UNIPROT:P51532 - FACTA Search

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Query: UNIPROT:P51532 (transcriptional activator)
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In Rhizobium meliloti the NifA protein plays a central role in the expression of genes involved in nitrogen fixation. The R. meliloti NifA protein has been found to be oxygen sensitive and therefore acts as a transcriptional activator only under microaerobic conditions. In order to generate oxygen-tolerant variants of the NifA protein a plasmid carrying the R. meliloti nifA gene was mutagenized in vitro with hydroxylamine. About 70 mutated nifA genes were isolated which mediated up to 12-fold increased NifA activity at high oxygen concentrations. A cloning procedure involving the combination of DNA fragments from mutated and wild-type nifA genes allowed mapping of the mutation sites within the central part of the nifA gene. For 17 mutated nifA genes the exact mutation sites were determined by DNA sequence analysis. It was found that all 17 mutated nifA genes carried identical guanosine--adenosine mutations resulting in a methionine--isoleucine exchange (M217I) near the putative nucleotide binding site within the central domain. Secondary structure predictions indicated that the conformation of the putative nucleotide binding site may be altered in the oxygen-tolerant NifA proteins. A model is proposed which assumes that at high oxygen concentrations the loss of activity of the R. meliloti NifA protein is due to a conformational change in the nucleotide binding site that may abolish binding or hydrolysis of the nucleotide. Such a conformational change may be blocked in the oxygen-tolerant NifA protein, thus allowing interaction with the nucleotide at high oxygen concentrations.
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PMID:A defined amino acid exchange close to the putative nucleotide binding site is responsible for an oxygen-tolerant variant of the Rhizobium meliloti NifA protein. 140 89

Inactivation of the centromere-binding factor 1 (CBF1) gene results in yeast strains that require methionine for growth. This auxotrophy is due to the inability of such strains to concentrate and assimilate sulfate from the medium. Northern (RNA) blot experiments reveal that the CBF1 protein is required for full induction of MET25 and MET16 gene transcription. However, we show that induction of the sulfate assimilation pathway is not achieved solely by CBF1. This induction also requires the integrity of a positive trans-acting factor, encoded by the MET4 gene. The MET4 gene was cloned, and its sequence reveals that it encodes a protein related to the family of the bZIP transcriptional activators. Evidence that MET4 is a transcriptional activator was provided by demonstrating that DNA-bound LexA-MET4 fusion proteins stimulate expression of a nearby promoter. The use of LexA-MET4 fusion proteins also reveals that the leucine zipper of MET4 is required for the recognition of the MET25 promoter. Moreover, an 18-bp fragment of the MET25 5' upstream region was found to confer S-adenosylmethionine-dependent regulation of a fusion gene. This regulation was shown to depend on both MET4 and CBF1. The obtained results suggest that the binding of CBF1 to its cognate sequences increases the ability of MET4 to stimulate transcription of the MET genes.
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PMID:MET4, a leucine zipper protein, and centromere-binding factor 1 are both required for transcriptional activation of sulfur metabolism in Saccharomyces cerevisiae. 154 23

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.
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PMID:GCD2, a translational repressor of the GCN4 gene, has a general function in the initiation of protein synthesis in Saccharomyces cerevisiae. 203 26

The centromere and promoter factor 1 (CPF1) binds specifically in vitro and in vivo to an octanucleotide (RTCACRTG). This sequence is found in the centromere DNA element I (CDEI) of yeast centromeres and upstream from a number of transcription units including MET25, GAL2 and TRP1. Inactivation of the CPF1 gene results in three phenotypes; slow growth, a partial loss of centromere function and methionine auxotrophy. These phenotypes correlate well with the known binding sites for CPF1 and have led to the suggestion that CPF1 functions as a kinetochore protein at centromeres and as a transcriptional activator at promoters such as MET25. By analysing transcription from the MET25, GAL2, and TRP1 genes in cpf1 strains, we demonstrate that CPF1 plays no direct role in their transcriptional regulation. Further evidence in support of this comes from the analysis of point mutations in the basic region of CPF1 that affect DNA binding. A strain expressing a non-DNA bound form of CPF1 is phenotypically Met+, shows normal growth rate but has sub-optimal centromere function. We conclude that a DNA-bound form of CPF1 is required for the kinetochore function but not for maintaining methionine prototrophy.
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PMID:DNA binding of CPF1 is required for optimal centromere function but not for maintaining methionine prototrophy in yeast. 205 54

