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Query: UNIPROT:P06889 (Mol)
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The GCN4 gene of the yeast Saccharomyces cerevisiae encodes a transcriptional activator of amino acid biosynthetic genes that is regulated at the translational level according to the availability of amino acids. GCN2 is a protein kinase required for increased translation of GCN4 mRNA in amino acid-starved cells. Centrifugation of cell extracts in sucrose gradients indicated that GCN2 comigrates with ribosomal subunits and polysomes. The fraction of GCN2 cosedimenting with polysomes was reduced under conditions in which polysomes were dissociated, suggesting that GCN2 is physically bound to these structures. When the association of 40S and 60S subunits was prevented by omitting Mg2+ from the gradient, almost all of the GCN2 comigrated with 60S ribosomal subunits, and it remained bound to these particles during gel electrophoresis under nondenaturing conditions. GCN2 could be dissociated from 60S subunits by 0.5 M KCl, suggesting that it is loosely associated with ribosomes rather than being an integral ribosomal protein. Accumulation of GCN2 on free 43S-48S particles and 60S subunits occurred during polysome runoff in vitro and under conditions of reduced growth rate in vivo. These observations, plus the fact that GCN2 shows preferential association with free ribosomal subunits during exponential growth, suggest that GCN2 interacts with ribosomes during the translation initiation cycle. The extreme carboxyl-terminal segment of GCN2 is essential for its interaction with ribosomes. These sequences are also required for the ability of GCN2 to stimulate GCN4 translation in vivo, leading us to propose that ribosome association by GCN2 is important for its access to substrates in the translational machinery or for detecting uncharged tRNA in amino acid-starved cells.
Mol Cell Biol 1991 Jun
PMID:Ribosome association of GCN2 protein kinase, a translational activator of the GCN4 gene of Saccharomyces cerevisiae. 203 14

The GCD2 protein is a translational repressor of GCN4, the transcriptional activator of multiple amino acid biosynthetic genes in Saccharomyces cerevisiae. We present evidence that GCD2 has a general function in the initiation of protein synthesis in addition to its gene-specific role in translational control of GCN4 expression. Two temperature-sensitive lethal gcd2 mutations result in sensitivity to inhibitors of protein synthesis at the permissive temperature, and the gcd2-503 mutation leads to reduced incorporation of labeled leucine into total protein following a shift to the restrictive temperature of 36 degrees C. The gcd2-503 mutation also results in polysome runoff, accumulation of inactive 80S ribosomal couples, and accumulation of at least one of the subunits of the general translation initiation factor 2 (eIF-2 alpha) in 43S-48S particles following a shift to the restrictive temperature. The gcd2-502 mutation causes accumulation of 40S subunits in polysomes, known as halfmers, that are indicative of reduced 40S-60S subunit joining at the initiation codon. These phenotypes suggest that GCD2 functions in the translation initiation pathway at a step following the binding of eIF-2.GTP.Met-tRNA(iMet) to 40S ribosomal subunits. consistent with this hypothesis, we found that inhibiting 40S-60S subunit joining by deleting one copy (RPL16B) of the duplicated gene encoding the 60S ribosomal protein L16 qualitatively mimics the phenotype of gcd2 mutations in causing derepression of GCN4 expression under nonstarvation conditions. However, deletion of RPL16B also prevents efficient derepression of GCN4 under starvation conditions, indicating that lowering the concentration of 60S subunits and reducing GCD2 function affect translation initiation at GCN4 in different ways. This distinction is in accord with a recently proposed model for GCN4 translational control in which ribosomal reinitiation at short upstream open reading frames in the leader of GCN4 mRNA is suppressed under amino acid starvation conditions to allow for increased reinitiation at the GCN4 start codon.
Mol Cell Biol 1991 Jun
PMID:GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae. 203 26

GCN4 is a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae whose expression is regulated by amino-acid availability at the translational level. GCD1 and GCD2 are negative regulators required for the repression of GCN4 translation under nonstarvation conditions that is mediated by upstream open reading frames (uORFs) in the leader of GCN4 mRNA. GCD factors are thought to be antagonized by the positive regulators GCN1, GCN2 and GCN3 in amino acid-starved cells to allow for increased GCN4 protein synthesis. Previous genetic studies suggested that GCD1, GCD2, and GCN3 have closely related functions in the regulation of GCN4 expression that involve translation initiation factor 2 (eIF-2). In agreement with these predictions, we show that GCD1, GCD2, and GCN3 are integral components of a high-molecular-weight complex of approximately 600,000 Da. The three proteins copurified through several biochemical fractionation steps and could be coimmunoprecipitated by using antibodies against GCD1 or GCD2. Interestingly, a portion of the eIF-2 present in cell extracts also cofractionated and coimmunoprecipitated with these regulatory proteins but was dissociated from the GCD1/GCD2/GCN3 complex by 0.5 M KCl. Incubation of a temperature-sensitive gcdl-101 mutant at the restrictive temperature led to a rapid reduction in the average size and quantity of polysomes, plus an accumulation of inactive 80S ribosomal couples; in addition, excess amounts of eIF-2 alpha, GCD1, GCD2, and GCN3 were found comigrating with free 40S ribosomal subunits. These results suggest that GCD1 is required for an essential function involving eIF-2 at a late step in the translation initiation cycle. We propose that lowering the function of this high-molecular-weight complex, or of eIF-2 itself, in amino acid-starved cells leads to reduced ribosomal recognition of the uORFs and increased translation initiation at the GCN4 start codon. Our results provide new insights into how general initiation factors can be regulated to affect gene-specific translational control.
Mol Cell Biol 1991 Jun
PMID:Complex formation by positive and negative translational regulators of GCN4. 203 27

