds.

To characterize the hisD3052 -1 frameshift allele of Salmonella typhimurium, we analyzed approximately 6000 spontaneous revertants (rev) for a 2-base deletion hotspot within the sequence (CG)4, and we sequenced approximately 500 nonhotspot rev. The reversion target is a minimum of 76 bases (nucleotides 843-918) that code for amino acids within a nonconserved region of the histidinol dehydrogenase protein. Only 0.4-3.9% were true rev. Of the following classes, 182 unique second-site mutations were identified: hotspot, complex frameshifts requiring DeltauvrB + pKM101 (TA98-specific) or not (concerted), 1-base insertions, duplications, and nonhotspot deletions. The percentages of hotspot mutations were 13.8% in TA1978 (wild type), 24.5% in UTH8413 (pKM101), 31.6% in TA1538 (DeltauvrB), and 41.0% in TA98 (DeltauvrB, pKM101). The DeltauvrB allele decreased by three times the mutant frequency (MF, rev/10(8) survivors) of duplications and increased by about two times the MF of deletions. Separately, the DeltauvrB allele or pKM101 plasmid increased by two to three times the MF of hotspot mutations; combined, they increased this MF by five times. The percentage of 1-base insertions was not influenced by either DeltauvrB or pKM101. Hotspot deletions and TA98-specific complex frameshifts are inducible by some mutagens; concerted complex frameshifts and 1-base insertions are not; and there is little evidence for mutagen-induced duplications and nonhotspot deletions. Except for the base substitutions in TA98-specific complex frameshifts, all spontaneous mutations of the hisD3052 allele are likely templated. The mechanisms may involve (1) the potential of direct and inverted repeats to undergo slippage and misalignment and to form quasi-palindromes and (2) the interaction of these sequences with DNA replication and repair proteins.

Alcohol Oxidoreductases↗
PubMed2000

The bleomycin amplification assay in V79 cells predicts frameshift mutagenicity of intercalative agents.

We have recently reported on the use of a cell-based bleomycin amplification assay for the detection of DNA intercalating agents. In order to further validate this assay, two series of proprietary compounds were evaluated for frameshift mutagenesis in the Ames bacterial reversion system and for bleomycin amplification in the Chinese hamster V79 micronucleus system. It is shown that 10 of 11 frameshift-positive compounds were bleomycin amplifiers. These studies indicate that positive frameshift mutagenicity findings are consistent with expectations from the results of the bleomycin amplification assay, providing additional validation of the amplification assay for the detection of DNA intercalating agents. The studies also demonstrate that intercalation is necessary but not sufficient for frameshift mutagenesis since bleomycin amplifiers lacking frameshift mutagenic activity were also identified.

Animals↗
PubMed1995

Prokaryotic ribosomes recode the HIV-1 gag-pol-1 frameshift sequence by an E/P site post-translocation simultaneous slippage mechanism.

The mechanism favoured for -1 frameshifting at typical retroviral sites is a pre-translocation simultaneous slippage model. An alternative post-translocation mechanism would also generate the same protein sequence across the frameshift site and therefore in this study the strategic placement of a stop codon has been used to distinguish between the two mechanisms. A 26 base pair frameshift sequence from the HIV-1 gag-pol overlap has been modified to include a stop codon immediately 3' to the heptanucleotide frameshift signal, where it often occurs naturally in retroviral recoding sites. Stop codons at the 3'-end of the heptanucleotide sequence decreased the frame-shifting efficiency on prokaryote ribosomes and the recording event was further depressed when the levels of the release factors in vivo were increased. In the presence of elevated levels of a defective release factor 2, frameshifting efficiency in vivo was increased in the constructs containing the stop codons recognized specifically by that release factor. These results are consistent with the last six nucleotides of the heptanucleotide slippery sequence occupying the ribosomal E and P sites, rather than the P and A sites, with the next codon occupying the A site and therefore with a post-translocation rather than a pre-translocation -1 slippage model.

Base Sequence↗
PubMed1996

Is the in-frame termination signal of the Escherichia coli release factor-2 frameshift site weakened by a particularly poor context?

