scholarly journals Translational Coupling to an Upstream Gene Promotes Folding of the Mycobacterial Plasmid pAL5000 Replication Protein RepB and Thereby Its Origin Binding Activity

2004 ◽  
Vol 186 (2) ◽  
pp. 335-342 ◽  
Author(s):  
Abhijit Basu ◽  
Sujoy Chatterjee ◽  
Sujoy K. Das Gupta
Keyword(s):  
Binding Activity ◽  
Coupled Systems ◽  
Open Reading Frame ◽  
Translational Coupling ◽  
Upstream Gene ◽  
Reading Frame ◽  
Active Structure ◽  
Significant Difference ◽  
Replication Region ◽  
And Function

ABSTRACT In the mycobacterial plasmid pAL5000 replication region, the replication genes repA and repB are organized in an operon. Earlier, a RepB-dependent origin binding activity was detected in Escherichia coli cells expressing the repA-repB operon. This activity was maximal when expression of the two genes was coupled (A. Basu, M. Chawla-Sarkar, S. Chakrabarti, and S. K. Das Gupta, J. Bacteriol. 184:2204-2214, 2002). In this study we have shown that translational coupling makes a significant difference in the structure and function of RepB. When repB expression was coupled to repA, the polypeptide folded into an active structure (referred to as RepB*), which possessed higher helical content than RepB expressed independently. RepB* could also be distinguished from the less active RepB on the basis of sensitivity to OmpT, an outer membrane protease of E. coli: RepB* was sensitive to the protease, whereas RepB was resistant. Similar conformational differences between RepB* and RepB could be observed when repA was replaced with an unrelated gene, malE (encoding maltose binding protein). These results show that translational coupling of repB to an upstream gene is necessary for better folding and origin binding activity. It is speculated that in coupled systems where translation machinery is passed on from the upstream to the downstream open reading frame, cotranslational folding of the polypeptide expressed from the downstream open reading frame is enhanced due to increased folding competence of translationally primed ribosomes.

scholarly journals Human Cytomegalovirus Open Reading Frame TRL11/IRL11 Encodes an Immunoglobulin G Fc-Binding Protein

2001 ◽  
Vol 75 (22) ◽  
pp. 11218-11221 ◽  
Author(s):  
Brendan N. Lilley ◽  
Hidde L. Ploegh ◽  
Rebecca S. Tirabassi
Keyword(s):  
Human Cytomegalovirus ◽  
Immunoglobulin G ◽  
Binding Protein ◽  
Fc Receptors ◽  
Binding Activity ◽  
Open Reading Frame ◽  
Membrane Glycoprotein ◽  
Type I ◽  
Reading Frame

ABSTRACT Several herpesviruses encode Fc receptors that may play a role in preventing antibody-mediated clearance of the virus in vivo. Human cytomegalovirus (HCMV) induces an Fc-binding activity in cells upon infection, but the gene that encodes this Fc-binding protein has not been identified. Here, we demonstrate that the HCMV AD169 open reading frame TRL11 and its identical copy, IRL11, encode a type I membrane glycoprotein that possesses IgG Fc-binding capabilities.

scholarly journals An Open Reading Frame Element Mediates Posttranscriptional Regulation of Tropoelastin and Responsiveness to Transforming Growth Factor β1

1999 ◽  
Vol 19 (11) ◽  
pp. 7314-7326 ◽  
Author(s):  
Mancong Zhang ◽  
Richard A. Pierce ◽  
Hiroshi Wachi ◽  
Robert P. Mecham ◽  
William C. Parks
Keyword(s):  
Growth Factor ◽  
Posttranscriptional Regulation ◽  
Mrna Decay ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Binding Activity ◽  
Open Reading Frame ◽  
Rnase Protection ◽  
Reading Frame ◽  
Tgf Β1