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.
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PMID:The degradation sequence of adenovirus E1A consists of the amino-terminal tetrapeptide Met-Arg-His-Ile. 214 91

Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) have two nonstructural trans-acting regulatory genes, tax and rex, located in the 3' region of the viral genome. The tax gene product (HTLV-I p40tax and HTLV-II p37tax) is the transcriptional activator of the viral long terminal repeat. The rex gene encodes two protein products, p27rex/p21rex and p26rex/p24rex in HTLV-I and HTLV-II, respectively. Rex acts posttranscriptionally to facilitate accumulation of full-length gag/pol and singly spliced env mRNA in the cytoplasm of HTLV-infected cells. Previous studies showed that the first ATG of the rex gene is critical for Rex production and function. The importance of the internal ATGs to Rex function is not known. However, in vitro mutagenesis of the HTLV-I rex gene has provided indirect evidence which suggests that p21rex, and by analogy HTLV-II p24rex, results from initiation at an internal AUG of the tax/rex mRNA. By using an infectious molecular clone of HTLV-II, we investigated the importance of the internal ATGs of the rex gene on Rex protein production and function. Our results indicate that p24rex of HTLV-II is not initiated at an internal AUG and that the internal methionine codons are not crucial to the function of the rex gene and, ultimately, the transforming properties of the virus.
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PMID:The internal methionine codons of human T-cell leukemia virus type II rex gene are not required for p24rex production or virus replication and transformation. 239 33

Nitric oxide (NO) reductase is an integral membrane component of the anaerobic respiratory chain of Pseudomonas stutzeri that transforms nitrate to dinitrogen (denitrification). The enzyme catalyzes the reduction of NO to nitrous oxide. The structural genes for the NO reductase complex, norC and norB, were sequenced and their organization established by primer extension and Northern blot analysis. The norCB genes encoding the cytochrome c and cytochrome b subunits of the enzyme are contiguous and transcribed as a single 2.0-kb transcript. The promoter region has a canonical recognition motif for the transcriptional activator protein Fnr, centered at -40.5 nucleotides from the initiation site of transcription. No similarity of the derived gene products to known cytochromes of b- or c-type was found in a data bank search. Post-translational processing of the two subunits was limited to the removal of the terminal methionine to leave an N-terminal serine in either subunit. The mature cytochrome c subunit (16508Da, 145 residues) is predicted to be a bitopic protein with a single membrane anchor. The mature cytochrome b subunit (53006Da, 473 residues) is a putatively polytopic, strongly hydrophobic membrane-bound protein with 12 potential transmembrane segments. Several histidine and proline residues were identified with potentially structural and/or functional importance. Mutational inactivation of NO reductase by deletion of norB or the norCB genes affected strongly the in vivo activity of respiratory nitrite reductase (cytochrome cd1), but to a much lesser extent the expression level of this enzyme. In turn, mutational inactivation of the structural gene for cytochrome cd1, nirS, or loss of in vivo nitrite reduction by mutation of the nirT gene, encoding a presumed tetraheme cytochrome, lowered the expression level of NO reductase to 5-20%, but hardly its catalytic activity. The cellular concentration of NO reductase increased again on restoration of nitrite reduction in the nirS::Tn5 mutant MK202 by complementation with nirS or with the heterologous nirK gene, encoding the Cu-containing nitrite reductase from Pseudomonas aureofaciens. Thus, NO may be required as an inducer for its own reductase. Our results show that the nitrite-reducing system and the NO-reducing system are not operating independently from each other but are interlaced by activity modulation and regulation of enzyme synthesis.
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PMID:Nitric oxide reductase from Pseudomonas stutzeri. Primary structure and gene organization of a novel bacterial cytochrome bc complex. 750 88

