Easy and Rapid Binding Assay for Functional Analysis of Disulfide-Containing Peptides by a Pull-Down Method Using a Puromycin-Linker and a Cell-Free Translation System

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.

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A murine fer testis-specific transcript (ferT) encodes a truncated Fer protein.

Molecular and Cellular Biology ◽

10.1128/mcb.10.1.146 ◽

1990 ◽

Vol 10 (1) ◽

pp. 146-153 ◽

Cited By ~ 56

Author(s):

K Fischman ◽

J C Edman ◽

G M Shackleford ◽

J A Turner ◽

W J Rutter ◽

...

Keyword(s):

Tyrosine Kinase ◽

Translation System ◽

Appearance Time ◽

Acid Protein ◽

Mouse Tissues ◽

Free Translation ◽

Alternatively Spliced ◽

A Cell ◽

Testis Cdna ◽

Carboxy Terminal

A cDNA for a potential tyrosine kinase-encoding mRNA was isolated from a mouse testis cDNA library. In a survey of eight mouse tissues, a transcript of 2.4 kilobases restricted to testis tissue was found. The mRNA encodes a 453-amino-acid protein of 51,383 daltons, the smallest tyrosine kinase protein ever described. RNA synthesized from the cDNA template directs the synthesis of a 51,000-Mr protein in a cell-free translation system. The carboxy-terminal 409 amino acids are 98 and 90% identical to the carboxy halves of the rat and human Fer proteins, respectively. This suggests that the cDNA represents an alternatively spliced testis-specific fer mRNA and is therefore termed by us ferT. On the basis of the appearance time of the fer mRNA in the testis of maturing neonatal mice, we speculate on the role played by this protein in the development of this organ.

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Chemical aminoacylation of tRNAs with fluorinated amino acids for in vitro protein mutagenesis

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.6.40 ◽

2010 ◽

Vol 6 ◽

Cited By ~ 8

Author(s):

Shijie Ye ◽

Allison Ann Berger ◽

Dominique Petzold ◽

Oliver Reimann ◽

Benjamin Matt ◽

...

Keyword(s):

Amino Acids ◽

Translation System ◽

Biologically Relevant ◽

Fluorinated Amino Acids ◽

Free Translation ◽

Trna Species ◽

A Cell ◽

Protein Environment ◽

Vitro Protein

This article describes the chemical aminoacylation of the yeast phenylalanine suppressor tRNA with a series of amino acids bearing fluorinated side chains via the hybrid dinucleotide pdCpA and ligation to the corresponding truncated tRNA species. Aminoacyl-tRNAs can be used to synthesize biologically relevant proteins which contain fluorinated amino acids at specific sites by means of a cell-free translation system. Such engineered proteins are expected to contribute to our understanding of discrete fluorines’ interaction with canonical amino acids in a native protein environment and to enable the design of fluorinated proteins with arbitrary desired properties.

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Studies of the Biosynthesis of Renin with a Cell-Free Translation System

Clinical Science ◽

10.1042/cs061241s ◽

1981 ◽

Vol 61 (s7) ◽

pp. 241s-243s ◽

Cited By ~ 3

Author(s):

V. J. Dzau ◽

A. Ouellette ◽

R. Pratt

Keyword(s):

Molecular Weight ◽

Polyacrylamide Gel Electrophoresis ◽

Submaxillary Gland ◽

Translation System ◽

Mouse Submaxillary Gland ◽

Free Translation ◽

Renin Mrna ◽

A Cell ◽

Deficient Mice ◽

Identical Fashion

1. Poly(A)+ mRNA from mouse submaxillary gland encodes a polypeptide of molecular weight 48 000 (48K polypeptide) which is abundant in the male. 2. This polypeptide is selectively absent in the translation products of mRNA from a strain of genetically renin-deficient mice C57 BL/10J. 3. The 48K polypeptide binds and co-elutes in identical fashion with pure authentic renin on pepstatin affinity chromatography. 4. Immunoprecipitation of translation products of male glandular mRNA with renin-specific antibody yielded this 48K band upon analysis by SDS/polyacrylamide gel electrophoresis and fluorography. Pure renin of molecular weight 37 000 blocked the binding of this polypeptide to antirenin antibody. 5. Mouse submaxillary gland synthesizes a renin precursor. The renin mRNA is androgenically regulated.

