scholarly journals Translation of a minigene in the 5′ leader sequence of the enterohaemorrhagic Escherichia coli LEE1 transcription unit affects expression of the neighbouring downstream gene

2011 ◽  
Vol 441 (1) ◽  
pp. 247-253 ◽  
Author(s):  
Md. Shahidul Islam ◽  
Robert K. Shaw ◽  
Gad Frankel ◽  
Mark J. Pallen ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Translation Initiation ◽  
Stop Codon ◽  
Leader Sequence ◽  
Open Reading Frame ◽  
Start Codon ◽  
Target Cells ◽  
Reading Frame ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Translation Start

The 5′ end of the major RNA transcript of the LEE1 operon of enterohaemorrhagic Escherichia coli contains ~170 bases before the AUG translation start codon of the first recognized gene, ler. This unusually long leader sequence carries three potential alternative AUG start codons. Using a lac fusion expression vector, we confirmed that the ler gene AUG is functional for translation initiation, and we checked for translation initiation at the three alternative AUG codons. Whereas two of the alternative AUG codons appear incompetent for translation initiation, we detected strong initiation at the third AUG, which is followed by one AAA codon and a UAG stop codon. The location of this very short two-codon open reading frame with respect to the ler translation start appears to be critical. Hence mutations that destroy the UAG stop codon, or short deletions between the UAG stop codon and the ler translation initiation region, result in big effects on ler expression. In the context of the full-length LEE1 operon leader sequence, translation of this very short two-codon open reading frame is necessary for optimal expression of the ler gene and for the subsequent interactions of enterohaemorrhagic Escherichia coli with host target cells.

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

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.

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

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.

scholarly journals A Translation-Aborting Small Open Reading Frame in the Intergenic Region Promotes Translation of a Mg2+ Transporter in Salmonella Typhimurium

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Eunna Choi ◽  
Yoontak Han ◽  
Shinae Park ◽  
Hyojeong Koo ◽  
Jung-Shin Lee ◽  
...  
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Translation Initiation ◽  
Intergenic Region ◽  
Ribosomal Subunit ◽  
Open Reading Frame ◽  
Start Codon ◽  
Stem Loop ◽  
Reading Frame ◽  
Ribosome Binding

ABSTRACT Bacterial mRNAs often harbor upstream open reading frames (uORFs) in the 5′ untranslated regions (UTRs). Translation of the uORF usually affects downstream gene expression at the levels of transcription and/or translation initiation. Unlike other uORFs mostly located in the 5′ UTR, we discovered an 8-amino-acid ORF, designated mgtQ, in the intergenic region between the mgtC virulence gene and the mgtB Mg2+ transporter gene in the Salmonella mgtCBRU operon. Translation of mgtQ promotes downstream mgtB Mg2+ transporter expression at the level of translation by releasing the ribosome-binding sequence of the mgtB gene that is sequestered in a translation-inhibitory stem-loop structure. Interestingly, mgtQ Asp2 and Glu5 codons that induce ribosome destabilization are required for mgtQ-mediated mgtB translation. Moreover, the mgtQ Asp and Glu codons-mediated mgtB translation is counteracted by the ribosomal subunit L31 that stabilizes ribosome. Substitution of the Asp2 and Glu5 codons in mgtQ decreases MgtB Mg2+ transporter production and thus attenuates Salmonella virulence in mice, likely by limiting Mg2+ acquisition during infection. IMPORTANCE Translation initiation regions in mRNAs that include the ribosome-binding site (RBS) and the start codon are often sequestered within a secondary structure. Therefore, to initiate protein synthesis, the mRNA secondary structure must be unfolded to allow the RBS to be accessible to the ribosome. Such unfolding can be achieved by various mechanisms that include translation of a small upstream open reading frame (uORF). In the intracellular pathogen Salmonella enterica serovar Typhimurium, translation of the Mg2+ transporter mgtB gene is enhanced by an 8-amino-acid upstream ORF, namely, mgtQ, that harbors Asp and Glu codons, which are likely to destabilize ribosome during translation. Translation of the mgtQ ORF promotes the formation of a stem-loop mRNA structure sequestering anti-RBS and thus releases the mgtB RBS. Because mgtQ-mediated MgtB Mg2+ transporter production is required for Salmonella virulence, this pathogen seems to control the virulence determinant production exquisitely via this uORF during infection.

