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

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.

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Protein synthesis and loss of viability of rice seeds: Effect of polyamines on in vitro translation

Physiologia Plantarum ◽

10.1111/j.1399-3054.1986.tb03379.x ◽

1986 ◽

Vol 68 (3) ◽

pp. 441-445 ◽

Cited By ~ 6

Author(s):

A. Mukhopadhyay ◽

B. Ghosh

Keyword(s):

Protein Synthesis ◽

In Vitro Translation ◽

Rice Seeds

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Polyribosome fractions and protein synthesis in stem rust infected wheat

Canadian Journal of Botany ◽

10.1139/b86-255 ◽

1986 ◽

Vol 64 (9) ◽

pp. 1916-1927 ◽

Cited By ~ 2

Author(s):

Andrew Greenland ◽

Michael Shaw

Keyword(s):

Protein Synthesis ◽

Stem Rust ◽

Rust Fungus ◽

High Molecular Mass ◽

In Vitro Translation ◽

Ribosomal Subunits ◽

Two Dimensional Electrophoresis ◽

Dimensional Electrophoresis

The effects of infection by stem-rust fungus on polyribosomal RNA fractions and protein synthesis in vitro and in vivo in near-isogenic resistant (Sr6) and susceptible (sr6) lines of wheat were determined. In infected resistant leaves the proportion of ribosomes present as polyribosomes was greater than that in healthy (uninfected) leaves at 1, 3, and 6 days and that in susceptible leaves at 1 and 3 days after inoculation. In the latter there were large increases in the pelletable RNA content (ribosomes, ribosomal subunits, and polyribosomes) and proportion of ribosomes present as polyribosomes from day 6. In vitro translation failed to detect any marked differences in polyribosomal translation products in resistant and susceptible leaves in response to infection. Labelling of polypeptides in vivo and separation by one- and two-dimensional electrophoresis showed that at 1 day after inoculation, two groups of high molecular mass polypeptides (80–96 and 100–110 kDa) were more heavily labelled and two novel polypeptides were present in resistant and susceptible leaves in response to infection. Synthesis of the high molecular weight and two novel polypeptides was maintained in infected resistant leaves up to 6 days after inoculation. In susceptible leaves the amount of radiolabel incorporated into these polypeptides and several proteins prominently labelled in uninfected controls declined rapidly from 3 days after inoculation.

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Purification and in Vitro Reconstitution of the Essential Protein Components of an Aromatic Polyketide Synthase†

Biochemistry ◽

10.1021/bi972919+ ◽

1998 ◽

Vol 37 (8) ◽

pp. 2084-2088 ◽

Cited By ~ 51

Author(s):

Christopher W. Carreras ◽

Chaitan Khosla

Keyword(s):

Polyketide Synthase ◽

Essential Protein ◽

In Vitro Reconstitution ◽

Protein Components

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Comparison of messenger RNA pools in active and dormant Artemia franciscana embryos: evidence for translational control

Journal of Experimental Biology ◽

10.1242/jeb.164.1.103 ◽

1992 ◽

Vol 164 (1) ◽

pp. 103-116 ◽

Cited By ~ 3

Author(s):

G. E. Hofmann ◽

S. C. Hand

Keyword(s):

Protein Synthesis ◽

Translational Control ◽

Messenger Rna ◽

Artemia Franciscana ◽

In Vitro Translation ◽

Translation Techniques ◽

Polysome Profile ◽

Anaerobic Dormancy ◽

Qualitative Changes

In response to environmental anoxia, embryos of the brine shrimp Artemia franciscana enter a dormant state during which energy metabolism and development are arrested. The intracellular acidification that correlates with this transition into anaerobic dormancy has been linked to the inhibition of protein synthesis in quiescent embryos. In this study, we have addressed the level of control at which a mechanism mediated by intracellular pH might operate to arrest protein synthesis. Two independent lines of evidence suggest that there is an element of translational control when protein synthesis is arrested in dormant embryos. First, as determined by in vitro translation techniques, there were no significant quantitative differences in mRNA pools in dormant as compared to actively developing embryos. In addition, fluorography of the translation products showed that there are no large qualitative changes in mRNA species when embryos become dormant. These data suggest that there was no net degradation of mRNA pools in dormant embryos and that protein synthesis may therefore be controlled more strongly at translation than at transcription. Second, polysome profile studies showed that dormant embryos possess reduced levels of polysomes relative to those found in cells or active embryos. The disaggregation of polysomes is an indication that the initiation step in protein synthesis is disrupted and is further evidence that the mechanism involved in protein synthesis arrest in dormant Artemia involves translational control.

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Translating Ribosomes Inhibit Poliovirus Negative-Strand RNA Synthesis

Journal of Virology ◽

10.1128/jvi.73.12.10104-10112.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 10104-10112 ◽

Cited By ~ 98

Author(s):

David J. Barton ◽

B. Joan Morasco ◽

James B. Flanegan

Keyword(s):

Protein Synthesis ◽

Polypeptide Chain ◽

Rna Replication ◽

Viral Rna ◽

In Vitro Translation ◽

Chain Elongation ◽

Protein Synthesis Inhibitors ◽

Positive Strand ◽

Negative Strand

ABSTRACT Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation “freeze” ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated with the natural clearance of translating ribosomes to avoid the dilemma of ribosome-polymerase collisions.

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