Dissection of a co-translational nascent chain separation event

2008 ◽  
Vol 36 (4) ◽  
pp. 712-716 ◽  
Author(s):  
Victoria A. Doronina ◽  
Pablo de Felipe ◽  
Cheng Wu ◽  
Pamila Sharma ◽  
Matthew S. Sachs ◽  
...  
Keyword(s):  
Protein Sequences ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Mrna Sequence ◽  
Yeast Genetics ◽  
Viral Genomes ◽  
Nascent Chain ◽  
Translation Systems ◽  
Chain Separation

Some RNA and protein sequences are capable of directing changes to the course of translation from that expected from the mRNA sequence, and this process is termed translational ‘recoding’. ‘CHYSEL’ peptides are ∼19-amino-acid sequences found in many viral genomes. When translated at internal portions of polypeptides, they yield co-translational separation of the nascent chain at their C-termini. We dissected the reaction promoted by CHYSEL sequences using yeast genetics and in vitro translation systems. Our results indicate that the reaction occurs within the peptidyltransferase centre of the ribosome where the nascent chain is hydrolytically released from tRNA despite the presence of further sense codons.

scholarly journals Probing self-regeneration of essential protein factors required for in vitro translation activity by serial transfer

2020 ◽  
Vol 56 (98) ◽  
pp. 15426-15429
Author(s):  
Kai Libicher ◽  
Hannes Mutschler
Keyword(s):  
Protein Synthesis ◽  
Serial Dilution ◽  
In Vitro Translation ◽  
Serial Transfer ◽  
Essential Protein ◽  
Protein Components ◽  
Translation Systems

Recombinant in vitro translation systems can regenerate essential protein components that maintain protein synthesis during serial dilution.

scholarly journals Identification of Mimotope Peptides Which Bind to the Mycotoxin Deoxynivalenol-Specific Monoclonal Antibody

1999 ◽  
Vol 65 (8) ◽  
pp. 3279-3286 ◽  
Author(s):  
Qiaoping Yuan ◽  
James J. Pestka ◽  
Brandon M. Hespenheide ◽  
Leslie A. Kuhn ◽  
John E. Linz ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Synthetic Peptide ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Translation System

ABSTRACT Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.

scholarly journals Differential Rescue of Poliovirus RNA Replication Functions by Genetically Modified RNA Polymerase Precursors

2004 ◽  
Vol 78 (23) ◽  
pp. 13007-13018 ◽  
Author(s):  
Christopher T. Cornell ◽  
Jo Ellen Brunner ◽  
Bert L. Semler
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Genetically Modified ◽  
Rna Replication ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Wild Type ◽  
Positive Strand ◽  
Positive Strand Rna

ABSTRACT We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

scholarly journals N-terminal binding domain of Gα subunits: involvement of amino acids 11–14 of Gα0 in membrane attachment

1997 ◽  
Vol 323 (1) ◽  
pp. 239-244 ◽  
Author(s):  
Liliana BUSCONI ◽  
Paula M. BOUTIN ◽  
Bradley M. DENKER
Keyword(s):  
Signal Transduction ◽  
Amino Acids ◽  
G Proteins ◽  
Membrane Binding ◽  
Amino Acid Sequences ◽  
Membrane Receptors ◽  
In Vitro Translation ◽  
Membrane Attachment ◽  
Membrane Components

Heterotrimeric guanine nucleotide binding proteins (G-proteins) transmit signals from membrane receptors to a variety of intracellular effectors. G-proteins reversibly associate with components of the signal transduction system, yet remain membrane attached throughout the cycle of activation. The Gα subunits remain attached to the plasma membrane through a combination of factors that are only partially defined. We now demonstrate that amino acids within the N-terminal domain of Gα subunits are involved in membrane binding. We used in vitro translation, a technique widely utilized to characterize functional aspects of G-proteins, and interactions with donor-acceptor membranes to demonstrate that amino acids 11-14 of Gαo contribute to membrane binding. The membrane binding of Gαo lacking amino acids 11-14 (D[11-14]) was significantly reduced at all membrane concentrations in comparison with wild-type Gαo. Several other N-terminal mutants of Gαo were characterized as controls, and these results indicate that differences in myristoylation, palmitoylation and βγ interactions do not account for the reduced membrane binding of D[11-14]. Furthermore, when membrane attachment of Gαo and mutants was characterized in transiently transfected 35S-labelled and [3H]myristate-labelled COS cells, amino acids 11-14 contributed to membrane binding. These studies reveal that membrane binding of Gα subunits occurs by a combination of factors that include lipids and amino acid sequences. These regions may provide novel sites for interaction with membrane components and allow additional modulation of signal transduction.

scholarly journals Computational approach for selection of epitope-based dengue vaccine targets

2018 ◽  
Vol 14 (4) ◽  
pp. 605-618
Author(s):  
Phuc Nguyen ◽  
Ly Le
Keyword(s):  
Amino Acids ◽  
T Cell ◽  
Dengue Virus ◽  
Rational Design ◽  
Protein Sequences ◽  
Computational Approach ◽  
Amino Acid Sequences ◽  
Universal Vaccine ◽  
E Protein