Abstract 022: A Short Open Reading Frame in the Angiotensin Type 1a Receptor 5' Leader Sequence Increases the Rate of Receptor Internalization

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Parnika Kadam ◽  
Susette Mueller ◽  
Hong Ji ◽  
Kathryn Sandberg
Keyword(s):  
Laser Scanning ◽  
Leader Sequence ◽  
Open Reading Frame ◽  
Receptor Internalization ◽  
Start Codon ◽  
Ang Ii ◽  
Exon 2 ◽  
Reading Frame ◽  
Short Open Reading Frame ◽  
Angiotensin Type

Background: We recently found that a seven amino acid peptide (PEP7) encoded within a short open reading frame (sORF) in exon 2 of the 5’ leader sequence (5'LS) of the angiotensin type 1a receptor (AT1aR) mRNA inhibits AT1aR-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) without having any effect on the AT1aR-inositol trisphosphate protein kinase C pathway. To investigate the mechanism by which PEP7 selectively inhibits the AT1aR-Erk1/2 signaling cascade, the start codon of the sORF was mutated at adenine -108 (A-108 to T-108) to create E1,2(-108T),3-AT1aR followed by cloning the E1,2,3-AT1aR and the mutant E1,2(-108T),3-AT1aR into the pEGFP-N2 plasmid. Methods: Human embryonic kidney 293 (HEK-293) cells were transfected with E1,2,3-AT1aR (intact sORF construct) or E1,2(-108T),3-AT1aR (disrupted sORF construct) by lipofectamine. Forty eight to seventy two hours later, live cell time lapse images were collected before and after treatment with Ang II (100 nM) using a TE300 Spinning disk laser scanning confocal microscope. Image analysis was performed using Velocity software. Results: Within 5 min of Ang II stimulation, punctae formed and moved throughout the cell membrane in cells transfected with both EGFP tagged receptors; however, even though the punctae represent the localized accumulation of identical AT1aR proteins, there were distinct differences in the intensity and time course of punctae. The rate of vesicle formation after Ang II treatment was markedly decreased by disrupting the PEP7 sORF [t1/2 (s): E1,2,3-AT1aR, 203s (N=21) vs E1,2(-108T),3-AT1aR, 328s (N=14); p<0.0001]. Conclusion: The PEP7 sORF facilitates Ang II-induced AT1R internalization. These findings suggest that we have uncovered a new mechanism governing agonist-induced AT1aR cellular trafficking that could have implications not only for regulation of AT1aR signaling cascades but also for other trafficking proteins that contain an upstream sORF within their 5'LS.

Cloning of a gar (Lepisosteus osseus) GH cDNA: trends in actinopterygian GH structure

1996 ◽  
Vol 16 (1) ◽  
pp. 73-80 ◽  
Author(s):  
D A Rubin ◽  
J H Youson ◽  
L E Marra ◽  
R M Dores
Keyword(s):  
Cdna Library ◽  
Untranslated Region ◽  
Signal Sequence ◽  
Leader Sequence ◽  
Open Reading Frame ◽  
Russian Sturgeon ◽  
Reading Frame ◽  
Terminal Codon

