Ribosomal -1 frameshifting during decoding of Bacillus subtilis cdd occurs at the sequence CGA AAG.
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During translation of the Bacillus subtilis cdd gene, encoding cytidine deaminase (CDA), a ribosomal -1 frameshift occurs near the stop codon, resulting in a CDA subunit extended by 13 amino acids. The frequency of the frameshift is approximately 16%, and it occurs both when the cdd gene is expressed from a multicopy plasmid in Escherichia coli and when it is expressed from the chromosomal copy in B. subtilis. As a result, heterotetrameric forms of the enzyme are formed in vivo along with the dominant homotetrameric species. The different forms have approximately the same specific activity. The cdd gene was cloned in pUC19 such that the lacZ' gene of the vector followed the cdd gene in the -1 reading frame immediately after the cdd stop codon. By using site-directed mutagenesis of the cdd-lacZ' fusion, it was shown that frameshifting occurred at the sequence CGA AAG, 9 bp upstream of the in-frame cdd stop codon, and that it was stimulated by a Shine-Dalgarno-like sequence located 14 bp upstream of the shift site. The possible function of this frameshift in gene expression is discussed. (+info)
Identification of putative programmed -1 ribosomal frameshift signals in large DNA databases.
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The cis-acting elements that promote efficient ribosomal frameshifting in the -1 (5') direction have been well characterized in several viral systems. Results from many studies have convincingly demonstrated that the basic molecular mechanisms governing programmed -1 ribosomal frameshifting are almost identical from yeast to humans. We are interested in testing the hypothesis that programmed -1 ribosomal frameshifting can be used to control cellular gene expression. Toward this end, a computer program was designed to search large DNA databases for consensus -1 ribosomal frameshift signals. The results demonstrated that consensus programmed -1 ribosomal frameshift signals can be identified in a substantial number of chromosomally encoded mRNAs and that they occur with frequencies from two- to sixfold greater than random in all of the databases searched. A preliminary survey of the databases resulting from the computer searches found that consensus frameshift signals are present in at least 21 homologous genes from different species, 2 of which are nearly identical, suggesting evolutionary conservation of function. We show that four previously described missense alleles of genes that are linked to human diseases would disrupt putative programmed -1 ribosomal frameshift signals, suggesting that the frameshift signal may be involved in the normal expression of these genes. We also demonstrate that signals found in the yeast RAS1 and the human CCR5 genes were able to promote significant levels of programmed -1 ribosomal frameshifting. The significance of these frameshifting signals in controlling gene expression is not known, however. (+info)
Transfer RNA modification: influence on translational frameshifting and metabolism.
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Transfer RNA modification improves the rate of aa-tRNA selection at the A-site and the fitness in the P-site and thereby prevents frameshifting according to a new model how frameshifting occurs [Qian et al. (1998) Mol. Cell 1, 471-482]. Evidence that the presence of various modified nucleosides in tRNA also influences central metabolism, thiamine metabolism, and bacterial virulence is reviewed. (+info)
Detecting and analyzing DNA sequencing errors: toward a higher quality of the Bacillus subtilis genome sequence.
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During the determination of a DNA sequence, the introduction of artifactual frameshifts and/or in-frame stop codons in putative genes can lead to misprediction of gene products. Detection of such errors with a method based on protein similarity matching is only possible when related sequences are available in databases. Here, we present a method to detect frameshift errors in DNA sequences that is based on the intrinsic properties of the coding sequences. It combines the results of two analyses, the search for translational initiation/termination sites and the prediction of coding regions. This method was used to screen the complete Bacillus subtilis genome sequence and the regions flanking putative errors were resequenced for verification. This procedure allowed us to correct the sequence and to analyze in detail the nature of the errors. Interestingly, in several cases in-frame termination codons or frameshifts were not sequencing errors but confirmed to be present in the chromosome, indicating that the genes are either nonfunctional (pseudogenes) or subject to regulatory processes such as programmed translational frameshifts. The method can be used for checking the quality of the sequences produced by any prokaryotic genome sequencing project. (+info)
Expression of the human immunodeficiency virus frameshift signal in a bacterial cell-free system: influence of an interaction between the ribosome and a stem-loop structure downstream from the slippery site.