Transcription initiation from the Bacillus subtilis phage phi 29 late A3 promoter requires the viral protein p4, a transcriptional activator. Protein p4 binds to a region of the A3 promoter, located between nucleotides -50 and -100 relative to the transcription start site, that presents a sequence-directed curvature. This curvature is enhanced when protein p4 binds to the promoter. A number of deletion mutants at the carboxyl end of protein p4 have been constructed and their behavior as transcriptional activators of the late A3 promoter has been investigated. The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation, DNase I footprinting, methylation interference and circular permutation assays. The results suggest that the last 12 amino acid residues of protein p4, six of which are positively charged, although not involved in the specific recognition of the promoter are responsible for part of the bend induced by protein p4 in its binding site. Evidence is presented which suggests that full induction of this curvature is needed for the transcription activation process. A model is proposed for protein p4 interaction with the A3 promoter, in which the bend is induced in two steps: first, two monomers of protein p4 bind to the inverted recognition sequences, subsequent interaction between them generating a bend between these sequences; second, the highly basic carboxyl terminus of protein p4 establishes non-specific electrostatic interactions with the DNA backbone inducing a bend at both ends of the protein p4 binding region.
J Mol Biol 1990 Feb 20
PMID:Bend induced by the phage phi 29 transcriptional activator in the viral late promoter is required for activation. 210 18

The mouse gene Krox-24 is transiently activated during cell cycle reentry. It encodes a protein with three zinc fingers similar to those of the transcription factor Sp1. Here we present a biochemical characterization of the gene products. Krox-24 mRNA is translated into two proteins of 82 and 88 kilodaltons, designated p82Krox-24 and p88Krox-24, respectively. p82Krox-24 is initiated at the first AUG codon of the open reading frame, whereas synthesis of p88Krox-24 starts at a non-AUG codon located upstream. Both proteins were synthesized in HeLa cells infected with recombinant vaccinia viruses expressing Krox-24 cDNAs. Under these conditions, they were found phosphorylated on serine residues and glycosylated. The availability of the proteins made possible the determination of the DNA recognition sequence. In vitro, Krox-24 bound specifically to the sequence 5'-GCG(C/G)GGGCG-3'. This sequence is similar but not identical to the Sp1 target sequence. Insertion of an oligomer for the binding site in cis, close to the herpes simplex virus thymidine kinase promoter, rendered this promoter responsive to Krox-24. Krox-24 is therefore a sequence-specific transcriptional activator. Krox-24-binding sites were found upstream of several serum-inducible genes, raising the possibility that Krox-24 is involved in the regulation of these genes.
Mol Cell Biol 1990 Jul
PMID:The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator. 211 74

The adenovirus E1A gene product is a potent transcriptional activator and nuclear oncoprotein. Like other regulatory proteins, E1A has a short half-life, in the range of 30 to 120 min. This short half-life, which was measured in cells synthesizing E1A, is not observed in cells injected with E1A protein made in bacteria or in vitro. In these cases, E1A is essentially refractory to degradation. In an attempt to reconcile this apparent paradox, we suggested that E1A was marked for degradation during its synthesis. Furthermore, we showed that a domain in the amino terminus of E1A was required for rapid degradation in cells translating E1A mRNA (J. M. Slavicek, N. C. Jones, and J. D. Richter, EMBO J. 7:3171-3180, 1988). In this study, we have used Xenopus laevis oocytes injected with mRNAs encoding altered E1A proteins to show that the amino-terminal tetrapeptide Met-Arg-His-Ile is required for E1A degradation. Even conservative amino acid substitutions in this degradation sequence render it nonfunctional. This degradation sequence can function as a transferable signal, since it induces instability when fused to another normally stable protein. Furthermore, the degradation sequence requires a proximity of no more than six residues from the amino terminus for activity. These data suggest that a trans-acting factor recognizes the amino terminus of E1A during the translation of its message to mark the protein for subsequent destruction.
Mol Cell Biol 1990 Nov
PMID:The degradation sequence of adenovirus E1A consists of the amino-terminal tetrapeptide Met-Arg-His-Ile. 214 91

