scholarly journals Suppression of a defect in the 5' untranslated leader of mitochondrial COX3 mRNA by a mutation affecting an mRNA-specific translational activator protein.

1993 ◽  
Vol 13 (8) ◽  
pp. 4806-4813 ◽  
Author(s):  
M C Costanzo ◽  
T D Fox
Keyword(s):  
Nuclear Gene ◽  
Gene Products ◽  
Wild Type ◽  
Large Deletions ◽  
Site Of Action ◽  
Genes Encoding ◽  
Cold Sensitive ◽  
Translational Activator ◽  
A Site ◽  
Dominant Suppressor

Translation of the Saccharomyces cerevisiae mitochondrial COX3 mRNA, encoding subunit III of cytochrome c oxidase, specifically requires the action of the nuclear gene products PET54, PET122, and PET494 at a site encoded in the 612-base 5' untranslated leader. To identify more precisely the site of action of the translational activators, we constructed two large deletions of the COX3 mRNA 5' untranslated leader. Both deletions blocked translation without affecting mRNA stability. However, one of the large deletions was able to revert to partial function by a small secondary deletion within the remaining 5' leader sequences. Translation of the resulting mutant (cox3-15) mRNA was still dependent on the nuclear-encoded specific activators but was cold sensitive. We selected revertants of this mitochondrial mutant at low temperature to identify genes encoding proteins that might interact with the COX3 mRNA 5' leader. One such revertant carried a missense mutation in the PET122 gene that was a strong and dominant suppressor of the cold-sensitive defect in the mRNA, indicating that the PET122 protein interacts functionally (possibly directly) with the COX3 mRNA 5' leader. The cox3-15 mutation was not suppressed by overproduction of the wild-type PET122 protein but was very weakly suppressed by overproduction of PET494 and slightly better suppressed by co-overproduction of PET494 and PET122.

Suppression of a defect in the 5' untranslated leader of mitochondrial COX3 mRNA by a mutation affecting an mRNA-specific translational activator protein

1993 ◽  
Vol 13 (8) ◽  
pp. 4806-4813
Author(s):  
M C Costanzo ◽  
T D Fox
Keyword(s):  
Nuclear Gene ◽  
Gene Products ◽  
Wild Type ◽  
Large Deletions ◽  
Site Of Action ◽  
Genes Encoding ◽  
Cold Sensitive ◽  
Translational Activator ◽  
A Site ◽  
Dominant Suppressor

Translation of the Saccharomyces cerevisiae mitochondrial COX3 mRNA, encoding subunit III of cytochrome c oxidase, specifically requires the action of the nuclear gene products PET54, PET122, and PET494 at a site encoded in the 612-base 5' untranslated leader. To identify more precisely the site of action of the translational activators, we constructed two large deletions of the COX3 mRNA 5' untranslated leader. Both deletions blocked translation without affecting mRNA stability. However, one of the large deletions was able to revert to partial function by a small secondary deletion within the remaining 5' leader sequences. Translation of the resulting mutant (cox3-15) mRNA was still dependent on the nuclear-encoded specific activators but was cold sensitive. We selected revertants of this mitochondrial mutant at low temperature to identify genes encoding proteins that might interact with the COX3 mRNA 5' leader. One such revertant carried a missense mutation in the PET122 gene that was a strong and dominant suppressor of the cold-sensitive defect in the mRNA, indicating that the PET122 protein interacts functionally (possibly directly) with the COX3 mRNA 5' leader. The cox3-15 mutation was not suppressed by overproduction of the wild-type PET122 protein but was very weakly suppressed by overproduction of PET494 and slightly better suppressed by co-overproduction of PET494 and PET122.

Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (11) ◽  
pp. 3990-3998
Author(s):  
S Harashima ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Lacz Gene ◽  
Gene Products ◽  
Wild Type ◽  
Double Mutation ◽  
Double Stranded Rna ◽  
Amino Acid Availability ◽  
Genes Encoding ◽  
New Alleles

GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

scholarly journals Atp10p Assists Assembly of Atp6p into the F0Unit of the Yeast Mitochondrial ATPase

2004 ◽  
Vol 279 (19) ◽  
pp. 19775-19780 ◽  
Author(s):  
Alexander Tzagoloff ◽  
Antoni Barrientos ◽  
Walter Neupert ◽  
Johannes M. Herrmann
Keyword(s):  
Nuclear Gene ◽  
Mitochondrial Atpase ◽  
Gene Products ◽  
Wild Type ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Inner Membrane ◽  
Atpase Subunits ◽  
Specific Factors

