An active variant of the prokaryotic transposable element IS903 carries an amber stop codon in the middle of an open reading frame

Murine cytomegalovirus mutant Rc29, with a premature stop codon mutation in the m29 open reading frame (ORF), produced no apparent phenotype in cell culture or following infection of BALB/c mice. In contrast, a similar mutant virus, Rc29.1, with a premature stop codon mutation in its m29.1 ORF, showed reduced virus yields (2–3 log10 p.f.u. ml−1) in tissue culture. Mutant virus yields in BALB/c mice were delayed, reduced (∼1 log10 p.f.u. per tissue) and persisted less well in salivary glands compared with wild-type (wt) and revertant (Rv29.1) virus. In severe combined immunodeficiency mice, Rc29.1 virus showed delayed and reduced replication initially in all tissues (liver, spleen, kidneys, heart, lung and salivary glands). This delayed death until 31 days post-infection (p.i.) compared with wt (23 days p.i.) but at death virus yields were similar to wt. m29 gene transcription was initiated at early times post-infection, while production of a transcript from ORF m29.1 in the presence of cycloheximide indicated that it was an immediate-early gene. ORFs m29.1 and M28 are expressed from a bicistronic message, which is spliced infrequently. However, it is likely that each ORF expresses its own protein, as antiserum derived in rabbits to the m29.1 protein expressed in bacteria from the m29.1 ORF detected only one protein in Western blot analysis of the size predicted for the m29.1 protein. Our results suggest that neither ORF is essential for virus replication but m29.1 is important for optimal viral growth in vitro and in vivo.

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IRES-mediated ribosome repositioning directs translation of a +1 overlapping ORF that enhances viral infection

10.1101/303388 ◽

2018 ◽

Author(s):

Craig H Kerr ◽

Qing S Wang ◽

Kyung-Mee Moon ◽

Kathleen Keatings ◽

Douglas W Allan ◽

...

Keyword(s):

Translation Initiation ◽

Stop Codon ◽

Open Reading Frame ◽

Wild Type Virus ◽

Infection Model ◽

Entry Site ◽

Internal Ribosome Entry ◽

Reading Frame ◽

Coding Capacity ◽

Paralysis Virus

AbstractRNA structures can interact with the ribosome to alter translational reading frame maintenance and promote recoding that result in alternative protein products. Here, we show that the internal ribosome entry site (IRES) from the dicistrovirus Cricket paralysis virus drives translation of the 0-frame viral polyprotein and an overlapping +1 open reading frame, called ORFx, via a novel mechanism whereby a subset of ribosomes recruited to the IRES bypasses downstream to resume translation at the +1-frame 13th non-AUG codon. A mutant of CrPV containing a stop codon in the +1 frame ORFx sequence, yet synonymous in the 0-frame, is attenuated compared to wild-type virus in a Drosophila infection model, indicating the importance of +1 ORFx expression in promoting viral pathogenesis. This work demonstrates a novel programmed IRES-mediated recoding strategy to increase viral coding capacity and impact virus infection, highlighting the diversity of RNA-driven translation initiation mechanisms in eukaryotes.Significance StatementViruses use alternate mechanisms to increase the coding capacity of their viral genomes. Here, we provide biochemical evidence that ribosomes recruited to the dicistrovirus cricket paralysis virus IRES undergo a bypass event to direct translation of a downstream +1 frame overlapping open reading frame, called ORFx. Mutations that block ORFx expression inhibit +1 frame translation and infection in fruit flies. These findings highlight the diversity of RNA-driven translation initiation mechanisms in eukaryotes.

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Evolutionarily conserved regions in Caenorhabditis transposable elements deduced by sequence comparison

Genome ◽

10.1139/g91-002 ◽

1991 ◽

Vol 34 (1) ◽

pp. 6-12 ◽

Cited By ~ 17

Author(s):

Shiv S. Prasad ◽

Linda J. Harris ◽

David L. Baillie ◽

Ann M. Rose

Keyword(s):

Amino Acid ◽

Transposable Element ◽

Sequence Comparison ◽

Horizontal Transmission ◽

Protein A ◽

Open Reading Frame ◽

Single Open Reading Frame ◽

Major Open Reading Frame ◽

Reading Frame ◽

Caenorhabditis Species

In this paper we present the sequence of an intact Caenorhabditis briggsae transposable element, Tcb2. Tcb2 is 1606 base pairs in length and contains 80 base pair imperfect terminal repeats and a single open reading frame. We have identified blocks of T-rich repeats in the regions 150–200 and 1421–1476 of this element which are conserved in the Caenorhabditis elegans element Tc1. The sequence conservation of these regions in elements from different Caenorhabditis species suggests that they are of functional importance. A single open reading frame corresponding to the major open reading frame of Tc1 is conserved among Tc1, Tcb1, and Tcb2. Comparison of the first 550 nucleotides of the sequence among the three elements has allowed the evaluation of a model proposing an extension of the major open reading frame. Our data support the suggestion that Tc1 is capable of producing a 335 amino acid protein. A comparison of the sequence coding for the amino and carboxy termini of the 273 amino acid transposase from Caenorhabditis Tc1-like elements and Drosophila HB1 showed different amounts of divergence for each of these regions, indicating that the two functional domains have undergone different amounts of selection. Our data are not compatible with the proposal that Tc1-related sequences have been acquired via horizontal transmission. The divergence of Tc1 from the two C. briggsae elements, Tcb1 and Tcb2, indicated that all three elements have been diverging from each other for approximately the same amount of time as the genomes of the two species.Key words: Caenorhabditis, transposable element, sequence comparison.

