A hierarchy of trans-acting factors modulates translation of an activator of amino acid biosynthetic genes in Saccharomyces cerevisiae

1985 ◽  
Vol 5 (9) ◽  
pp. 2349-2360 ◽  
Author(s):  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Fusion Transcript ◽  
Transcript Level ◽  
Amino Acid Starvation ◽  
Lacz Fusion ◽  
Recessive Mutation ◽  
Translational Efficiency ◽  
Biosynthetic Genes ◽  
Fusion Enzyme

The GCN4 gene encodes a positive effector of amino acid biosynthetic genes in Saccharomyces cerevisiae. Genetic analysis has suggested that GCN4 is regulated by a hierarchy of interacting positive and negative effectors in response to amino acid starvation. Results presented here for a GCN4-lacZ gene fusion support this regulatory model and suggest that the regulators of GCN4 exert their effects primarily at the level of translation of GCN4 mRNA. Both the GCN2 and GCN3 products appear to stimulate translation of GCN4 mRNA in response to amino acid starvation, because a recessive mutation in either gene blocked derepression of GCN4-lacZ fusion enzyme levels but did not reduce the fusion transcript level relative to that in wild-type cells grown in the same conditions. The GCD1 product appears to inhibit translation of GCN4 mRNA because under certain growth conditions, the gcd1-101 mutation led to derepression of the GCN4-lacZ fusion enzyme level in the absence of any increase in the fusion transcript level. In addition, the gcd1-101 mutation suppressed the low translational efficiency of GCN4-lacZ mRNA observed in gcn2- and gcn3- cells. A deletion of four small open reading frames in the 5' leader of GCN4-lacZ mRNA mimicked the effect of a gcd1 mutation and derepressed translation of the fusion transcript in the absence of either starvation conditions or the GCN2 and GCN3 products. By contrast, in a gcd1- strain, the deletion resulted in little additional increase in the translational efficiency of the fusion transcript. These results suggest that GCD1 mediates the translational repression normally exerted by the GCN4 leader sequences and that GCN2 and GCN3 antagonize these negative elements in response to amino acid starvation. The effects of the trans-acting mutations on the translation of GCN4-lacZ mRNA remained intact even when transcription of the fusion gene was placed under the control of the S. cerevisiae GAL1 transcriptional control element.

scholarly journals A hierarchy of trans-acting factors modulates translation of an activator of amino acid biosynthetic genes in Saccharomyces cerevisiae.

1985 ◽  
Vol 5 (9) ◽  
pp. 2349-2360 ◽  
Author(s):  
A G Hinnebusch
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Fusion Transcript ◽  
Transcript Level ◽  
Amino Acid Starvation ◽  
Lacz Fusion ◽  
Recessive Mutation ◽  
Translational Efficiency ◽  
Biosynthetic Genes ◽  
Fusion Enzyme

The GCN4 gene encodes a positive effector of amino acid biosynthetic genes in Saccharomyces cerevisiae. Genetic analysis has suggested that GCN4 is regulated by a hierarchy of interacting positive and negative effectors in response to amino acid starvation. Results presented here for a GCN4-lacZ gene fusion support this regulatory model and suggest that the regulators of GCN4 exert their effects primarily at the level of translation of GCN4 mRNA. Both the GCN2 and GCN3 products appear to stimulate translation of GCN4 mRNA in response to amino acid starvation, because a recessive mutation in either gene blocked derepression of GCN4-lacZ fusion enzyme levels but did not reduce the fusion transcript level relative to that in wild-type cells grown in the same conditions. The GCD1 product appears to inhibit translation of GCN4 mRNA because under certain growth conditions, the gcd1-101 mutation led to derepression of the GCN4-lacZ fusion enzyme level in the absence of any increase in the fusion transcript level. In addition, the gcd1-101 mutation suppressed the low translational efficiency of GCN4-lacZ mRNA observed in gcn2- and gcn3- cells. A deletion of four small open reading frames in the 5' leader of GCN4-lacZ mRNA mimicked the effect of a gcd1 mutation and derepressed translation of the fusion transcript in the absence of either starvation conditions or the GCN2 and GCN3 products. By contrast, in a gcd1- strain, the deletion resulted in little additional increase in the translational efficiency of the fusion transcript. These results suggest that GCD1 mediates the translational repression normally exerted by the GCN4 leader sequences and that GCN2 and GCN3 antagonize these negative elements in response to amino acid starvation. The effects of the trans-acting mutations on the translation of GCN4-lacZ mRNA remained intact even when transcription of the fusion gene was placed under the control of the S. cerevisiae GAL1 transcriptional control element.