ABSTRACT Elastin, an extracellular component of arteries, lung, and skin, is produced during fetal and neonatal growth. We reported previously that the cessation of elastin production is controlled by a posttranscriptional mechanism. Although tropoelastin pre-mRNA is transcribed at the same rate in neonates and adults, marked instability of the fully processed transcript bars protein production in mature tissue. Using RNase protection, we identified a 10-nucleotide sequence in tropoelastin mRNA near the 5′ end of the sequences coded by exon 30 that interacts specifically with a developmentally regulated cytosolic 50-kDa protein. Binding activity increased as tropoelastin expression dropped, being low in neonatal fibroblasts and high in adult cells, and treatment with transforming growth factor β1 (TGF-β1), which stimulates tropoelastin expression by stabilizing its mRNA, reduced mRNA-binding activity. No other region of tropoelastin mRNA interacted with cellular proteins, and no binding activity was detected in nuclear extracts. The ability of the exon-30 element to control mRNA decay and responsiveness to TGF-β1 was assessed by three distinct functional assays: (i) insertion of exon 30 into a heterologous gene conferred increased reporter activity after exposure to TGF-β1; (ii) addition of excess exon 30 RNA slowed tropoelastin mRNA decay in an in vitro polysome degradation assay; and (iii) a mutant tropoelastin cDNA lacking exon 30, compared to wild-type cDNA, produced a stable transcript whose levels were not affected by TGF-β1. These findings demonstrate that posttranscriptional regulation of elastin production in mature tissue is conferred by a specific element within the open reading frame of tropoelastin mRNA.

scholarly journals The Protein Encoded at the 3′ End of the Serine Protease Gene of Aeromonas sobria Functions as a Chaperone in the Production of the Protease

2002 ◽  
Vol 184 (24) ◽  
pp. 7058-7061 ◽  
Author(s):  
Tomohiko Nomura ◽  
Yoshio Fujii ◽  
Hiroyasu Yamanaka ◽  
Hidetomo Kobayashi ◽  
Keinosuke Okamoto
Keyword(s):  
Serine Protease ◽  
Open Reading Frame ◽  
Protease Gene ◽  
Reading Frame ◽  
Active Structure ◽  
Aeromonas Sobria ◽  
Orf2 Protein ◽  
Open Reading Frame 2 ◽  
Serine Protease Gene ◽  
Successful Production

ABSTRACT For the successful production of Aeromonas sobria serine protease (ASP), open reading frame 2 (ORF2) protein, encoded at the 3′ end of the protease operon, is required. In this study, we examined the action of ORF2 protein. The results showed that the protein associated with ASP in the periplasm and helped ASP to form an active structure.

scholarly journals Regulation of Melanocortin-5 Receptor Pharmacology by Two Isoforms of MRAP2 in Ricefield Eel (Monopterus albus)

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A508-A508
Author(s):  
Ting Liu ◽  
Ti-Lin Yi ◽  
Dai-Qin Yang ◽  
Ya-Xiong Tao
Keyword(s):  
Amino Acids ◽  
Binding Affinity ◽  
Transmembrane Domain ◽  
Open Reading Frame ◽  
Acid Oxidation ◽  
Intracellular Camp ◽  
Acth Stimulation ◽  
Monopterus Albus ◽  
Reading Frame ◽  
Significant Difference