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A-1 frameshift event is required for expression of the pol gene when ribosomes translate the mRNA of human immunodeficiency virus type-1 (HIV-1). In this study, we inserted the frameshift region of HIV-1 (a slippery heptanucleotide motif followed by a stem-loop) in a reporter gene coding for firefly luciferase. The ability of the corresponding mRNA, generated by in vitro transcription, to be translated in an Escherichia coli cell-free extract is the first demonstration that the HIV-1 frameshift can be reproduced in a bacterial cell-free extract, providing a powerful approach for analysis of the frameshift mechanism. The responses of the frameshift signal to chloramphenicol, an inhibitor of peptide bond formation, and spectinomycin, an inhibitor of translocation, suggest that the frameshift complies with the same rules found in eukaryotic translation systems. Furthermore, when translation was performed in the presence of streptomycin and neamine, two error-inducing antibiotics, or with hyperaccurate ribosomes mutated in S12, the frameshift efficiency was increased or decreased, respectively, but only in the presence of the stem-loop, suggesting that the stem-loop can influence the frameshift through a functional interaction with the ribosomes. (+info)
Translational suppressors and antisuppressors alter the efficiency of the Ty1 programmed translational frameshift.
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Certain viruses, transposons, and cellular genes have evolved specific sequences that induce high levels of specific translational errors. Such "programmed misreading" can result in levels of frameshifting or nonsense codon readthrough that are up to 1,000-fold higher than normal. Here we determine how a number of mutations in yeast affect the programmed misreading used by the yeast Ty retrotransposons. These mutations have previously been shown to affect the general accuracy of translational termination. We find that among four nonsense suppressor ribosomal mutations tested, one (a ribosomal protein mutation) enhanced the efficiency of the Tyl frameshifting, another (an rRNA mutation) reduced frameshifting, and two others (another ribosomal protein mutation and another rRNA mutation) had no effect. Three antisuppressor rRNA mutations all reduced Tyl frameshifting; however the antisuppressor mutation in the ribosomal protein did not show any effect. Among nonribosomal mutations, the allosuppressor protein phosphatase mutation enhanced Tyl frameshifting, whereas the partially inactive prion form of the release factor eRF3 caused a slight decrease, if any effect. A mutant form of the other release factor, eRF1, also had no effect on frameshifting. Our data suggest that Ty frameshifting is under the control of the cellular translational machinery. Surprisingly we find that translational suppressors can affect Ty frameshifting in either direction, whereas antisuppressors have either no effect or cause a decrease. (+info)
Inhibition of in vitro and ex vivo translation by a transplatin-modified oligo(2'-O-methylribonucleotide) directed against the HIV-1 gag-pol frameshift signal.
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A 2'-O-methylribooligonucleotide containing a G1.U.G3 triad modified by trans-diamminedichloro-platinum(II) was targeted to the RNA region responsible for the gag-pol frameshifting during translation of the HIV-1 mRNA. The binding of the platinated oligonucleotide to its target RNA induced a rearrangement of the (G1, G3)-intrastrand crosslink, leading to the formation of an intermolecular oligonucleotide-RNA G-A crosslink. This resulted in the selective arrest of translation of a luciferase gene placed downstream of the HIV-1 frameshift signal both in a cell-free extract (rabbit reticulocyte lysate) and in RNA-transfected cells. A specific inhibition of luciferase activity was still observed when the oligonucleotide-RNA complex was not pre-formed prior to either translation or transfection. Moreover, a selective inhibition was also observed when the oligonucleotide and the plasmid DNA encoding the luciferase and bearing the RNA gag- pol frameshifting signal were co-transfected in NIH 3T3 cultured cells. Therefore the intra-strand-->interstrand conversion of the platinum crosslink kinetically competes with the translation machinery and blocks the polypeptide elongation. These transplatin-modified oligonucleotides which operate within a live cell on a 'real-time' basis and do not need an external triggering signal constitute a promising new class of selective reactive probes. (+info)
Near-cognate peptidyl-tRNAs promote +1 programmed translational frameshifting in yeast.
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Translational frameshifting is a ubiquitous, if rare, form of alternative decoding in which ribosomes spontaneously shift reading frames during translation elongation. In studying +1 frameshifting in Ty retrotransposons of the yeast S. cerevisiae, we previously showed that unusual P site tRNAs induce frameshifting. The frameshift-inducing tRNAs we show here are near-cognates for the P site codon. Their abnormal decoding induces frameshifting in either of two ways: weak codon-anticodon pairing allows the tRNA to disengage from the mRNA and slip +1, or an unusual codon-anticodon structure interferes with cognate in-frame decoding allowing out-of-frame decoding in the A site. We draw parallels between this mechanism and a proposed mechanism of frameshift suppression by mutant tRNAs. (+info)