scholarly journals Assembly of the ribonucleoprotein complex containing the mRNA of the β-subunit of the mitochondrial H+-ATP synthase requires the participation of two distal cis-acting elements and a complex set of cellular trans-acting proteins

2002 ◽  
Vol 365 (2) ◽  
pp. 417-428 ◽  
Author(s):  
Javier RICART ◽  
José M. IZQUIERDO ◽  
Carlo M. Di LIEGRO ◽  
José M. CUEZVA
Keyword(s):  
Atp Synthase ◽  
Sucrose Concentration ◽  
Β Subunit ◽  
Reading Frame ◽  
Ribonucleoprotein Complex ◽  
Cis Acting ◽  
Translational Machinery ◽  
F1 Atpase ◽  
Reticulocyte Lysate

The mRNA encoding the β-subunit of the mitochondrial H+-ATP synthase (β-F1-ATPase) is localized in an approx. 150nm structure of the hepatocyte of mammals. In the present study, we have investigated the cis- and trans-acting factors involved in the generation of the ribonucleoprotein complex containing β-F1-ATPase mRNA. Two cis-acting elements (β1.2 and 3′β) have been identified. The β1.2 element is placed in the open reading frame, downstream of the region encoding the mitochondrial pre-sequence of the protein. The 3′β element is the 3′ non-translated region of the mRNA. Complex sets of proteins from the soluble and non-soluble fractions of the liver interact with the β1.2 and 3′β elements. A soluble p88, present also in reticulocyte lysate, displays binding specificity for both the cis-acting elements. Sedimentation and high-resolution in situ hybridization experiments showed that the structure containing the rat liver β-F1-ATPase mRNA is found in fractions of high sucrose concentration, where large polysomes sediment. Treatment of liver extracts with EDTA promoted the mobilization of β-F1-ATPase mRNA to fractions of lower sucrose concentration, suggesting that the structure containing β-F1-ATPase mRNA is a large polysome. Finally, in vitro reconstitution experiments with reticulocyte lysate, using either the full-length, mutant or chimaeric versions of β-F1-ATPase mRNA, reveal that the assembly of the β-F1-ATPase mRNA polysome requires the co-operation of both the cis-acting mRNA determinants. The present study illustrates the existence of an intramolecular RNA cross-talking required for the association of the mRNA with the translational machinery.

cis-acting translational effects of the 5' noncoding region of c-myc mRNA

1988 ◽  
Vol 8 (7) ◽  
pp. 2875-2883
Author(s):  
N Parkin ◽  
A Darveau ◽  
R Nicholson ◽  
N Sonenberg
Keyword(s):  
Inhibitory Effect ◽  
Noncoding Region ◽  
Translational Efficiency ◽  
Cis Acting ◽  
Cell Extracts ◽  
Negative Effect ◽  
Reticulocyte Lysate ◽  
Established Cell

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.

scholarly journals Unusual ciliate-specific codons in Tetrahymena mRNAs are translated correctly in a rabbit reticulocyte lysate supplemented with a subcellular fraction from Tetrahymena

1987 ◽  
Vol 244 (2) ◽  
pp. 331-335 ◽  
Author(s):  
P H Andreasen ◽  
H Dreisig ◽  
K Kristiansen
Keyword(s):  
Codon Usage ◽  
Genetic Code ◽  
Subcellular Fraction ◽  
Tetrahymena Thermophila ◽  
Rabbit Reticulocyte Lysate ◽  
Reading Frame ◽  
Universal Genetic Code ◽  
Reticulocyte Lysate ◽  
Translational Termination

The codon usage of Tetrahymena thermophila and other ciliates deviates from the ‘universal genetic code’ in that UAA and probably UAG are not translational termination signals but code for glutamine. Therefore, translation in vitro of mRNA from Tetrahymena in a reticulocyte lysate is prematurely terminated if a UAA or UAG triplet is present in the reading frame of the mRNA. We show that the addition of a subcellular fraction from Tetrahymena thermophila enables a rabbit reticulocyte lysate to translate Tetrahymena mRNAs into full-sized proteins. The activity of the subcellular fraction is shown to depend on the combined function of a protein component(s) and a tRNA(s). The subcellular fraction is easily prepared and its usefulness for the identification of isolated mRNAs from Tetrahymena by their translation products in vitro is demonstrated.

scholarly journals A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth.

