A genetic link between an mRNA-specific translational activator and the translation system in yeast mitochondria.

Genetics ◽  
1990 ◽  
Vol 125 (3) ◽  
pp. 495-503 ◽  
Author(s):  
P Haffter ◽  
T W McMullin ◽  
T D Fox
Keyword(s):  
Mitochondrial Gene ◽  
Mitochondrial Protein ◽  
Open Reading Frame ◽  
Null Alleles ◽  
Translation System ◽  
Amino Acid Residues ◽  
Mitochondrial Translation ◽  
Reading Frame ◽  
Large Deletions ◽  
Translational Activator

Abstract Translation of the Saccharomyces cerevisiae mitochondrial mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the products of at least three nuclear genes, PET122, PET494 and PET54. pet122 mutations that remove 24-67 amino acid residues from the carboxyterminus of the gene product were found to be suppressed by unlinked nuclear mutations. These unlinked suppressors fail to suppress both a pet122 missense mutation and a complete pet122 deletion. One of the suppressor mutations causes a heat-sensitive nonrespiratory growth phenotype in an otherwise wild-type strain and reduces translation of all mitochondrial gene products in cells grown at high temperature. This suppressor maps to a newly identified gene on chromosome XV termed PET123. The sequence of a DNA fragment carrying PET123 contains one major open reading frame encoding a basic protein of 318 amino acids. Inactivation of the chromosomal copy of PET123 by interruption of this open reading frame causes cells to become rho- (sustain large deletions in their mtDNA). This phenotype is characteristic for null alleles of genes whose products are essential for general mitochondrial protein synthesis. Thus our data strongly suggest that the PET123 protein is a component of the mitochondrial translation apparatus that interacts directly with the coxIII-mRNA-specific translational activator PET122.

scholarly journals Mutation avoidance and DNA repair proficiency in Ustilago maydis are differentially lost with progressive truncation of the REC1 gene product.

1995 ◽  
Vol 15 (10) ◽  
pp. 5329-5338 ◽  
Author(s):  
K Onel ◽  
M P Thelen ◽  
D O Ferguson ◽  
R L Bennett ◽  
W K Holloman
Keyword(s):  
Amino Acid ◽  
Mutation Rate ◽  
Spontaneous Mutation ◽  
Ustilago Maydis ◽  
Radiation Sensitivity ◽  
Open Reading Frame ◽  
Exonuclease Activity ◽  
Amino Acid Residues ◽  
Spontaneous Mutation Rate ◽  
Reading Frame

The REC1 gene of Ustilago maydis has an uninterrupted open reading frame, predicted from the genomic sequence to encode a protein of 522 amino acid residues. Nevertheless, an intron is present, and functional activity of the gene in mitotic cells requires an RNA processing event to remove the intron. This results in a change in reading frame and production of a protein of 463 amino acid residues. The 3'-->5' exonuclease activity of proteins derived from the REC1 genomic open reading frame, the intronless open reading frame, and several mutants was investigated. The mutants included a series of deletions constructed by removing restriction fragments at the 3' end of the cloned REC1 gene and a set of mutant alleles previously isolated in screens for radiation sensitivity. All of these proteins were overproduced in Escherichia coli as N-terminal polyhistidine-tagged fusions that were subsequently purified by immobilized metal affinity chromatography and assayed for 3'-->5' exonuclease activity. The results indicated that elimination of the C-terminal third of the protein did not result in a serious reduction in 3'-->5' exonuclease activity, but deletion into the midsection caused a severe loss of activity. The biological activity of the rec1-1 allele, which encodes a truncated polypeptide with full 3'-->5' exonuclease activity, and the rec1-5 allele, which encodes a more severely truncated polypeptide with no exonuclease activity, was investigated. The two mutants were equally sensitive to the lethal effect of UV light, but the spontaneous mutation rate was elevated 10-fold over the wild-type rate in the rec1-1 mutant and 100-fold in the rec1-5 mutant. The elevated spontaneous mutation rate correlated with the ablation of exonuclease activity, but the radiation sensitivity did not. These results indicate that the C-terminal portion of the Rec1 protein is not essential for exonuclease activity but is crucial in the role of REC1 in DNA damage repair.

scholarly journals Mitochondrial OXPHOS Biogenesis: Co-Regulation of Protein Synthesis, Import, and Assembly Pathways

2020 ◽  
Vol 21 (11) ◽  
pp. 3820 ◽  
Author(s):  
Jia Xin Tang ◽  
Kyle Thompson ◽  
Robert W. Taylor ◽  
Monika Oláhová
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Protein Import ◽  
Acyl Carrier Protein ◽  
Early Stage ◽  
Mitochondrial Gene ◽  
Mitochondrial Protein ◽  
Fine Tuning ◽  
Mitochondrial Translation ◽  
Oxphos Complex