High antigenic variability in the envelope (E) protein of different virus strains has been a major obstacle in designing effective vaccines for Dengue virus (DENV). To maintain their biological function, some parts of viral proteins remain stable during evolution thus one possible approach to solve this problem is to recognize specific regions within different protein sequences of E that have the tendency to stay constant through evolution. These regions may possess some special attributes to become a vaccine candidate against dengue virus. In this study, a computational approach was utilized to identify and analyze highly conserved amino acid sequences of the DENV E protein. Sequences of 9 amino acids or more were specifically focused due to their immune-relevant as T-cell determinants. Different bioinformatics tools were responsible for revealing conserved regions in the DENV E protein and constructing the phylogenetic tree from the sequence database. The tools also predicted immunogenicity of the identified vaccine targets. Ultimately, two peptide regions of at least 9 amino acids were chosen due to their high conserved attribute in more than 95% of all collected DENV sequences. Moreover, both of them was found to be immune-relevant by their correspondence to known or putative HLA-restricted T cell determinants. The conserved attribute of these sequences through the entire analysis of this study supports their potential as candidates for further in vitro experiments for rational design a universal vaccine which has longer and broader impact.

Cryo-electron Tomography of in vitro Translation Systems

2005 ◽  
Vol 2005 (Spring) ◽  
Author(s):  
Karoline Bopp ◽  
Markus Stemp ◽  
Friedrich F�rster ◽  
Julio Ortiz ◽  
Vishwas Agashe ◽  
...  
Keyword(s):  
Electron Tomography ◽  
In Vitro Translation ◽  
Cryo Electron Tomography ◽  
Translation Systems

Ubiquitin is a heat shock protein in chicken embryo fibroblasts

1985 ◽  
Vol 5 (5) ◽  
pp. 949-956
Author(s):  
U Bond ◽  
M J Schlesinger
Keyword(s):  
Heat Shock ◽  
Chicken Embryo ◽  
Recombinant Dna ◽  
Protein Product ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Bovine Ubiquitin ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.

scholarly journals A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation.

1991 ◽  
Vol 112 (5) ◽  
pp. 809-821 ◽  
Author(s):  
R N Thrift ◽  
D W Andrews ◽  
P Walter ◽  
A E Johnson
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Secretory Protein ◽  
In Vitro Translation ◽  
Nascent Chain ◽  
Transmembrane Sequence ◽  
Er Membrane

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.

Nucleotide (cDNA) sequence encoding the horse gonadotrophin α-subunit

1987 ◽  
Vol 115 (2) ◽  
pp. 341-346 ◽  
Author(s):  
F. Stewart ◽  
J. A. Thomson ◽  
S. E. A. Leigh ◽  
J. M. Warwick
Keyword(s):  
Amino Acid ◽  
Peptide Sequencing ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Northern Hybridization ◽  
Cdna Clones ◽  
Subunit Gene ◽  
Α Subunit ◽  
Pituitary Glands

ABSTRACT Several cDNA clones corresponding to mRNA for the α-subunit of the horse (Equus caballus) pituitary and placental (chorionic) gonadotrophic hormones have been isolated and sequenced. Polyadenylated mRNA was purified from horse pituitary glands (the source of FSH and LH) and horse placental tissues (the source of chorionic gonadotrophin; CG). The mRNA preparations were characterized by in-vitro translation and Northern hybridization techniques using human and ovine gonadotrophin cDNA clones as probes. Complementary DNA libraries were created from the pituitary and placental mRNAs and a human CG α-subunit probe was used to isolate several horse α-subunit cDNA clones. The α-subunit nucleotide sequence from both sources of tissue was identical, thereby indicating that in the horse (as in man) the same gonadotrophin α-subunit gene is expressed in the pituitary and placenta. Our results are consistent with transcription of a single α-subunit gene for all the glycoprotein hormones in the horse, and we suggest that the reported differences between the horse CG and FSH α-subunit amino acid sequences determined by conventional peptide sequencing methods arose due to errors in the FSH α-subunit sequence. Comparison of the deduced amino acid sequence of the horse α-subunit with that of other α-subunit sequences indicated a number of significant differences which may be related to the unusual receptor-binding properties of the equine gonadotrophins. J. Endocr. (1987) 115, 341–346

Inhibitory Effect of Naked Neural BC1 RNA or BC200 RNA on Eukaryotic in vitro Translation Systems is Reversed by Poly(A)-binding Protein (PABP)

2005 ◽  
Vol 353 (1) ◽  
pp. 88-103 ◽  
Author(s):  
Alexander V. Kondrashov ◽  
Martin Kiefmann ◽  
Klaus Ebnet ◽  
Tasneem Khanam ◽  
Ravi Sondekoppa Muddashetty ◽  
...  
Keyword(s):  
Binding Protein ◽  
Inhibitory Effect ◽  
In Vitro Translation ◽  
Translation Systems
Sign in / Sign up

Export Citation Format

Share Document