The synthesis of release factor-2 (RF-2) in bacteria is regulated by a high efficiency +1 frameshifting event at an in-frame UGA stop codon. The stop codon does not specify the termination of synthesis efficiently because of several upstream stimulators for frameshifting. This study focusses on whether the particular context of the stop codon within the frameshift site of the Escherichia coli RF-2 mRNA contributes to the poor efficiency of termination. The context of UGA in this recoding site is rare at natural termination sites in E.coli genes. We have evaluated how the three nucleotides downstream from the stop codon (+4, +5 and +6 positions) in the native UGACUA sequence affect the competitiveness of the termination codon against the frameshifting event. Changing the C in the +4 position and, separately, the A in the +6 position significantly increase the termination signal strength at the frameshift site, whereas the nucleotide in the +5 position had little influence. The efficiency of particular termination signals as a function of the +4 or +6 nucleotides correlates with how often they occur at natural termination sites in E.coli; strong signals occur more frequently and weak signals are less common.

Base Sequence↗
PubMed1992

Sequence requirements for efficient translational frameshifting in the Escherichia coli dnaX gene and the role of an unstable interaction between tRNA(Lys) and an AAG lysine codon.

Synthesis of the gamma-subunit of DNA polymerase III holoenzyme depends on precise and efficient translational frameshifting to the -1 frame at a specific site in the dnaX gene of Escherichia coli. In vitro mutagenesis of this frameshift site demonstrated the importance of an A AAA AAG heptanucleotide sequence, which allows two adjacent tRNAs to retain a stable interaction with mRNA after they slip to the -1 position. The AAG lysine codon present in the 3' half of this heptanucleotide was a key element for highly efficient frameshifting. A tRNA(Lys) with a CUU anticodon, which has a strong affinity for AAG lysine codons, is present in eukaryotic cells but absent in E. coli. Expression in E. coli of a mutant tRNA(Lys) with a CUU anticodon specifically inhibited the frameshifting at the AAG codon, suggesting that the absence of this tRNA in E. coli contributes to the efficiency of the dnaX frameshift.

Base Sequence↗
PubMed1992

Ribosomal frameshifting requires a pseudoknot in the Saccharomyces cerevisiae double-stranded RNA virus.

The large double-stranded RNA of the Saccharomyces cerevisiae (yeast) virus has two large overlapping open reading frames on the plus strand, one of which is translated via a -1 ribosomal frameshift. Sequences including the overlapping region, placed in novel contexts, can direct ribosomes to make a -1 frameshift in wheat germ extract, Escherichia coli and S. cerevisiae. This sequence includes a consensus slippery sequence, GGGUUUA, and has the potential to form a pseudoknot 3' to the putative frameshift site. Based on deletion analysis, a region of 71 nucleotides including the potential pseudoknot and the putative slippery sequence is sufficient for frameshifting. Site-directed mutagenesis demonstrates that the pseudoknot is essential for frameshifting.

Base Composition↗
PubMed1992

Ribosomal frameshifting efficiency and gag/gag-pol ratio are critical for yeast M1 double-stranded RNA virus propagation.

About 1.9% of ribosomes translating the gag open reading frame of the yeast L-A double-stranded RNA virus positive strand undergo a -1 frameshift and continue translating in the pol open reading frame to make a 170-kDa gag-pol fusion protein. The importance of frameshifting efficiency for viral propagation was tested in a system where the M1 (killer toxin-encoding) satellite RNA is supported by a full-length L-A cDNA clone. Either increasing or decreasing the frameshift efficiency more than twofold by alterations in the slippery site disrupted viral propagation. A threefold increase caused by a chromosomal mutation, hsh1 (high shifter), had the same effect. Substituting a +1 ribosomal frameshift site from Ty1 with the correct efficiency also allowed support of M1 propagation. The normal -1 frameshift efficiency is similar to the observed molar ratio in viral particles of the 170-kDa gag-pol protein to the 70-kDa gag gene product, the major coat protein. The results are interpreted in terms of a packaging model for L-A.

Base Sequence↗
PubMed1993

Characterization of ribosomal frameshifting for expression of pol gene products of human T-cell leukemia virus type I.