ABSTRACT A cDNA containing the sequence of GH was cloned and sequenced from a pituitary cDNA library for the holostean fish Lepisosteus osseus (common name: gar). The gar GH cDNA contained an open reading frame of 633 nucleotides and a 3′ untranslated region (including the terminal codon TAG) of 1058 nucleotides. The overall length of the gar GH cDNA including leader sequence, signal sequence, hormone sequence and 3′ untranslated region was 1713 nucleotides. Thus, the gar GH cDNA is the largest vertebrate GH cDNA yet cloned. A comparison of GH sequences from ancient (holostean fishes — gar and bowfin; one chondrostean fish — the Russian sturgeon) and more modern (27 species of teleosts) members of class Actinopterygii indicate that members of this class have maintained many of the invariant residues deemed necessary for GH folding motifs (intramolecular relationships) observed in mammals.

scholarly journals Regulation of the nfsA Gene in Escherichia coli by SoxS

2002 ◽  
Vol 184 (1) ◽  
pp. 51-58 ◽  
Author(s):  
E. Suzanne Paterson ◽  
Sherri E. Boucher ◽  
I. B. Lambert
Keyword(s):  
Escherichia Coli ◽  
Promoter Sequence ◽  
Open Reading Frame ◽  
Base Sequence ◽  
Start Site ◽  
Transcription Start ◽  
Band Shift ◽  
Reading Frame ◽  
Shift Analysis ◽  
Small Open Reading Frame

ABSTRACT In Escherichia coli, the response to oxidative stress due to elevated levels of superoxide is mediated, in part, by the soxRS regulon. One member of the soxRS regulon, nfsA, encodes the major oxygen-insensitive nitroreductase in Escherichia coli which catalyzes the reduction of nitroaromatic and nitroheterocyclic compounds by NADPH. In this study we investigate the regulation of nfsA in response to the superoxide generating compound paraquat. The transcription start site (TSS) of nfsA was located upstream of the ybjC gene, a small open reading frame of unknown function located directly upstream of nfsA, suggesting that these two genes form an operon. The activity of the promoter associated with this TSS was confirmed with lacZ fusions and was shown to be inducible by paraquat. Footprinting and band shift analysis showed that purified His-tagged SoxS protein binds to a 20-base sequence 10 bases upstream of the −35 promoter sequence in the forward orientation, suggesting that the ybjC-nfsA promoter is a class I SoxS-dependent promoter.

scholarly journals Translationskopplung via Termination-Reinitiation in Archaea und Bacteria

2021 ◽  
Author(s):  
◽  
Madeleine Huber
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Stop Codon ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Directed Mutagenesis ◽  
Ribosome Display ◽  
E Coli

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.

The extrachromosomal replication of Dictyostelium plasmid Ddp2 requires a cis-acting element and a plasmid-encoded trans-acting factor

1990 ◽  
Vol 10 (7) ◽  
pp. 3727-3736
Author(s):  
B Leiting ◽  
I J Lindner ◽  
A A Noegel
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Wild Strain ◽  
Open Reading Frame ◽  
Mobility Shift ◽  
Reading Frame ◽  
Cis Acting ◽  
Transformation Vector ◽  
Cis And Trans ◽  
Electrophoretic Mobility Shift Assays

Dictyostelium discoideum plasmid Ddp2 from the wild strain WS380B is a 5.8-kilobase (kb) supercoiled circle with a copy number of 300 per haploid genome. We previously described the construction of an extrachromosomally replicating transformation vector pnDeI carrying 4.7 kb of Ddp2 sequences (B. Leiting, and A. Noegel, Plasmid 20:241-248, 1988). In order to reduce the sequences required for extrachromosomal maintenance in D. discoideum, we characterized Ddp2 by sequence analysis, by deletion experiments, by transcription mapping, by electrophoretic mobility shift assays, and by expression of its single open reading frame in Escherichia coli. Two elements were involved in replication of Ddp2: a cis-acting sequence located on a 592-base-pair (bp) fragment that consisted of 220 bp of essential and 372 bp of auxiliary sequences, and a 2.7-kb open reading frame which most likely encodes a trans-acting factor. The cis- and trans-acting elements did not overlap and were shown to act independently from the location of the sequences encoding the trans-acting factor.

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