Abstract The melanocortin-5 receptor (MC5R) has been implicated in the regulation of exocrine gland secretion, immune regulation, and muscle fatty acid oxidation. However, its function in fish is not well established. Melanocortin-2 receptor accessory protein 2 (MRAP2) can modulate trafficking, ligand binding, and signaling of melanocortin receptors. Ricefield eel (Monopterus albus) is an economically and evolutionarily important fish widely distributed in tropics and subtropics. To explore potential interaction between eel MC5R and MRAP2, herein we cloned ricefield eel mc5r, mrap2X1 and mrap2X2. Eel mc5r consisted of a 1056 bp open reading frame encoding a protein of 351 amino acids. Eel mrap2X1 consisted of a 708 bp open reading frame encoding a protein of 235 amino acids, while eel mrap2X2consisted of a 567 bp open reading frame encoding a protein of 188 amino acids. Multiple sequence alignment showed that maMRAP2X1 and maMRAP2X2 shared 90.43% identity. Interestingly, maMRAP2X2 lost the transmembrane domain. Phylogenetic analysis showed that maMC5R and maMRAP2s were closely related to piscine MC5Rs and MRAP2s, respectively. The maMC5R was further demonstrated to be a functional receptor and could be modulated by maMRAP2s in pharmacological studies. Three agonists, [Nle4, D-Phe7]-alpha-melanocyte stimulating hormone (NDP-MSH), alpha-MSH, and adrenocorticotropin (ACTH), could bind to maMC5R and induce intracellular cAMP production dose-dependently. Compared to human MC5R (hMC5R), maMC5R displayed a significantly decreased Bmax but higher binding affinity to alpha-MSH or ACTH. No significant difference in constitutive activity was observed between hMC5R and maMC5R. When stimulated with α-MSH and ACTH, maMC5R showed significantly lower EC50 and Rmax than that of hMC5R. Eel MRAP2s had no effect on cell surface and total expression of maMC5R, whereas they significantly increased Bmax. Only maMRAP2X2 significantly decreased the binding affinity of ACTH. Both maMRAP2X1 and maMRAP2X2 significantly reduced Rmax but did not affect EC50 in response to alpha-MSH or ACTH stimulation. The availability of maMC5R pharmacological characteristics and the modulation by maMRAP2s will facilitate the investigation of its function in regulating diverse physiological processes in ricefield eel.

scholarly journals A novel vector for positive selection of inserts harboring an open reading frame by translational coupling

BioTechniques ◽  
2007 ◽  
Vol 43 (6) ◽  
pp. 751-754 ◽  
Author(s):  
Sumiko Ohashi-Kunihiro ◽  
Masafumi Yohda ◽  
Haruhiko Masaki ◽  
Masayuki Machida
Keyword(s):  
Positive Selection ◽  
Open Reading Frame ◽  
Translational Coupling ◽  
Reading Frame ◽  
Selection Of

scholarly journals Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Hongmarn Park ◽  
Louise C. McGibbon ◽  
Anastasia H. Potts ◽  
Helen Yakhnin ◽  
Tony Romeo ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Translation Initiation ◽  
Binding Sites ◽  
Sigma Factor ◽  
Rna Structure ◽  
Rna Binding ◽  
Open Reading Frame ◽  
Translational Coupling ◽  
Reading Frame ◽  
Ribosome Binding

ABSTRACT CsrA is a global regulatory RNA binding protein that has important roles in regulating carbon metabolism, motility, biofilm formation, and numerous other cellular processes. IraD functions as an antiadapter protein that inhibits RssB-mediated degradation of RpoS, the general stress response and stationary-phase sigma factor of Escherichia coli . Here we identified a novel mechanism in which CsrA represses iraD translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and lacZ fusions demonstrated that CsrA represses iraD expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the iraD leader transcript, all of which are far upstream of the iraD ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the iraD Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the iraD leader region. Two of these ORFs do not affect iraD expression. However, the translation initiation region of the third ORF contains three of the CsrA binding sites, one of which overlaps its SD sequence. Furthermore, the ORF stop codon overlaps the iraD start codon, a sequence arrangement indicative of translational coupling. In vivo expression and in vitro translation studies with wild-type and mutant reporter fusions demonstrated that bound CsrA directly represses translation initiation of this ORF. We further established that CsrA-dependent repression of iraD translation occurs entirely via translational coupling with this ORF, leading to accelerated iraD mRNA decay. IMPORTANCE CsrA posttranscriptionally represses gene expression associated with stationary-phase bacterial growth, often in opposition to the transcriptional effects of the stationary-phase sigma factor RpoS. We show that CsrA employs a novel regulatory mechanism to repress translation of iraD , which encodes an antiadapter protein that protects RpoS against proteolysis. CsrA binds to four sites in the iraD leader transcript but does not directly occlude ribosome binding to the iraD SD sequence. Instead, CsrA represses translation of a short open reading frame encoded upstream of iraD , causing repression of iraD translation via translational coupling. This finding offers a novel mechanism of gene regulation by the global regulator CsrA, and since RpoS can activate csrA transcription, this also highlights a new negative-feedback loop within the complex Csr and RpoS circuitry.