A novel mammalian regulatory system was created by using the Escherichia coli lac repressor. The lac repressor was converted into a mammalian transcriptional activator by modifying the lac repressor coding region to include a nuclear localization signal from the simian virus 40 (SV40) large tumor antigen and the transcription activation domain from the herpes simplex virus type 1 virion protein 16. The lac activator protein (LAP) fusions were potent activators of several promoters containing lac operator sequences positioned either upstream or downstream of the transcription unit. A single lac operator allowed for transactivation, whereas multiple operators acted synergistically when separated by a small distance. Promoters containing 14 or 21 operator sequences were induced at least 1,000-fold in response to LAP, reaching levels of activity 20 to 30 times greater than that of the SV40 early promoter in HeLa cells. Activation was strongly inhibited by isopropyl-beta-D-thiogalactoside (IPTG), indicating that LAP retained the functions needed for allosteric regulation. LAP was bifunctional, also acting as a repressor of expression of an SV40 promoter containing an operator immediately downstream of the TATA box. Finally, genetic selection schemes were developed such that LAP-expressing cell lines can be generated at high frequency from either established or primary cells in culture.
Mol Cell Biol 1990 Jul
PMID:Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells. 216 73

Endogenous mouse mammary tumor virus (MMTV) proviral transcripts are up regulated during the normal course of B-lymphocyte differentiation. We report here that the regulatory mechanisms which lead to increased levels of MMTV transcripts in differentiating, lipopolysaccharide (LPS)-stimulated normal B cells and in the inducible B-cell lymphoma line CH12 are at least partially distinct from those controlling increases in immunoglobulin and J-chain gene expression. In studies designed to characterize the stimulatory pathways leading to MMTV expression in CH12 cells, we found that stimulation with either LPS or dexamethasone (Dex), a transcriptional activator of MMTV genes, induced not only MMTV expression but also differentiation to antibody secretion. Only Dex-induced and not LPS-induced MMTV expression and differentiation were inhibited by the glucocorticoid antagonist RU486, demonstrating that Dex and LPS stimulate B cells by distinct molecular pathways. Therefore, in B cells, MMTV expression can be regulated via either the conventional hormone receptor-dependent pathway or a hormone receptor-independent pathway. Furthermore, these results suggest that steroid stimulation of B cells can lead to alterations in the expression of other results suggest that steroid stimulation of B cells can lead to alterations in the expression of other steroid-responsive genes that can become involved in the process of B-cell differentiation.
Mol Cell Biol 1990 Aug
PMID:Lipopolysaccharide and dexamethasone induce mouse mammary tumor proviral gene expression and differentiation in B lymphocytes through distinct regulatory pathways. 216 35

CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene. In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc). Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc. Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc. A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2. This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein. Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.
Mol Cell Biol 1990 Sep
PMID:A single amino acid change in CUP2 alters its mode of DNA binding. 216 39

GCN4 is a transcriptional activator of amino acid-biosynthetic genes in the yeast Saccharomyces cerevisiae. GCN2, a translational activator of GCN4 expression, contains a domain homologous to the catalytic subunit of eucaryotic protein kinases. Substitution of a highly conserved lysine residue in the kinase domain abolished GCN2 regulatory function in vivo and its ability to autophosphorylate in vitro, indicating that GCN2 acts as a protein kinase in stimulating GCN4 expression. Elevated GCN2 gene dosage led to derepression of GCN4 under nonstarvation conditions; however, we found that GCN2 mRNA and protein levels did not increase in wild-type cells in response to amino acid starvation. Therefore, it appears that GCN2 protein kinase function is stimulated posttranslationally in amino acid-starved cells. Three dominant-constitutive GCN2 point mutations were isolated that led to derepressed GCN4 expression under nonstarvation conditions. Two of the GCN2(Con) mutations mapped in the kinase domain itself. The third mapped just downstream from a carboxyl-terminal segment homologous to histidyl-tRNA synthetase (HisRS), which we suggested might function to detect uncharged tRNA in amino acid-starved cells and activate the adjacent protein kinase moiety. Deletions and substitutions in the HisRS-related sequences and in the carboxyl-terminal segment in which one of the GCN2(Con) mutation mapped abolished GCN2 positive regulatory function in vivo without lowering autophosphorylation activity in vitro. These results suggest that sequences flanking the GCN2 protein kinase moiety are positive-acting domains required to increase recognition of physiological substrates or lower the requirement for uncharged tRNA to activate kinase activity under conditions of amino acid starvation.
Mol Cell Biol 1990 Jun
PMID:Identification of positive-acting domains in GCN2 protein kinase required for translational activation of GCN4 expression. 218


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