For study of the pol gene expression of human T-cell leukemia virus type I (HTLV-I), RNA was transcribed in vitro from proviral DNA and translated in rabbit reticulocyte lysates. This cell-free translation resulted in two major translation products representing the Gag and Gag-Pro polyproteins. By contrast, the Gag-Pro-Pol polyprotein could be readily observed only when translation was performed with mutant mRNA in which the protease (pro) reading frame was aligned to gag to eliminate the frameshifting event in the gag-pro overlap. The results indicated that two independent ribosomal frameshifting events are required for expression of the HTLV-I pol gene product. Studies with mutant DNAs facilitated the characterization of the primary structure of the HTLV-I mRNA responsible for the ribosomal frameshift in the pro-pol overlap and demonstrated that the frameshift occurs at the signal sequence UUUAAAC. Direct amino acid sequencing of the transframe protein localized the site of the frameshift to the asparagine codon AAC.

Amino Acid Sequence↗
PubMed1993

A heptanucleotide sequence mediates ribosomal frameshifting in mammalian cells.

Ribosomal frameshifting is an essential requirement for replication of many viruses and retrovirus-like elements. It is regarded as a potential target for antiretroviral therapy. It has been shown that the frameshifting event takes place in the -1 direction within a sequence, the slippery sequence, which is usually followed by structured RNA. To distinguish between the basic sequence requirements and the modulating elements in intact cells, we have established a sensitive assay system for quantitative determination of ribosomal frameshifting in mammalian cell culture. In this assay system, the gag and pol genes of human immunodeficiency virus type 1 are replaced by the genes for the functional enzymes beta-galactosidase and luciferase, respectively. The sensitivity of the test system allows us to demonstrate for the first time that the slippery sequence, a heptanucleotide, is sufficient to mediate a basal level of ribosomal frameshifting independent of its position within a gene. The stem-loop sequence serves only as a positive modulator. These data indicate that frameshifting could also occur during translation of cellular genes in which a slippery sequence is present within the reading frame. The resulting putative transframe proteins might have a functional importance for cellular processes.

Animals↗
PubMed1995

Ribosomal frameshifting during translation of measles virus P protein mRNA is capable of directing synthesis of a unique protein.

Members of the Paramyxoviridae family utilize a variety of different strategies to increase coding capacity within their P cistrons. Translation initiation at alternative 5'-proximal AUG codons is used by measles virus (MV) to express the virus-specific P and C proteins from overlapping reading frames on their mRNAs. Additional species of mRNAs are transcribed from the MV P cistron by the insertion of extra nontemplated G residues at a specific site within the P transcript. Addition of only a single nontemplated G residue results in the expression of the V protein, which contains a unique carboxyl terminus. We have used an Escherichia coli system to express MV P cistron-related mRNAs and proteins. We have found that ribosomal frameshifting on the MV P protein mRNA is capable of generating a previously unrecognized P cistron-encoded protein that we have designated R. Some ribosomes which have initiated translation of the P protein mRNA use the sequence TCC CCG AG (24 nucleotides upstream of the V protein stop codon) to slip into the -1 reading frame, thus translating the sequence as TC CCC GAG. The resulting R protein terminates five codons downstream of the frameshift site at the V protein stop codon. We have gone on to use a chloramphenicol acetyltransferase reporter system to demonstrate that this MV-specific sequence is capable of directing frameshifting during in vivo translation in eukaryotic cells. Analysis of immunoprecipitated proteins from MV-infected cells by two-dimensional gel electrophoresis allowed detection of a protein species consistent with R protein in MV-infected cells. Quantitation of this protein species allowed a rough estimation of frameshift frequency of approximately 1.8%. Significant stimulation of ribosomal frameshift frequency at this locus of the MV P mRNA was mediated by a downstream stimulator element which, although not yet fully defined, appeared to be neither a conventional stem-loop nor an RNA pseudoknot structure.

Amino Acid Sequence↗
PubMed1996

Possible origin of murine AIDS (MAIDS) virus: conversion of an endogenous retroviral p12gag sequence to a MAIDS-inducing sequence by frameshift mutations.