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Reconstitution of functional mRNA-protein complexes in a rabbit reticulocyte cell-free translation system.

Molecular and Cellular Biology ◽

10.1128/mcb.5.2.342 ◽

1985 ◽

Vol 5 (2) ◽

pp. 342-351 ◽

Cited By ~ 28

Author(s):

J R Greenberg ◽

E Carroll

Keyword(s):

Mammalian Cells ◽

Uv Light ◽

Protein Complexes ◽

Micrococcal Nuclease ◽

Translation System ◽

Cytoplasmic Process ◽

Free Translation ◽

Associated Proteins ◽

A Cell ◽

And Function

A variety of evidence suggests that the cytoplasmic mRNA-associated proteins of eucaryotic cells are derived from the cytoplasm and function there, most likely in protein synthesis or some related process. Furthermore, the evidence suggests that protein-free mRNA added to a cell-free translation system should become associated with a set of proteins similar to those associated with mRNA in native polyribosomes. To test this hypothesis, we added deproteinized rabbit reticulocyte mRNA to a homologous cell-free translation system made dependent on exogenous mRNA by treatment with micrococcal nuclease. The resulting reconstituted complexes were irradiated with UV light to cross-link the proteins to mRNA, and the proteins were analyzed by gel electrophoresis. The proteins associated with polyribosomal mRNA in the reconstituted complexes were indistinguishable from those associated with polyribosomal mRNA in intact reticulocytes. Furthermore, reticulocyte mRNA-associated proteins were very similar to those of cultured mammalian cells. The composition of the complexes varied with the translational state of the mRNA; that is, certain proteins present in polyribosomal mRNA-protein complexes were absent or reduced in amount in 40S to 80S complexes and in complexes formed in the absence of translation. However, other proteins, including a 78-kilodalton protein associated with polyadenylate, were present irrespective of translational state, or else they were preferentially associated with untranslated mRNA. These findings are in agreement with previous data suggesting that proteins associated with cytoplasmic mRNA are derived from the cytoplasm and that they function in translation or some other cytoplasmic process, rather than transcription, RNA processing, or transport from the nucleus to the cytoplasm.

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Synthesis of rat muscle carbonic anhydrase III in a cell-free translation system

FEBS Letters ◽

10.1016/0014-5793(82)81256-8 ◽

1982 ◽

Vol 148 (1) ◽

pp. 122-126 ◽

Cited By ~ 2

Author(s):

Alan Shiels ◽

Ian Phillips ◽

Stephen Jeffrey ◽

Elizabeth Shephard ◽

Nicholas Carter

Keyword(s):

Carbonic Anhydrase ◽

Translation System ◽

Rat Muscle ◽

Carbonic Anhydrase Iii ◽

Free Translation ◽

A Cell

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A New Internal Ribosomal Entry Site 5′ Boundary Is Required for Poliovirus Translation Initiation in a Mouse System

Journal of Virology ◽

10.1128/jvi.72.3.2398-2405.1998 ◽

1998 ◽

Vol 72 (3) ◽

pp. 2398-2405 ◽

Cited By ~ 11

Author(s):

Toshihiko Ishii ◽

Kazuko Shiroki ◽

Duck-Hee Hong ◽

Takahiro Aoki ◽

Yoshihiro Ohta ◽

...

Keyword(s):

Internal Ribosomal Entry Site ◽

Translation System ◽

Entry Site ◽

Stem Loop ◽

Free Translation ◽

A Cell ◽

Mouse Cells ◽

Ribosomal Entry ◽

Reduced Activity ◽

Mouse System

ABSTRACT Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1–8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.

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Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination

10.1101/2020.11.11.377739 ◽

2020 ◽

Author(s):

Alexey Shuvalov ◽

Ekaterina Shuvalova ◽

Nikita Biziaev ◽

Elizaveta Sokolova ◽

Konstantin Evmenov ◽

...

Keyword(s):

Stop Codon ◽

Translation Termination ◽

Translation System ◽

Release Factor ◽

Viral Mrnas ◽

Codon Recognition ◽

Free Translation ◽

Stop Codon Recognition ◽

A Cell

ABSTRACTThe Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.

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