The assembly of mitochondrial oxidative phosphorylation (OXPHOS) complexes is an intricate process, which—given their dual-genetic control—requires tight co-regulation of two evolutionarily distinct gene expression machineries. Moreover, fine-tuning protein synthesis to the nascent assembly of OXPHOS complexes requires regulatory mechanisms such as translational plasticity and translational activators that can coordinate mitochondrial translation with the import of nuclear-encoded mitochondrial proteins. The intricacy of OXPHOS complex biogenesis is further evidenced by the requirement of many tightly orchestrated steps and ancillary factors. Early-stage ancillary chaperones have essential roles in coordinating OXPHOS assembly, whilst late-stage assembly factors—also known as the LYRM (leucine–tyrosine–arginine motif) proteins—together with the mitochondrial acyl carrier protein (ACP)—regulate the incorporation and activation of late-incorporating OXPHOS subunits and/or co-factors. In this review, we describe recent discoveries providing insights into the mechanisms required for optimal OXPHOS biogenesis, including the coordination of mitochondrial gene expression with the availability of nuclear-encoded factors entering via mitochondrial protein import systems.

Molecular cloning, characterization, and distribution of the gerbil angiotensin II AT2 receptor

2003 ◽  
Vol 285 (6) ◽  
pp. R1373-R1383 ◽  
Author(s):  
Kwang-Lae Hoe ◽  
Ines Armando ◽  
Gustavo Baiardi ◽  
Taduru Sreenath ◽  
Ashok Kulkarni ◽  
...  
Keyword(s):  
Open Reading Frame ◽  
Late Gestation ◽  
At2 Receptor ◽  
Ang Ii ◽  
Amino Acid Residues ◽  
Receptor Ligands ◽  
Reading Frame ◽  
Receptor Mrna ◽  
Dental Papilla

We isolated a cDNA clone encoding the gerbil AT2 receptor (gAT2) gene from a gerbil adrenal gland cDNA library. The full-length cDNA contains a 1,089-bp open reading frame encoding 363 amino acid residues with 90.9, 96.1, and 95.6% identity with the human (hAT2), rat (rAT2), and mouse AT2 (mAT2) receptors, respectively. There are at least seven nonconserved amino acids in the NH2-terminal domain and in positions Val196, Val217, and Met293, important for angiotensin (ANG) II but not for CGP-42112 binding. Displacement studies in adrenal sections revealed that affinity of the gAT2 receptor was 10-20 times lower for ANG II, ANG III, and PD-123319 than was affinity of the rAT2 receptor. The affinity of each receptor remained the same for CGP-42112. When transfected into COS-7 cells, the gAT2 receptor shows affinity for ANG II that is three times lower than that shown by the hAT2 receptor, whereas affinities for ANG III and the AT2 receptor ligands CGP-42112 and PD-123319 were similar. Autoradiography in sections of the gerbil head showed higher binding in muscles, retina, skin, and molars at embryonic day 19 than at 1 wk of age. In situ hybridization and emulsion autoradiography revealed that at embryonic day 19 the gAT2 receptor mRNA was highly localized to the base of the dental papilla of maxillary and mandibular molars. Our results suggest selective growth-related functions in late gestation and early postnatal periods for the gAT2 receptor and provide an essential basis for future mutagenesis studies to further define structural requirements for agonist binding.

Molecular cloning of an S-adenosylmethionine synthase gene from pigeon pea (Cajanus cajan) and its expression analysis under arbuscularmycorrhizae fungi colonization and drought stress

2017 ◽  
Author(s):  
Guang Qiao ◽  
Bingxue Zhang ◽  
Xiaopeng Wen
Keyword(s):  
Drought Stress ◽  
Expression Analysis ◽  
Cajanus Cajan ◽  
Gene Expression Analysis ◽  
Pigeon Pea ◽  
Open Reading Frame ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Am Symbiosis ◽  
Cloned Cdna

An S-adenosylmethionine synthase (SAMS) gene associated with the drought responsiveness was isolated and characterized from pigeon pea. It was designated CcSAMS and contained an open reading frame of 1,182 bp, which encoded 394 amino acid residues. Sequence analysis of the cloned cDNA showed 94% identity with SAMS from other plant species, suggesting that this gene was considerably conserved in plants. Gene expression analysis demonstrated that CcSAMS was highly expressed in the leaves of AM-colonized plants, irrespective of exposure to either drought or drought-free. Rather, the expression levels of AM plants were significantly higher than that of NAM plants as subjected to drought stress. Therefore, AM symbiosis might enhance the expression of CcSAMS, and the elevated tolerance of AM- colonized pigeon pea to drought-stress was at least partially ascribed to the overexpression of SAMS gene.

scholarly journals A novel small-subunit ribosomal protein of yeast mitochondria that interacts functionally with an mRNA-specific translational activator.