How Optimized Is the Translational Machinery in Escherichia coli, Salmonella typhimurium and Saccharomyces cerevisiae?

Genetics ◽  
1998 ◽  
Vol 149 (1) ◽  
pp. 37-44 ◽  
Author(s):  
Xuhua Xia
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Salmonella Typhimurium ◽  
Codon Usage ◽  
Translational Efficiency ◽  
Square Root ◽  
Translational Machinery ◽  
Mutual Adaptation ◽  
Trna Species

Abstract The optimization of the translational machinery in cells requires the mutual adaptation of codon usage and tRNA concentration, and the adaptation of tRNA concentration to amino acid usage. Two predictions were derived based on a simple deterministic model of translation which assumes that elongation of the peptide chain is rate-limiting. The highest translational efficiency is achieved when the codon recognized by the most abundant tRNA reaches the maximum frequency. For each codon family, the tRNA concentration is optimally adapted to codon usage when the concentration of different tRNA species matches the square-root of the frequency of their corresponding synonymous codons. When tRNA concentration and codon usage are well adapted to each other, the optimal content of all tRNA species carrying the same amino acid should match the square-root of the frequency of the amino acid. These predictions are examined against empirical data from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae.

Association of RAP1 binding sites with stringent control of ribosomal protein gene transcription in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (5) ◽  
pp. 2723-2735 ◽  
Author(s):  
C M Moehle ◽  
A G Hinnebusch
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Ribosomal Protein ◽  
Binding Sites ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Ribosomal Protein Genes ◽  
Biosynthetic Genes ◽  
Stringent Control

An amino acid limitation in bacteria elicits a global response, called stringent control, that leads to reduced synthesis of rRNA and ribosomal proteins and increased expression of amino acid biosynthetic operons. We have used the antimetabolite 3-amino-1,2,4-triazole to cause histidine limitation as a means to elicit the stringent response in the yeast Saccharomyces cerevisiae. Fusions of the yeast ribosomal protein genes RPL16A, CRY1, RPS16A, and RPL25 with the Escherichia coli lacZ gene were used to show that the expression of these genes is reduced by a factor of 2 to 5 during histidine-limited exponential growth and that this regulation occurs at the level of transcription. Stringent regulation of the four yeast ribosomal protein genes was shown to be associated with a nucleotide sequence, known as the UASrpg (upstream activating sequence for ribosomal protein genes), that binds the transcriptional regulatory protein RAP1. The RAP1 binding sites also appeared to mediate the greater ribosomal protein gene expression observed in cells growing exponentially than in cells in stationary phase. Although expression of the ribosomal protein genes was reduced in response to histidine limitation, the level of RAP1 DNA-binding activity in cell extracts was unaffected. Yeast strains bearing a mutation in any one of the genes GCN1 to GCN4 are defective in derepression of amino acid biosynthetic genes in 10 different pathways under conditions of histidine limitation. These Gcn- mutants showed wild-type regulation of ribosomal protein gene expression, which suggests that separate regulatory pathways exist in S. cerevisiae for the derepression of amino acid biosynthetic genes and the repression of ribosomal protein genes in response to amino acid starvation.

scholarly journals Negative regulatory gene for general control of amino acid biosynthesis in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (9) ◽  
pp. 3150-3155 ◽  
Author(s):  
P L Myers ◽  
R C Skvirsky ◽  
M L Greenberg ◽  
H Greer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Regulatory Gene ◽  
Negative Regulator ◽  
Single Amino Acid ◽  
Mrna Levels ◽  
General Control ◽  
Biosynthetic Genes ◽  
Isolation And Characterization ◽  
Steady State Levels

In Saccharomyces cerevisiae, many amino acid biosynthetic pathways are coregulated by a complex general control system: starvation for a single amino acid results in the derepression of amino acid biosynthetic genes in multiple pathways. Derepression of these genes is mediated by positive (GCN) and negative (GCD) regulatory genes. In this paper we describe the isolation and characterization of a previously unreported negative regulatory gene, GCD3. A gcd3 mutation is recessive to wild type, confers resistance to multiple amino acid analogs, and results in overproduction and partially constitutive elevation of mRNA levels for amino acid biosynthetic genes. Furthermore, a gcd3 mutation can overcome the derepression-deficient phenotype of mutations in the positive regulatory GCN1, GCN2, and GCN3 genes. However, the gcd3 mutation cannot overcome the derepression-deficient phenotype of a gcn4 mutation, suggesting that GCD3 acts as a negative regulator of the important GCN4 gene. Northern blot analysis confirmed this conclusion, in that the steady-state levels of GCN4 mRNA are greatly increased in a gcd3 mutant. Thus, the negative regulatory gene GCD3 plays a central role in derepression of amino acid biosynthetic genes.

