scholarly journals Reconstitution of Prenyltransferase Activity on Nanodiscs by Components of the Rubber Synthesis Machinery of the Para Rubber Tree and Guayule

2021 ◽  
Author(s):  
Fu Kuroiwa ◽  
Akira Nishino ◽  
Yasuko Mandal ◽  
Masataka Honzawa ◽  
Miki Suenaga-Hiromori ◽  
...  
Keyword(s):  
Lipid Membrane ◽  
Rubber Tree ◽  
Translation System ◽  
Structure Modeling ◽  
Parthenium Argentatum ◽  
Free Translation ◽  
A Cell ◽  
Synthesis Activity ◽  
Biosynthetic Machinery ◽  
Protein Reconstitution

Abstract Natural rubber of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity. The proteins HRT1, HRT2, and HRBP have been identified as candidate components of the rubber biosynthetic machinery. To clarify the contribution of these proteins to prenyltransferase activity, we established a cell-free translation system for nanodisc-based protein reconstitution and measured the enzyme activity of the protein-nanodisc complexes. Co-expression of HRT1 and HRBP in the presence of nanodiscs yielded marked polyisoprene synthesis activity. By contrast, neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP manifested such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (PaCPT1–3) manifested prenyltransferase activity only if co-expressed with PaCBP, the homolog of HRBP. Our results thus indicate that two heterologous subunits form the core prenyltransferase of the rubber biosynthetic machinery. A recently developed structure modeling program predicted the structure of such heterodimer complexes including HRT1/HRBP and PaCPT2/PaCBP. HRT and PaCPT proteins were also found to possess affinity for a lipid membrane in the absence of HRBP or PaCBP, and structure modeling implicated an amphipathic α-helical domain of HRT1 and PaCPT2 in membrane binding of these proteins.

scholarly journals Reconstitution of prenyltransferase activity on nanodiscs by components of the rubber synthesis machinery of the Para rubber tree and guayule

2021 ◽  
Author(s):  
Fu Kuroiwa ◽  
Akira Nishino ◽  
Yasuko Mandal ◽  
Miki Suenaga-Hiromori ◽  
Kakeru Suzuki ◽  
...  
Keyword(s):  
Rubber Tree ◽  
Translation System ◽  
Parthenium Argentatum ◽  
The Core ◽  
Free Translation ◽  
A Cell ◽  
Synthesis Activity ◽  
Living Organisms ◽  
Biosynthetic Machinery ◽  
Protein Reconstitution

AbstractPrenyltransferases mediate the biosynthesis of various types of polyisoprene compound in living organisms. Natural rubber (NR) of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity, with the proteins HRT1, HRT2, and HRBP having been identified as candidate components of the rubber biosynthetic machinery. To clarify the contribution of these proteins to prenyltransferase activity, we established a cell-free translation system for nanodisc-based protein reconstitution and measured the enzyme activity of the protein-nanodisc complexes. Cell-free synthesis of HRT1, HRT2, and HRBP in the presence of asolectin nanodiscs revealed that all three proteins were membrane associated. A complex of HRT1 and HRBP formed as a result of co-expression of the two proteins in the presence of nanodiscs manifested marked polyisoprene synthesis activity, whereas neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP exhibited such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (CPT1–3) manifested prenyltransferease activity only if co-expressed with the homolog of HRBP (CBP). Our results thus indicate that the core prenyltransferase of the rubber biosynthetic machinery of both the Para rubber tree and guayule is formed by the assembly of heterologous subunits (HRT1 and HRBP in the former species).

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

1993 ◽  
Vol 13 (6) ◽  
pp. 3340-3349 ◽  
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.

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

1990 ◽  
Vol 10 (1) ◽  
pp. 146-153 ◽  
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.

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

2010 ◽  
Vol 6 ◽  
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.

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

1981 ◽  
Vol 61 (s7) ◽  
pp. 241s-243s ◽  
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.

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

1985 ◽  
Vol 5 (2) ◽  
pp. 342-351 ◽  
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.

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

FEBS Letters ◽  
1982 ◽  
Vol 148 (1) ◽  
pp. 122-126 ◽  
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

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

1998 ◽  
Vol 72 (3) ◽  
pp. 2398-2405 ◽  
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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