1990 ◽  
Vol 10 (9) ◽  
pp. 4590-4595 ◽  
Author(s):  
T W McMullin ◽  
P Haffter ◽  
T D Fox
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Mitochondrial Gene ◽  
Ribosomal Subunit ◽  
Nuclear Gene ◽  
Small Subunit ◽  
Rrna Gene ◽  
Mitochondrial Translation ◽  
Activator Proteins ◽  
Translational Activator

Mitochondrial translation of the mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the action of three position activator proteins encoded in the nucleus of Saccharomyces cerevisiae. Some mutations affecting one of these activators, PET122, can be suppressed by mutations in an unlinked nuclear gene termed PET123. PET123 function was previously demonstrated to be required for translation of all mitochondrial gene products. We have now generated an antibody against the PET123 protein and have used it to demonstrate that PET123 is a mitochondrial ribosomal protein of the small subunit. PET123 appears to be present at levels comparable to those of other mitochondrial ribosomal proteins, and its accumulation is dependent on the presence of the 15S rRNA gene in mitochondria. Taken together with the previous genetic data, these results strongly support a model in which the mRNA-specific translational activator PET122 works by directly interacting with the small ribosomal subunit to promote translation initiation on the coxIII mRNA.

scholarly journals Isolation, characterization and sequence analysis of a full-length cDNA clone encoding acetohydroxy acid reductoisomerase from spinach chloroplasts

1991 ◽  
Vol 277 (2) ◽  
pp. 469-475 ◽  
Author(s):  
R Dumas ◽  
M Lebrun ◽  
R Douce
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Single Gene ◽  
Amino Acid Sequences ◽  
Full Length ◽  
Open Reading Frame ◽  
Amino Acid Residues ◽  
Protein Precursor ◽  
Reading Frame ◽  
Full Length Cdna

Acetohydroxy acid reductoisomerase (AHRI), the second enzyme in the parallel isoleucine/valine-biosynthetic pathway, catalyses an unusual two-step reaction in which the substrate, either 2-acetolactate or 2-aceto-2-hydroxybutyrate, is converted via an alkyl migration and an NADPH-dependent reduction to give 2,3-dihydroxy-3-methylbutyrate or 2,3-dihydroxy-3-methylvalerate respectively. We have isolated and characterized a full-length cDNA from a lambda gt11 spinach library encoding the complete acetohydroxy acid reductoisomerase protein precursor. The 2050-nucleotide sequence contains a 1785-nucleotide open reading frame. The derived amino acid sequence indicates that the protein precursor consists of 595 amino acid residues including a presequence peptide of 72 amino acid residues. The N-terminal sequence of the first 16 amino acid residues of the purified AHRI confirms the identity of the cDNA. The derived amino acid sequence from this open reading frame shows 23% identity with the deduced amino acid sequences of the Escherichia coli and Saccharomyces cerevisiae AHRI proteins. There are two blocks of conserved amino acid residues in these three proteins. One of these is a sequence similar to the ‘fingerprint’ region of the NAD(P)H-binding site found in a large number of NAD(P)H-dependent oxidoreductases. The other, a short sequence (Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Ser-His-Gly-Phe) containing the amino acids lysine and histidine, could well be the catalytic site of the first step of the AHRI reaction. Southern-blot analysis indicated that AHRI is encoded by a single gene per haploid genome of about 7.5 kbp containing at least four introns.

scholarly journals Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5' leader.

1987 ◽  
Vol 7 (8) ◽  
pp. 2728-2734 ◽  
Author(s):  
C A Strick ◽  
T D Fox
Keyword(s):  
Amino Acids ◽  
Regulatory Gene ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Amino Terminal ◽  
Interesting Pattern ◽  
Reading Frames

The yeast nuclear gene PET111 is required specifically for translation of the mitochondrion-coded mRNA for cytochrome c oxidase subunit II. We have determined the nucleotide sequence of a 3-kilobase segment of DNA that carries PET111. The sequence contains a single long open reading frame that predicts a basic protein of 718 amino acids. The PET111 gene product is a mitochondrial protein, since a hybrid protein which includes the amino-terminal 154 amino acids of PET111 fused to beta-galactosidase is specifically associated with mitochondria. PET111 is translated from a 2.9-kilobase mRNA which, interestingly, has an extended 5'-leader sequence containing four short open reading frames upstream of the long open reading frame. These open reading frames exhibit an interesting pattern of overlap with each other and with the PET111 reading frame.