The yeast ILV2 gene is under general amino acid control

Genome ◽  
1988 ◽  
Vol 30 (6) ◽  
pp. 984-986 ◽  
Author(s):  
W. Xiao ◽  
G. H. Rank
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Key Words ◽  
Acetolactate Synthase ◽  
Amino Acid Starvation ◽  
Sulfometuron Methyl ◽  
General Amino Acid Control ◽  
Acid Control ◽  
High Level ◽  
Level Resistance

The yeast ILV2 gene encodes acetolactate synthase, the first enzyme in the biosynthesis of isoleucine and valine. Its multiple regulation has precluded the clear demonstration of whether ILV2 is under general amino acid control. Nonderepressible gcn4 strains were used as recipients for transformation with a YCp plasmid carrying GCN4. Parental gcn4 cells and their isogenic GCN4 transformants were evaluated for ALS derepression following induced amino acid starvation. GCN4 cells showed 1.5-to 1.7-fold derepression but no derepression was observed in isogenic control gcn4 strains. A similar depression of ILV2 mRNA was also observed. Genetic evidence for general amino acid control was the gcn4 suppression of high level resistance to sulfometuron methyl by the SMR1-410 allele of ILV2.Key words: Saccharomyces cerevisiae, ILV2 gene, general amino acid control, multiple regulators.

GCD11, a negative regulator of GCN4 expression, encodes the gamma subunit of eIF-2 in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (1) ◽  
pp. 506-520
Author(s):  
E M Hannig ◽  
A M Cigan ◽  
B A Freeman ◽  
T G Kinzy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Metal Binding ◽  
Critical Role ◽  
Cross Reactivity ◽  
Translation Initiation Factor ◽  
Negative Regulator ◽  
Initiation Factor ◽  
Translational Efficiency ◽  
Gamma Subunit

The eukaryotic translation initiation factor eIF-2 plays a critical role in regulating the expression of the yeast transcriptional activator GCN4. Mutations in genes encoding the alpha and beta subunits of eIF-2 alter translational efficiency at the GCN4 AUG codon and constitutively elevate GCN4 translation. Mutations in the yeast GCD11 gene have been shown to confer a similar phenotype. The nucleotide sequence of the cloned GCD11 gene predicts a 527-amino-acid polypeptide that is similar to the prokaryotic translation elongation factor EF-Tu. Relative to EF-Tu, the deduced GCD11 amino acid sequence contains a 90-amino-acid N-terminal extension and an internal cysteine-rich sequence that contains a potential metal-binding finger motif. We have identified the GCD11 gene product as the gamma subunit of eIF-2 by the following criteria: (i) sequence identities with mammalian eIF-2 gamma peptides; (ii) increased eIF-2 activity in extracts prepared from cells cooverexpressing GCD11, eIF-2 alpha, and eIF-2 beta; and (iii) cross-reactivity of antibodies directed against the GCD11 protein with the 58-kDa polypeptide present in purified yeast eIF-2. The predicted GCD11 polypeptide contains all of the consensus elements known to be required for guanine nucleotide binding, suggesting that, in Saccharomyces cerevisiae, the gamma subunit of eIF-2 is responsible for GDP-GTP binding.

scholarly journals Transcriptional-translational regulatory circuit in Saccharomyces cerevisiae which involves the GCN4 transcriptional activator and the GCN2 protein kinase.

1988 ◽  
Vol 8 (5) ◽  
pp. 2132-2139 ◽  
Author(s):  
I Roussou ◽  
G Thireos ◽  
B M Hauge
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Protein Kinase ◽  
Dna Sequences ◽  
Transcriptional Activation ◽  
Translational Regulation ◽  
Catalytic Domain ◽  
Amino Acid Starvation ◽  
Protein Kinase Activity ◽  
Regulatory Circuit

GCN4 protein mediates the transcriptional activation of amino acid biosynthetic genes in Saccharomyces cerevisiae by specifically binding to DNA sequences in their 5'-regulatory regions. GCN4 expression is regulated at the level of translation, with translational derepression occurring under conditions of amino acid starvation. The product of the GCN2 gene is essential for translational derepression of GCN4. Sequence analysis of the GCN2 gene reveals that the GCN2 protein has a domain highly homologous to the catalytic domain of all known protein kinases. Furthermore, gcn2 strains are deficient in a protein kinase activity corresponding to a protein with the calculated molecular weight deduced from the GCN2 open reading frame. Therefore it is likely that GCN2 encodes a protein kinase, which may be directly involved in translational regulation of the GCN4 mRNA. Transcription of the GCN2 gene is increased when cells are cultured in amino acid starvation medium. This transcriptional activation is mediated by the GCN4 protein, which binds to the promoter region of the GCN2 gene. Thus, this system is modulated by a transcriptional-translational regulatory circuit, which is activated by amino acid starvation. Activation is not the result of a simple quantitative increase of either one of the identified components of the circuit.