The F0F1-ATPase complex of yeast mitochondria contains three mitochondrial and at least 17 nuclear gene products. The coordinate assembly of mitochondrial and cytosolic translation products relies on chaperones and specific factors that stabilize the pools of some unassembled subunits. Atp10p was identified as a mitochondrial inner membrane component necessary for the biogenesis of the hydrophobic F0sector of the ATPase. Here we show that, following its synthesis on mitochondrial ribosomes, subunit 6 of the ATPase (Atp6p) can be cross-linked to Atp10p. This interaction is required for the integration of Atp6p into a partially assembled subcomplex of the ATPase. Pulse labeling and chase of mitochondrial translation productsin vivoindicate that Atp6p is less stable and more rapidly degraded in anatp10null mutant than in wild type. Based on these observations, we propose Atp10p to be an Atp6p-specific chaperone that facilitates the incorporation of Atp6p into an intermediate subcomplex of ATPase subunits.

scholarly journals Product of Saccharomyces cerevisiae nuclear gene PET494 activates translation of a specific mitochondrial mRNA.

1986 ◽  
Vol 6 (11) ◽  
pp. 3694-3703 ◽  
Author(s):  
M C Costanzo ◽  
T D Fox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
Mitochondrial Genes ◽  
Wild Type ◽  
Site Of Action ◽  
Flanking Sequences ◽  
Untranslated Leaders ◽  
Beta Galactosidase

The product of Saccharomyces cerevisiae nuclear gene PET494 is known to be required for a posttranscriptional step in the accumulation of one mitochondrial gene product, subunit III of cytochrome c oxidase (coxIII). Here we show that the PET494 protein probably acts in mitochondria by demonstrating that both a PET494-beta-galactosidase fusion protein and unmodified PET494 are specifically associated with mitochondria. To define the PET494 site of action, we isolated mutations that suppress a pet494 deletion. These mutations were rearrangements of the mitochondrial gene oxi2 that encodes coxIII. The suppressor oxi2 genes had acquired the 5'-flanking sequences of other mitochondrial genes and gave rise to oxi2 transcripts carrying the 5'-untranslated leaders of their mRNAs. These results demonstrate that in wild-type cells PET494 specifically promotes coxIII translation, probably by interacting with the 5'-untranslated leader of the oxi2 mRNA.

scholarly journals Dominant Mutations in Three Different Subunits of Replication Factor C Suppress Replication Defects in Yeast PCNA Mutants

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1617-1628
Author(s):  
Neelam S Amin ◽  
K Michelle Tuffo ◽  
Connie Holm
Keyword(s):  
Nuclear Antigen ◽  
Replication Factor C ◽  
Replication Factor ◽  
Suppressor Mutations ◽  
Intimate Association ◽  
Cold Sensitive ◽  
C Complex ◽  
Factor C ◽  
A Site ◽  
Dominant Suppressor

Abstract To identify proteins that interact with the yeast proliferating cell nuclear antigen (PCNA), we used a genetic approach to isolate mutations that compensate for the defects in cold-sensitive (Cs−) mutants of yeast PCNA (POL30). Because the cocrystal structure of human PCNA and a p21WAF1/CIP1 peptide shows that the interdomain region of PCNA is a site of p21 interaction, we specifically looked for new mutations that suppress mutations in the equivalent region of yeast PCNA. In independent screens using three different Cs− mutants, we identified spontaneously arising dominant suppressor mutations in the RFC3 gene. In addition, dominant suppressor mutations were identified in the RFC1 and RFC2 genes using a single pol30 mutant. An intimate association between PCNA and RFC1p, RFC2p, and RFC3p is suggested by the allele-restricted suppression of 10 different pol30 alleles by the RFC suppressors. RFC1, RFC2, and RFC3 encode three of the five subunits of the replication factor C complex, which is required to load PCNA onto DNA in reconstituted DNA replication reactions. Genomic sequencing reveals a common region in RFC1p, RFC2p, and RFC3p that is important for the functional interaction with PCNA. Biochemical analysis of the wild type and mutant PCNA and RFC3 proteins shows that mutant RFC3p enhances the production of long DNA products in pol δ-dependent DNA synthesis, which is consistent with an increase in processivity.

scholarly journals Multiple Translational Control Sequences in the 5′ Leader of the Chloroplast psbC mRNA Interact With Nuclear Gene Products in Chlamydomonas reinhardtii