The murine AIDS (MAIDS) virus has a unique sequence in its p12gag region, which is responsible for MAIDS development. A transcript hybridizing with this sequence is expressed in normal C57BL/6 mice. The transcript, designated Edv, has been previously cloned and sequenced (Y. Kubo, Y. Nakagawa, K. Kakimi, H. Matsui, K. Higo, L. Wang, H. Kobayashi, T. Hirama, and A. Ishimoto, J. Gen. Virol. 75:881-888, 1994). Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic replication-defective MAIDS virus has a 16-bp deletion and a 1-bp insertion in the 5' and 3' regions of the p12gag sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the defective MAIDS virus p12gag region is not homologous to that of the helper virus and the Edv transcript because of the frameshift. To determine whether the amino acid sequence resulting from the frameshift is critical for MAIDS development, we constructed chimeric viruses that contained the p12gag regions of the helper virus and the Edv transcript, respectively, with and without the same frame as the defective MAIDS virus by the artificial frameshift mutations. The mutant viruses with the frameshift mutations induced MAIDS in inoculated mice, but the viruses without the mutations did not. These results suggested that the MAIDS virus was generated by frameshift mutations in the p12gag region of Edv or a related sequence.

Animals↗
PubMed2002

Analysis of natural variants of the human immunodeficiency virus type 1 gag-pol frameshift stem-loop structure.

Human immunodeficiency virus type 1 uses ribosomal frameshifting for translation of the Gag-Pol polyprotein. Frameshift activities are thought to be tightly regulated. Analysis of gag p1 sequences from 270 plasma virions identified in 64% of the samples the occurrence of polymorphism that could lead to changes in thermodynamic stability of the stem-loop. Expression in Saccharomyces cerevisiae of p1-beta-galactosidase fusion proteins from 10 representative natural stem-loop variants and three laboratory mutant constructs (predicted the thermodynamic stability [Delta G degrees] ranging from -23.0 to -4.3 kcal/mol) identified a reduction in frameshift activity of 13 to 67% compared with constructs with the wild-type stem-loop (Delta G degrees, -23.5 kcal/mol). Viruses carrying stem-loops associated with greater than 60% reductions in frameshift activity presented profound defects in viral replication. In contrast, viruses with stem-loop structures associated with 16 to 42% reductions in frameshift efficiency displayed no significant viral replication deficit.

Base Sequence↗
PubMed2003

Decreased peptidyltransferase activity correlates with increased programmed -1 ribosomal frameshifting and viral maintenance defects in the yeast Saccharomyces cerevisiae.

Increased efficiencies of programmed -1 ribosomal frameshifting in yeast cells expressing mutant forms of ribosomal protein L3 are unable to maintain the dsRNA "Killer" virus. Here we demonstrate that changes in frameshifting and virus maintenance in these mutants correlates with decreased peptidyltransferase activities. The mutants did not affect Ty1-directed programmed +1 ribosomal frameshifting or nonsense-mediated mRNA decay. Independent experiments demonstrate similar programmed -1 ribosomal frameshifting specific defects in cells lacking ribosomal protein L41, which has previously been shown to result in peptidyltransferase defects in yeast. These findings are consistent with the hypothesis that decreased peptidyltransferase activity should result in longer ribosome pause times after the accommodation step of the elongation cycle, allowing more time for ribosomal slippage at programmed -1 ribosomal frameshift signals.

Alleles↗
PubMed2006

Comparative study of the effects of heptameric slippery site composition on -1 frameshifting among different eukaryotic systems.

Studies of programmed -1 ribosomal frameshifting (-1 PRF) have been approached over the past two decades by many different laboratories using a diverse array of virus-derived frameshift signals in translational assay systems derived from a variety of sources. Though it is generally acknowledged that both absolute and relative -1 PRF efficiency can vary in an assay system-dependent manner, no methodical study of this phenomenon has been undertaken. To address this issue, a series of slippery site mutants of the SARS-associated coronavirus frameshift signal were systematically assayed in four different eukaryotic translational systems. HIV-1 promoted frameshifting was also compared between Escherichia coli and a human T-cell line expression systems. The results of these analyses highlight different aspects of each system, suggesting in general that (1) differences can be due to the assay systems themselves; (2) phylogenetic differences in ribosome structure can affect frameshifting efficiency; and (3) care must be taken to employ the closest phylogenetic match between a specific -1 PRF signal and the choice of translational assay system.

Base Sequence↗
PubMed2000

Lack of frameshift mutations at coding mononucleotide repeats in hepatocellular carcinoma in Japanese patients.