scholarly journals Transcriptional Profiling of Cross Pathway Control in Neurospora crassa and Comparative Analysis of the Gcn4 and CPC1 Regulons

2007 ◽  
Vol 6 (6) ◽  
pp. 1018-1029 ◽  
Author(s):  
Chaoguang Tian ◽  
Takao Kasuga ◽  
Matthew S. Sachs ◽  
N. Louise Glass
Keyword(s):  
Amino Acid ◽  
Neurospora Crassa ◽  
Regulatory Networks ◽  
Transcriptional Profiling ◽  
Trna Synthetase ◽  
Network Evolution ◽  
Amino Acid Starvation ◽  
Bzip Transcription Factor ◽  
Biosynthetic Genes ◽  
Data Set

ABSTRACT Identifying and characterizing transcriptional regulatory networks is important for guiding experimental tests on gene function. The characterization of regulatory networks allows comparisons among both closely and distantly related species, providing insight into network evolution, which is predicted to correlate with the adaptation of different species to particular environmental niches. One of the most intensely studied regulatory factors in the yeast Saccharomyces cerevisiae is the bZIP transcription factor Gcn4p. Gcn4p is essential for a global transcriptional response when S. cerevisiae experiences amino acid starvation. In the filamentous ascomycete Neurospora crassa, the ortholog of GCN4 is called the cross pathway control-1 (cpc-1) gene; it is required for the ability of N. crassa to induce a number of amino acid biosynthetic genes in response to amino acid starvation. Here, we deciphered the CPC1 regulon by profiling transcription in wild-type and cpc-1 mutant strains with full-genome N. crassa 70-mer oligonucleotide microarrays. We observed that at least 443 genes were direct or indirect CPC1 targets; these included 67 amino acid biosynthetic genes, 16 tRNA synthetase genes, and 13 vitamin-related genes. Comparison among the N. crassa CPC1 transcriptional profiling data set and the Gcn4/CaGcn4 data sets from S. cerevisiae and Candida albicans revealed a conserved regulon of 32 genes, 10 of which are predicted to be directly regulated by Gcn4p/CPC1. The 32-gene conserved regulon comprises mostly amino acid biosynthetic genes. The comparison of regulatory networks in species with clear orthology among genes sheds light on how gene interaction networks evolve.

scholarly journals Association of RAP1 binding sites with stringent control of ribosomal protein gene transcription in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (5) ◽  
pp. 2723-2735 ◽  
Author(s):  
C M Moehle ◽  
A G Hinnebusch
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Ribosomal Protein ◽  
Binding Sites ◽  
Protein Gene ◽  
Ribosomal Protein Gene ◽  
Ribosomal Protein Genes ◽  
Biosynthetic Genes ◽  
Stringent Control

An amino acid limitation in bacteria elicits a global response, called stringent control, that leads to reduced synthesis of rRNA and ribosomal proteins and increased expression of amino acid biosynthetic operons. We have used the antimetabolite 3-amino-1,2,4-triazole to cause histidine limitation as a means to elicit the stringent response in the yeast Saccharomyces cerevisiae. Fusions of the yeast ribosomal protein genes RPL16A, CRY1, RPS16A, and RPL25 with the Escherichia coli lacZ gene were used to show that the expression of these genes is reduced by a factor of 2 to 5 during histidine-limited exponential growth and that this regulation occurs at the level of transcription. Stringent regulation of the four yeast ribosomal protein genes was shown to be associated with a nucleotide sequence, known as the UASrpg (upstream activating sequence for ribosomal protein genes), that binds the transcriptional regulatory protein RAP1. The RAP1 binding sites also appeared to mediate the greater ribosomal protein gene expression observed in cells growing exponentially than in cells in stationary phase. Although expression of the ribosomal protein genes was reduced in response to histidine limitation, the level of RAP1 DNA-binding activity in cell extracts was unaffected. Yeast strains bearing a mutation in any one of the genes GCN1 to GCN4 are defective in derepression of amino acid biosynthetic genes in 10 different pathways under conditions of histidine limitation. These Gcn- mutants showed wild-type regulation of ribosomal protein gene expression, which suggests that separate regulatory pathways exist in S. cerevisiae for the derepression of amino acid biosynthetic genes and the repression of ribosomal protein genes in response to amino acid starvation.

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