1988 ◽  
Vol 8 (8) ◽  
pp. 3094-3103 ◽  
Author(s):  
C K Shih ◽  
R Wagner ◽  
S Feinstein ◽  
C Kanik-Ennulat ◽  
N Neff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Atp Synthase ◽  
Resistance Mutation ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Reading Frame ◽  
F0f1 Atp Synthase ◽  
General Inhibitor

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.

scholarly journals Subcellular structure containing mRNA for β subunit of mitochondrial H+-ATP synthase in rat hepatocytes is translationally active

1997 ◽  
Vol 324 (2) ◽  
pp. 635-643 ◽  
Author(s):  
Javier RICART ◽  
Gustavo EGEA ◽  
José M. IZQUIERDO ◽  
SAN MARTÍN Carmen ◽  
José M. CUEZVA
Keyword(s):  
Atp Synthase ◽  
Rat Hepatocytes ◽  
Translational Efficiency ◽  
Β Subunit ◽  
F1 Atpase ◽  
Close Proximity ◽  
Show Evidence ◽  
Positive Hybridization ◽  
Cytoplasmic Location

We have recently reported that the nuclear-encoded mRNA for the β subunit of mitochondrial H+-ATP synthase (β-mRNA) is localized in rounded, electron-dense clusters in the cytoplasm of rat hepatocytes. Clusters of β-mRNA are often found in close proximity to mitochondria. These findings suggested a role for these structures in controlling the cytoplasmic expression and sorting of the encoded mitochondrial precursor. Here we have addressed the question of whether the structures containing β-mRNA are translationally active. For this purpose a combination of high-resolution in situ hybridization and immunocytochemical procedures was used. Three different co-localization criteria showed that β-mRNA-containing structures always revealed positive immunoreactive signals for mitochondrial H+-ATP synthase (F1-ATPase), ribosomal and hsc70 proteins. Furthermore, clusters show evidence in situ of developmental changes in the translational efficiency of the β-mRNA. These findings suggest that structures containing β-mRNA are translationally active irrespective of their cytoplasmic location. The immunocytochemical quantification of the cytoplasmic presentation of hsc70 in the hepatocyte reveals that approx. 86% of the protein has a dispersed distribution pattern. However, the remaining hsc70 is presented in clusters of which only half reveal positive hybridization for β-mRNA. The interaction of hsc70 with the β-F1-ATPase precursor protein is documented by the co-localization of F1-ATPase immunoreactive material within cytoplasmic clusters of hsc70 and by the co-immunoprecipitation of hsc70 with the β-subunit precursor from liver post-mitochondrial supernatants. Taken together, these results suggest a role for hsc70 in the translation/sorting pathway of the mammalian precursor of the β-F1-ATPase protein.

scholarly journals cis-acting translational effects of the 5' noncoding region of c-myc mRNA.

1988 ◽  
Vol 8 (7) ◽  
pp. 2875-2883 ◽  
Author(s):  
N Parkin ◽  
A Darveau ◽  
R Nicholson ◽  
N Sonenberg
Keyword(s):  
Inhibitory Effect ◽  
Noncoding Region ◽  
Translational Efficiency ◽  
Cis Acting ◽  
Cell Extracts ◽  
Negative Effect ◽  
Reticulocyte Lysate ◽  
Established Cell

We have previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte lysate (A. Darveau, J. Pelletier, and N. Sonenberg, Proc. Natl. Acad. Sci. USA 82:2315-2319, 1985). We wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. Here we report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, we report that the cis-inhibitory effect is also exhibited in microinjected Xenopus oocytes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.