BACKGROUND: Microsatellite instability occurs frequently in hereditary nonpolyposis colorectal carcinoma, in sporadic gastrointestinal carcinoma, and in other tumors. In these tumors, slippage-related frameshift mutations have been detected at coding mononucleotide repeats in genes such as those for transforming growth factor-beta receptor type II (TGFbetaRII), mannose 6-phosphate/insulinlike growth factor II receptor (M6P/IGFIIR), hMSH3, hMSH6, and Bcl-2-associated X protein (BAX). Because these genes regulate cell growth or repair DNA mismatches, loss of their function is thought to promote tumor development. The authors screened for these frameshift mutations and investigated the incidence of microsatellite instability (MI) in hepatocellular carcinoma (HCC) in Japan. METHODS: Fifty HCC samples were analyzed in this study. The authors used polymerase chain reactions to screen for frameshift mutation at the TGFbetaRII (A)(10) tract, the M6P/IGFIIR (G)(8) tract, the hMSH3 (A)(8) tract, the hMSH6 (C)(8) tract, and the BAX (G)(8) tract. For MI analysis, matched tumor and nontumor liver DNA were investigated with respect to 10 microsatellite loci. RESULTS: No frameshift mutation was detected in any case, and only 4% of these cancers exhibited MI in comparisons between tumor and nontumor liver specimens. CONCLUSIONS: This study suggests that frameshift mutation at coding mononucleotide repeats within TGFbetaRII, M6P/IGFIIR, hMSH3, hMSH6, and BAX genes did not seem to be involved in hepatocarcinogenesis in the Japanese population studied.

Adult↗
PubMed2004

Immunogenic peptides generated by frameshift mutations in DNA mismatch repair-deficient cancer cells.

About 15% of all human colorectal, gastric, and endometrial tumors, and the majority of tumors in patients suffering from hereditary nonpolyposis colorectal cancer syndrome, are caused by loss of DNA mismatch repair functions. In the affected cancer cells, this results in insertion or deletion mutations at short, repetitive DNA sequences referred to as microsatellites. Such mutations in coding microsatellites (cMS) cause translational frameshifts that may destroy gene function. These frameshift mutations could also cause the translation of immunogenic neopeptides at the carboxy terminus. Several such mutations have been identified recently. However, since none of the frameshift-induced neopeptides identified so far is generated in all cancer cells with microsatellite instability (MSI), we aim to define a broad but comprehensive set of frameshift peptides (FSPs) that might be combined in a multivalent vaccine for MSI+ cancers. Here, we characterize the immunogenic properties of five additional HLA-A0201-restricted frameshift-induced neopeptides derived from mutations in three cMS-containing genes (Caspase-5, TAF-1b, and HT001) that are frequently hit in MSI+ cancer cells. One Caspase-5-derived FSP, (67)-FLIIWQNTM (FSP26), was identified as a novel HLA-A0201-restricted CTL epitope. FSP26-specific CTLs efficiently lysed colon carcinoma cells expressing HLA-A0201 and the underlying (-1) mutation. This mutation in an A(10) cMS is observed in up to 66% of MSI+ colorectal cancers. Thus, this newly identified CTL epitope may be another essential component of a multivalent vaccine against cancers with MSI.

Antigens, Neoplasm↗
PubMed1993

On the mechanism of frameshift (deletion) mutagenesis in vitro.

An experimental system has been developed to quantify frameshift deletions and base substitutions formed during DNA synthesis in vitro. Oligodeoxynucleotides, modified site-specifically with acetylaminofluorene or other adducts and lesions, were used as templates in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The influence of DNA sequence context on frameshift mutagenesis was determined by modifying systematically the bases flanking the lesion. Frequencies of nucleotide insertion opposite the lesion and chain extension from the 3'-primer terminus were established by steady state kinetic analysis. The ability of a damaged nucleotide to generate one-base and two-base frameshift deletions was determined primarily by two parameters: the nature of the base inserted opposite the adduct with respect to the sequence context in which the lesion is embedded and the overall rate of translesional DNA synthesis. Frameshift deletions generated during DNA synthesis were greatly enhanced in the absence of proofreading exonuclease. Misinsertion of bases opposite the lesion precedes misalignment of the template-primer. Extending on earlier studies (Kunkel, T. A. (1990) Biochemistry 29, 8003-8011), a model has been proposed and used in various sequence contexts to predict the propensity of aminofluorene adducts, exocyclic DNA adducts, 8-oxopurines, and synthetic abasic sites to generate frameshift deletions in vitro.

Base Sequence↗