scholarly journals Pneumococcal Bacteriophage Cp-1 Encodes Its Own Protease Essential for Phage Maturation

1998 ◽  
Vol 72 (4) ◽  
pp. 3491-3494 ◽  
Author(s):  
Ana C. Martín ◽  
Rubens López ◽  
Pedro García
Keyword(s):  
Escherichia Coli ◽  
Major Capsid Protein ◽  
Open Reading Frame ◽  
Posttranslational Processing ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Gram Positive Bacteria ◽  
First Case ◽  
Key Enzyme ◽  
Head Protein

ABSTRACT The major capsid protein of the pneumococcal phage Cp-1 that accounts for 90% of the total protein found in the purified virions is synthesized by posttranslational processing of the product of the open reading frame (ORF) orf9. Cloning of different ORFs of the Cp-1 genome in Escherichia coli and Streptococcus pneumoniae combined with Western blot analysis of the expressed products led to the conclusion that the product oforf13 is an endoprotease that cleaves off the first 48 amino acid residues of the major head protein. This protease appears to be a key enzyme in the morphopoietic pathway of the Cp-1 phage head. To our knowledge, this is the first case of a bacteriophage infecting gram-positive bacteria that encodes a protease involved in phage maturation.

Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5' leader

1987 ◽  
Vol 7 (8) ◽  
pp. 2728-2734
Author(s):  
C A Strick ◽  
T D Fox
Keyword(s):  
Amino Acids ◽  
Regulatory Gene ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Amino Terminal ◽  
Interesting Pattern ◽  
Reading Frames

The yeast nuclear gene PET111 is required specifically for translation of the mitochondrion-coded mRNA for cytochrome c oxidase subunit II. We have determined the nucleotide sequence of a 3-kilobase segment of DNA that carries PET111. The sequence contains a single long open reading frame that predicts a basic protein of 718 amino acids. The PET111 gene product is a mitochondrial protein, since a hybrid protein which includes the amino-terminal 154 amino acids of PET111 fused to beta-galactosidase is specifically associated with mitochondria. PET111 is translated from a 2.9-kilobase mRNA which, interestingly, has an extended 5'-leader sequence containing four short open reading frames upstream of the long open reading frame. These open reading frames exhibit an interesting pattern of overlap with each other and with the PET111 reading frame.

Isolation and incubation conditions to study heart mitochondrial protein synthesis

1990 ◽  
Vol 258 (3) ◽  
pp. E492-E502 ◽  
Author(s):  
E. E. McKee ◽  
B. L. Grier ◽  
G. S. Thompson ◽  
J. D. McCourt
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Adenine Nucleotide ◽  
Isolated Heart ◽  
Mitochondrial Gene ◽  
Mitochondrial Protein ◽  
Specific Radioactivity ◽  
Companion Paper ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation

Although much is now known with regard to the processes of mammalian mitochondrial gene expression, relatively little is known concerning the quantitative regulation of this pathway in response to hormones or other physiological stimuli. This has been caused, in large part, by the lack of adequate assay systems in which such processes can be meaningfully measured. The purpose of this and the companion paper [E. E. McKee, B. L. Grier, G. S. Thompson, A. C. F. Leung, and J. D. McCourt. Am. J. Physiol. 258 [Endocrinol. Metab. 21):E503-E510, 1990] is to describe a system in which the quantitative regulation of mitochondrial protein synthesis in rat heart can be investigated. In this report the conditions for mitochondrial isolation and labeling are described, and the importance of isolating intact, tightly coupled mitochondria in obtaining high and reliable rates of protein synthesis is demonstrated. The highest levels of protein synthesis are obtained in mitochondria isolated from hearts perfused and homogenized in the presence of subtilisin, conditions in which the fastest rates of state 3 respiration and the highest respiratory control ratios are also observed. Analysis of the free amino acid pools indicates that isolated heart mitochondria have a negligible level of endogenous methionine as well as other amino acids. As a result, the concentration and specific radioactivity of the [35S]methionine pool serving protein synthesis could be easily determined. Optimal translation occurred at 30 degrees C at a pH of 7.0-7.2 and required the addition of methionine (20 microM), the other 19 amino acids (0.1 mM each), K+ (60-90 mM), Cl- (30-90 mM), Mg2+ (0.5-5 mM), and bovine serum albumin (1 mg/ml). As shown in the companion paper, adenine nucleotide (0.5-4.0 mM) and oxidizable substrate (10-20 mM glutamate) are also required for isolated heart mitochondrial protein synthesis. Analysis of labeled mitochondrial translation products demonstrated that bona fide mitochondrial peptides were synthesized.

Sign in / Sign up

Export Citation Format

Share Document