scholarly journals A Fibrinogen-Binding Protein ofStaphylococcus epidermidis

1998 ◽  
Vol 66 (6) ◽  
pp. 2666-2673 ◽  
Author(s):  
Martin Nilsson ◽  
Lars Frykberg ◽  
Jan-Ingmar Flock ◽  
Lei Pei ◽  
Martin Lindberg ◽  
...  
Keyword(s):  
Phage Display ◽  
Binding Protein ◽  
Phage Display Library ◽  
Binding Activity ◽  
Surface Proteins ◽  
Open Reading Frame ◽  
Pcr Analysis ◽  
Sequence Comparisons ◽  
Reading Frame ◽  
Different Strains

ABSTRACT The present study reports on fibrinogen (Fg) binding ofStaphylococcus epidermidis. Adhesion of differentS. epidermidis strains to immobilized Fg was found to vary significantly between different strains, and the component responsible was found to be proteinaceous in nature. To further characterize the Fg-binding activity, a shotgun phage display library covering the S. epidermidis chromosome was constructed. By affinity selection (panning) against immobilized Fg, a phagemid clone, pSEFG1, was isolated, which harbors an insert with an open reading frame of ∼1.7 kilobases. Results from binding and inhibition experiments demonstrated that the insert of pSEFG1 encodes a specific Fg-binding protein. Furthermore, affinity-purified protein encoded by pSEFG1 completely inhibited adhesion of S. epidermidis to immobilized Fg. By additional cloning and DNA sequence analyses, the complete gene, termed fbe, was found to consist of an open reading frame of 3,276 nucleotides encoding a protein, called Fbe, with a deduced molecular mass of ∼119 kDa. With a second phage display library made from another clinical isolate ofS. epidermidis, it was possible to localize the Fg-binding region to a 331-amino-acid-long fragment. PCR analysis showed that the fbe gene was found in 40 of 43 clinical isolates of S. epidermidis. The overall organization of Fbe resembles those of other extracellular surface proteins of staphylococci and streptococci. Sequence comparisons with earlier known proteins revealed that this protein is related to an Fg-binding protein of Staphylococcus aureus called clumping factor.

scholarly journals Disorder at the Start: The Contribution of Dysregulated Translation Initiation to Cancer Therapy Resistance

2021 ◽  
Vol 2 ◽  
Author(s):  
Gulshan Sunavala-Dossabhoy
Keyword(s):  
Amino Acids ◽  
Cancer Therapy ◽  
Translation Initiation ◽  
Translational Control ◽  
Treatment Resistance ◽  
Therapy Resistance ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Cellular Processes ◽  
And Function

Translation of cellular RNA to protein is an energy-intensive process through which synthesized proteins dictate cellular processes and function. Translation is regulated in response to extracellular effectors and availability of amino acids intracellularly. Most eukaryotic mRNA rely on the methyl 7-guanosine (m7G) nucleotide cap to recruit the translation machinery, and the uncoupling of translational control that occurs in tumorigenesis plays a significant role in cancer treatment response. This article provides an overview of the mammalian translation initiation process and the primary mechanisms by which it is regulated. An outline of how deregulation of initiation supports tumorigenesis and how initiation at a downstream open reading frame (ORF) of Tousled-like kinase 1 (TLK1) leads to treatment resistance is discussed.

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