Genetics ◽  
2003 ◽  
Vol 163 (3) ◽  
pp. 895-904
Author(s):  
William Zerges ◽  
Andrea H Auchincloss ◽  
Jean-David Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Reporter Gene ◽  
Translational Control ◽  
Nuclear Gene ◽  
Gene Products ◽  
Wild Type ◽  
Translation Initiation Region ◽  
Mrna Stabilization ◽  
In The Wild ◽  
Nuclear Loci

Abstract Translation of the chloroplast psbC mRNA in the unicellular eukaryotic alga Chlamydomonas reinhardtii is controlled by interactions between its 547-base 5′ untranslated region and the products of the nuclear loci TBC1, TBC2, and possibly TBC3. In this study, a series of site-directed mutations in this region was generated and the ability of these constructs to drive expression of a reporter gene was assayed in chloroplast transformants that are wild type or mutant at these nuclear loci. Two regions located in the middle of the 5′ leader and near the initiation codon are important for translation. Other deletions still allow for partial expression of the reporter gene in the wild-type background. Regions with target sites for TBC1 and TBC2 were identified by estimating the residual translation activity in the respective mutant backgrounds. TBC1 targets include mostly the central part of the leader and the translation initiation region whereas the only detected TBC2 targets are in the 3′ part. The 5′-most 93 nt of the leader are required for wild-type levels of transcription and/or mRNA stabilization. The results indicate that TBC1 and TBC2 function independently and further support the possibility that TBC1 acts together with TBC3.

scholarly journals Multiple GCD genes required for repression of GCN4, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (11) ◽  
pp. 3990-3998 ◽  
Author(s):  
S Harashima ◽  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Lacz Gene ◽  
Gene Products ◽  
Wild Type ◽  
Double Mutation ◽  
Double Stranded Rna ◽  
Amino Acid Availability ◽  
Genes Encoding ◽  
New Alleles

GCN4 encodes a positive regulator of multiple unlinked genes encoding amino acid biosynthetic enzymes in Saccharomyces cerevisiae. Expression of GCN4 is coupled to amino acid availability by a control mechanism involving GCD1 as a negative effector and GCN1, GCN2, and GCN3 as positive effectors of GCN4 expression. We used reversion of a gcn2 gcn3 double mutation to isolate new alleles of GCD1 and mutations in four additional GCD genes which we designate GCD10, GCD11, GCD12, and GCD13. All of the mutations lead to constitutive derepression of HIS4 transcription in the absence of the GCN2+ and GCN3+ alleles. By contrast, the gcd mutations require the wild-type GCN4 allele for their derepressing effect, suggesting that each acts by influencing the level of GCN4 activity in the cell. Consistent with this interpretation, mutations in each GCD gene lead to constitutive derepression of a GCN4::lacZ gene fusion. Thus, at least five gene products are required to maintain the normal repressed level of GCN4 expression in nonstarvation conditions. Interestingly, the gcd mutations are pleiotropic and also affect growth rate in nonstarvation conditions. In addition, certain alleles lead to a loss of M double-stranded RNA required for the killer phenotype. This pleiotropy suggests that the GCD gene products contribute to an essential cellular function, in addition to, or in conjunction with, their role in GCN4 regulation.

scholarly journals Catabolism of Benzoate and Phthalate in Rhodococcus sp. Strain RHA1: Redundancies and Convergence

2005 ◽  
Vol 187 (12) ◽  
pp. 4050-4063 ◽  
Author(s):  
Marianna A. Patrauchan ◽  
Christine Florizone ◽  
Manisha Dosanjh ◽  
William W. Mohn ◽  
Julian Davies ◽  
...  
Keyword(s):  
Polychlorinated Biphenyl ◽  
Carbon Sources ◽  
Growth Conditions ◽  
Lag Phase ◽  
Gene Products ◽  
Wild Type ◽  
Bacterial Genomes ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Proteomic Analyses

