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

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.

Download Full-text

Induction of cytochrome P-450 by pregnenolone-16 alpha-carbonitrile in primary monolayer cultures of adult rat hepatocytes and in a cell-free translation system.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)69128-3 ◽

1981 ◽

Vol 256 (12) ◽

pp. 6060-6068

Author(s):

N.A. Elshourbagy ◽

J.L. Barwick ◽

P.S. Guzelian

Keyword(s):

Rat Hepatocytes ◽

Translation System ◽

Adult Rat ◽

Monolayer Cultures ◽

Adult Rat Hepatocytes ◽

Free Translation ◽

A Cell ◽

Primary Monolayer ◽

Cytochrome P 450 ◽

Primary Monolayer Cultures

Download Full-text

The 3' untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3340 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3340-3349 ◽

Cited By ~ 68

Author(s):

X Danthinne ◽

J Seurinck ◽

F Meulewaeter ◽

M Van Montagu ◽

M Cornelissen

Keyword(s):

Tobacco Necrosis Virus ◽

Translation System ◽

Coding Region ◽

Necrosis Virus ◽

Untranslated Sequence ◽

Virus Rna ◽

Free Translation ◽

Order Of Magnitude ◽

A Cell

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.

Download Full-text

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

Biology ◽

10.3390/biology4010161 ◽

2015 ◽

Vol 4 (1) ◽

pp. 161-172 ◽

Cited By ~ 2

Author(s):

Yutaro Tanemura ◽

Yuki Mochizuki ◽

Shigefumi Kumachi ◽

Naoto Nemoto

Keyword(s):

Functional Analysis ◽

Binding Assay ◽

Translation System ◽

Free Translation ◽

A Cell ◽

Rapid Binding

Download Full-text

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.

Download Full-text

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.

Download Full-text

Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors

Biochemical Journal ◽

10.1042/bj3230415 ◽

1997 ◽

Vol 323 (2) ◽

pp. 415-419 ◽

Cited By ~ 94

Author(s):

Lakshmi KASTURI ◽

Hegang CHEN ◽

Susan H. SHAKIN-ESHLEMAN

Keyword(s):

Amino Acid ◽

Amino Acid Residue ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Free Translation ◽

A Cell ◽

And Function ◽

A Site ◽

The Impact

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa = Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa = Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

Download Full-text

Regulation of microRNA biogenesis and function

Thrombosis and Haemostasis ◽

10.1160/th11-12-0836 ◽

2012 ◽

Vol 107 (04) ◽

pp. 605-610 ◽

Cited By ~ 113

Author(s):

Thomas Treiber ◽

Nora Treiber ◽

Gunter Meister

Keyword(s):

Mammalian Cells ◽

Untranslated Region ◽

Protein Complexes ◽

Mature Mirnas ◽

Review Article ◽

Cellular Functions ◽

Target Sites ◽

Cellular Pathways ◽

And Function ◽

Site Binding

SummaryMicroRNAs (miRNAs) are considered as key regulators of literally all cellular pathways. Therefore, miRNA biosynthesis and their individual cellular functions must be tightly regulated as well. MiRNAs are transcribed as primary transcripts, which are processed to mature miRNAs in two consecutive maturation steps. Finally, the mature miRNA is incorporated into a miRNA-protein complex, where it directly interacts with a member of the Argonaute (Ago) protein family. The miRNA guides such protein complexes to partial complementary target sites, which are typically located in the 3’ untranslated region (UTR) of mRNAs leading to inhibition of gene expression. MiRNA activity and abundance is regulated on various levels ranging from transcription and processing to target site binding and miRNA stability. Recent advances in our understanding of how miRNA activity is regulated in mammalian cells are summarised and discussed in this review article.

Download Full-text

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.

Download Full-text