The DNA genome of caulimoviruses contains a set of essential genes: I (movement gene), IV (major capsid protein gene), V (reverse transcriptase gene), and VI (gene coding for a post-transcriptional activator of the expression of other virus genes). In peanut chlorotic streak caulimovirus (PCISV), three ORFs, A, B, and C, are located between genes I and IV. They are dissimilar to other caulimovirus ORFs. ORF VII of PCISV is a homolog of ORF VII of soybean chlorotic mottle caulimovirus (SoCMV), but is not similar to the nonconserved ORF VII in other caulimoviruses. The sequence complementary to a portion of tRNA(Met), thought to be essential for the priming of minus-strand DNA synthesis in caulimoviruses, is located within the coding sequence of ORF A. To explore the functional significance of ORFs VII, A, B, and C, various mutations were engineered into an infectious DNA clone of PCISV. ORFs VII and B are shown to be dispensable, while ORFs A and C are essential. ORF C is a possible functional equivalent of gene III in other caulimoviruses. Sequences within ORF A that are required for efficient priming of minus-strand synthesis are likely to extend beyond the 12-bp tRNA-binding site. Complete deletion of ORF VII was correlated with severe symptoms, notably with the necrosis of apical meristems. Significance of these observations for the understanding of replication and pathogenesis of plant pararetroviruses and for the improvement of caulimovirus-based expression vectors is discussed.
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PMID:Molecular analysis of the essential and nonessential genetic elements in the genome of peanut chlorotic streak caulimovirus. 753 17

GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. Previous work suggested that the principal activation domain of GCN4 is a highly acidic segment of approximately 40 amino acids located in the center of the protein. We conducted a mutational analysis of GCN4 with a single-copy allele expressed under the control of the native promoter and translational control elements. Our results indicate that GCN4 contains two activation domains of similar potency that can function independently to promote high-level transcription of the target genes HIS3 and HIS4. One of these domains is coincident with the acidic activation domain defined previously; the other extends over the N-terminal one-third of the protein. Both domains are partially dependent on the coactivator protein ADA2. Each domain appears to be composed of two or more small subdomains that have additive effects on transcription and that can cooperate in different combinations to promote high-level expression of HIS3 and HIS4. At least three of these subdomains are critically dependent on bulky hydrophobic amino acids for their function. Five of the important hydrophobic residues, Phe-97, Phe-98, Met-107, Tyr-110, and Leu-113, fall within a region of proposed sequence homology between GCN4 and the herpesvirus acidic activator VP16. The remaining three residues, Trp-120, Leu-123, and Phe-124, are highly conserved between GCN4 and its Neurospora counterpart, cpc-1. Because of the functional redundancy in the activation domain, mutations at positions 97 and 98 must be combined with mutations at positions 120 to 124 to observe a substantial reduction in activation by full-length GCN4, and substitution of all eight hydrophobic residues was required to inactivate full-length GCN4. These hydrophobic residues may mediate important interactions between GCN4 and one or more of its target proteins in the transcription initiation complex.
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PMID:The transcriptional activator GCN4 contains multiple activation domains that are critically dependent on hydrophobic amino acids. 786 16

Active E1 component of Pseudomonas putida branched-chain-oxoacid dehydrogenase was purified from P. putida strains carrying pJRS84 which contains bkdR (encoding the transcriptional activator) and bkdA1 and bkdA2 (encoding the alpha and beta subunits). Expression was inducible, however, 45-, 39- and 37-kDa proteins were produced instead of the expected 45-kDa and 37-kDa proteins. The 45-kDa protein was identified as E1 alpha and the 37-kDa and 39-kDa proteins were identified as separate translational products of bkdA2 by their N-terminal sequences. The N-terminal amino acid of the 39-kDa protein was leucine instead of methionine. The 45-, 39- and 37-kDa proteins were also produced in wild-type P.putida. Translation of bkdA1 and bkdA2 from an Escherichia coli expression plasmid produced only 45-kDa and 39-kDa proteins, with N-terminal methionine on the 39-kDa protein. The insertion of guanine residues 5' to the first ATG of bkdA2 did not affect expression of E1 beta in P. putida including the N-terminal leucine which appears to eliminate the possibility of ribosome jumping. The Z-average molecular mass of the E1 component was determined by sedimentation equilibrium to be 172 +/- 9 kDa compared to a calculated value of 166 kDa for the heterotetramer and a Stokes radius of 5.1 nm. E1 alpha Ser313, which is homologous to the phosphorylated residue of rat liver E1 alpha, was converted to alanine resulting in about a twofold increase in Km, but no change in Kcat. S315A and S319A mutations had no effect on Km or Kcat indicating that these residues do not play a major part in catalysis of E1 alpha beta 2.
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PMID:Purification of active E1 alpha 2 beta 2 of Pseudomonas putida branched-chain-oxoacid dehydrogenase. 852 48

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