ABSTRACT Genomic and proteomic approaches were used to investigate phthalate and benzoate catabolism in Rhodococcus sp. strain RHA1, a polychlorinated biphenyl-degrading actinomycete. Sequence analyses identified genes involved in the catabolism of benzoate (ben) and phthalate (pad), the uptake of phthalate (pat), and two branches of the β-ketoadipate pathway (catRABC and pcaJIHGBLFR). The regulatory and structural ben genes are separated by genes encoding a cytochrome P450. The pad and pat genes are contained on a catabolic island that is duplicated on plasmids pRHL1 and pRHL2 and includes predicted terephthalate catabolic genes (tpa). Proteomic analyses demonstrated that the β-ketoadipate pathway is functionally convergent. Specifically, the pad and pat gene products were only detected in phthalate-grown cells. Similarly, the ben and cat gene products were only detected in benzoate-grown cells. However, pca-encoded enzymes were present under both growth conditions. Activity assays for key enzymes confirmed these results. Disruption of pcaL, which encodes a fusion enzyme, abolished growth on phthalate. In contrast, after a lag phase, growth of the mutant on benzoate was similar to that of the wild type. Proteomic analyses revealed 20 proteins in the mutant that were not detected in wild-type cells during growth on benzoate, including a CatD homolog that apparently compensated for loss of PcaL. Analysis of completed bacterial genomes indicates that the convergent β-ketoadipate pathway and some aspects of its genetic organization are characteristic of rhodococci and related actinomycetes. In contrast, the high redundancy of catabolic pathways and enzymes appears to be unique to RHA1 and may increase its potential to adapt to new carbon sources.

scholarly journals PET111, a Saccharomyces cerevisiae Nuclear Gene Required for Translation of the Mitochondrial mRNA Encoding Cytochrome c Oxidase Subunit II

Genetics ◽  
1987 ◽  
Vol 115 (4) ◽  
pp. 637-647
Author(s):  
Candace G Poutre ◽  
Thomas D Fox
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Mitochondrial Gene ◽  
Nuclear Gene ◽  
State Level ◽  
Mitochondrial Genes ◽  
Chimeric Proteins ◽  
Wild Type ◽  
Oxidase Subunit ◽  
A Site

ABSTRACT Mutations in the nuclear gene PET111 are recessive and specifically block accumulation of cytochrome c oxidase subunit II (coxII), the product of a mitochondrial gene. However, the coxII mRNA is present in pet111 mutants at a level approximately one-third that of wild type. The simplest explanation for this phenotype is that PET111 is required for translation of the coxII mRNA. The reduced steady-state level of this mRNA is probably a secondary effect, caused by increased degradation of the untranslated transcript. Mitochondrial suppressors of pet111, carried on rho- mtDNAs, bypass the requirement for PET111 in coxII translation. Three suppressors are fusions between the coxII structural gene and other mitochondrial genes, that encode chimeric proteins consisting of the N-terminal portions of other mitochondrially coded proteins fused to the coxII precursor protein. When present together with rho  + mtDNA in a heteroplasmic state, these suppressors allow coxII synthesis in pet111 mutants. Thus in wild type, the PET111 product, or something under its control, probably acts at a site coded in the proximal portion of the gene for coxII to promote translation of the mRNA. PET111 was isolated by molecular cloning and genetically mapped to a position approximately midway between rna1 and SUP8 on chromosome XIII.

scholarly journals Purification of the Formate-Tetrahydrofolate Ligasefrom Methylobacterium extorquens AM1 and Demonstrationof Its Requirement for MethylotrophicGrowth

2003 ◽  
Vol 185 (24) ◽  
pp. 7169-7175 ◽  
Author(s):  
Christopher J. Marx ◽  
Markus Laukel ◽  
Julia A. Vorholt ◽  
Mary E. Lidstrom
Keyword(s):  
Mutant Strain ◽  
Gene Products ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Type Conversion ◽  
Purification And Characterization ◽  
Formaldehyde Oxidation ◽  
Genes Encoding

ABSTRACT The serine cycle methylotroph Methylobacterium extorquens AM1 contains two pterin-dependent pathways for C1 transfers, the tetrahydrofolate (H4F) pathway and the tetrahydromethanopterin (H4MPT) pathway, and both are required for growth on C1 compounds. With the exception of formate-tetrahydrofolate ligase (FtfL, alternatively termed formyl-H4F synthetase), all of the genes encoding the enzymes comprising these two pathways have been identified, and the corresponding gene products have been purified and characterized. We present here the purification and characterization of FtfL from M. extorquens AM1 and the confirmation that this enzyme is encoded by an ftfL homolog identified previously through transposon mutagenesis. Phenotypic analyses of the ftfL mutant strain demonstrated that FtfL activity is required for growth on C1 compounds. Unlike mutants defective for the H4MPT pathway, the ftfL mutant strain does not exhibit phenotypes indicative of defective formaldehyde oxidation. Furthermore, the ftfL mutant strain remained competent for wild-type conversion of [14C]methanol to [14C]CO2. Collectively, these data confirm our previous presumptions that the H4F pathway is not the key formaldehyde oxidation pathway in M. extorquens AM1. Rather, our data suggest an alternative model for the role of the H4F pathway in this organism in which it functions to convert formate to methylene H4F for assimilatory metabolism.

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