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Bioinformatic Prediction of an tRNASec Gene Nested inside an Elongation Factor SelB Gene in Alphaproteobacteria

International Journal of Molecular Sciences ◽

10.3390/ijms22094605 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4605

Author(s):

Takahito Mukai

Keyword(s):

Insertion Sequence ◽

Trna Gene ◽

Stop Codon ◽

Elongation Factor ◽

Selenium Deficiency ◽

Open Reading Frame ◽

Start Codon ◽

Sequence Element ◽

Reading Frame ◽

Insertion Sequence Element

In bacteria, selenocysteine (Sec) is incorporated into proteins via the recoding of a particular codon, the UGA stop codon in most cases. Sec-tRNASec is delivered to the ribosome by the Sec-dedicated elongation factor SelB that also recognizes a Sec-insertion sequence element following the codon on the mRNA. Since the excess of SelB may lead to sequestration of Sec-tRNASec under selenium deficiency or oxidative stress, the expression levels of SelB and tRNASec should be regulated. In this bioinformatic study, I analyzed the Rhizobiales SelB species because they were annotated to have a non-canonical C-terminal extension. I found that the open reading frame (ORF) of diverse Alphaproteobacteria selB genes includes an entire tRNASec sequence (selC) and overlaps with the start codon of the downstream ORF. A remnant tRNASec sequence was found in the Sinorhizobium melilotiselB genes whose products have a shorter C-terminal extension. Similar overlapping traits were found in Gammaproteobacteria and Nitrospirae. I hypothesized that once the tRNASec moiety is folded and processed, the expression of the full-length SelB may be repressed. This is the first report on a nested tRNA gene inside a protein ORF in bacteria.

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Characterization and Role of tbuX in Utilization of Toluene by Ralstonia pickettii PKO1

Journal of Bacteriology ◽

10.1128/jb.182.5.1232-1242.2000 ◽

2000 ◽

Vol 182 (5) ◽

pp. 1232-1242 ◽

Cited By ~ 66

Author(s):

Hyung-Yeel Kahng ◽

Armando M. Byrne ◽

Ronald H. Olsen ◽

Jerome J. Kukor

Keyword(s):

Amino Acid ◽

Outer Membrane ◽

Stop Codon ◽

Outer Membrane Proteins ◽

Open Reading Frame ◽

Predicted Amino Acid Sequence ◽

Reading Frame ◽

Ralstonia Pickettii ◽

Degrading Bacteria

ABSTRACT The tbu regulon of Ralstonia pickettii PKO1 encodes enzymes involved in the catabolism of toluene, benzene, and related alkylaromatic hydrocarbons. The first operon in this regulon contains genes that encode the tbu pathway's initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional activator for the entire regulon. It has been previously shown that the organization of tbuT, which is located immediately downstream of tbuA1UBVA2C, and the associated promoter (PtbuA1) is unique in that it results in a cascade type of up-regulation of tbuT in response to a variety of effector compounds. In our efforts to further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded on the strand opposite that of tbuT, 63 bp downstream of the tbuT stop codon. The 1,374-bp open reading frame, encoding a 458-amino-acid peptide, was designatedtbuX. The predicted amino acid sequence of TbuX exhibited significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids inEscherichia coli. Based on sequence analysis, transcriptional and expression studies, and deletion analysis, TbuX seems to play an important role in the catabolism of toluene inR. pickettii PKO1. In addition, the expression oftbuX appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene-3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.

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Studies on p40, the leucine zipper motif-containing protein encoded by the first open reading frame of an active human LINE-1 transposable element.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)88618-0 ◽

1992 ◽

Vol 267 (28) ◽

pp. 19765-19768

Author(s):

S.E. Holmes ◽

M.F. Singer ◽

G.D. Swergold

Keyword(s):

Transposable Element ◽

Leucine Zipper ◽

Open Reading Frame ◽

Leucine Zipper Motif ◽

Reading Frame ◽

Human Line

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Tissue-specific dynamic codon redefinition in Drosophila

10.1101/2020.06.22.165522 ◽

2020 ◽

Author(s):

Andrew M. Hudson ◽

Gary Loughran ◽

Nicholas L. Szabo ◽

Norma M. Wills ◽

John F. Atkins ◽

...

Keyword(s):

High Efficiency ◽

Stop Codon ◽

Open Reading Frame ◽

Stem Loop ◽

Tissue Specific ◽

Reading Frame ◽

Cis Acting ◽

A Subunit ◽

Specific Regulation ◽

Stop Codon Readthrough

Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3’ of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue- and cell-specific dynamic stop codon redefinition in Drosophila.

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