A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth

1988 ◽  
Vol 8 (8) ◽  
pp. 3094-3103 ◽  
Author(s):  
C K Shih ◽  
R Wagner ◽  
S Feinstein ◽  
C Kanik-Ennulat ◽  
N Neff
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Atp Synthase ◽  
Resistance Mutation ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Reading Frame ◽  
F0f1 Atp Synthase ◽  
General Inhibitor

The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.

scholarly journals The Rubella Virus Nonstructural Protease Requires Divalent Cations for Activity and Functions in trans

1998 ◽  
Vol 72 (5) ◽  
pp. 4463-4466 ◽  
Author(s):  
Xin Liu ◽  
Susan L. Ropp ◽  
Richard J. Jackson ◽  
Teryl K. Frey
Keyword(s):  
Rubella Virus ◽  
Divalent Cations ◽  
Three Dimensional ◽  
Catalytic Site ◽  
Binding Motif ◽  
Reading Frame ◽  
Rabbit Reticulocyte Lysate System ◽  
Reticulocyte Lysate

ABSTRACT The rubella virus (RUB) nonstructural (NS) protease is a papain-like cysteine protease (PCP) located in the NS-protein open reading frame (NSP-ORF) that cleaves the NSP-ORF translation product at a single site to produce two products, P150 (the N-terminal product) and P90 (the C-terminal product). The RUB NS protease was found not to function following translation in vitro in a standard rabbit reticulocyte lysate system, although all of the other viral PCPs do so. However, in the presence of divalent cations such as Zn2+, Cd2+, and Co2+, the RUB NS protease functioned efficiently, indicating that these cations are required either as direct cofactors in catalytic activity or for correct acquisition of three-dimensional conformation of the protease. Since other viral and cell PCPs do not require cations for activity and the RUB NS protease contains a putative zinc binding motif, the latter possibility is more likely. Previous in vivo expression studies of the RUB NS protease failed to demonstrate trans cleavage activity (J.-P. Chen et al., J. Virol. 70:4707–4713, 1996). To study whethertrans cleavage could be detected in vitro, a protease catalytic site mutant and a mutant in which the C-terminal 31 amino acids of P90 were deleted were independently introduced into plasmid constructs that express the complete NSP-ORF. Cotranslation of these mutants in vitro yielded both the native and the mutated forms of P90, indicating that the protease present in the mutated construct cleaved the catalytic-site mutant precursor. Thus, RUB NS protease can function in trans.

ATP Synthase from Bovine Heart Mitochondria In Vitro Assembly of a Stalk Complex in the Presence of F1-ATPase and in its Absence

1994 ◽  
Vol 242 (4) ◽  
pp. 408-421 ◽  
Author(s):  
I COLLINSON ◽  
M VANRAAIJ ◽  
M RUNSWICK ◽  
I FEARNLEY ◽  
J SKEHEL ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Heart Mitochondria ◽  
F1 Atpase ◽  
Bovine Heart ◽  
In Vitro Assembly

scholarly journals The Mitochondrial Genome Integrity Gene, MGI1, of Kluyveromyces lactis Encodes the β-Subunit of F1-ATPase

Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1445-1454 ◽  
Author(s):  
Xin Jie Chen ◽  
G Desmond Clark-Walker
Keyword(s):  
Mitochondrial Genome ◽  
Kluyveromyces Lactis ◽  
Three Dimensional ◽  
Genome Integrity ◽  
Dimensional Structure ◽  
Deletion Mutants ◽  
Β Subunit ◽  
Gain Of Function ◽  
F1 Atpase ◽  
Γ Subunit

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the β-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the “gain-of-function” phenotype found in mgi1 point mutants. The location of Arg435 in the β-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the β- and α- (MGI2) subunits with the γ-subunit (MGI5) is likely to be affected by the mutations.

Sign in / Sign up

Export Citation Format

Share Document