Adenovirus E1A proteins prepare the host cell for viral replication, stimulating cell cycling and viral transcription through interactions with critical cellular regulatory proteins such as RB and CBP. Here we show that the E1A zinc-finger domain that is required to activate transcription of viral early genes binds to a host-cell multiprotein complex containing homologues of yeast Srb/Mediator proteins. This occurs through a stable interaction with the human homologue of Caenorhabditis elegans SUR-2, a protein required for many developmental processes in the nematode. This human Srb/Mediator complex stimulates transcription in vitro in response to both the E1A zinc-finger and the herpes simplex virus VP16 activation domains. Interaction with human Sur-2 is also required for transcription to be activated by the activation domain of a transcription factor of the ETS-family in response to activated mitogen-activated protein (MAP) kinase.
Host-range mutants of adenovirus 5 that contain a defect in region ElA (0-4.5 units) fail to replicate in HeLa cells and to transform rodent cells. In HeLa cells, these mutants synthesize only the two RNAs from EIA that share the same 5' and 3' termini but differ in length by the amount of internal sequence removed by splicing. RNA In eukaryotic cells and their viruses, modulation ofgene expression by a specific gene product has been shown in only a few instances. Examples of these include early RNA transcription in papovaviruses (1) and the regulation of galactose catabolism in yeast (2). In this paper, we report the characterization of adenovirus mutants and describe a specific gene product that regulates the expression of other viral genes.Early in adenovirus infection, five transcription regions (E1A and E1B and E2-E4) of the 35-kilobase genome are independently promoted (3) and produce one or more overlapping RNAs, most ofwhich have had internal sequences removed by splicing (4). Studies using wild-type and mutant viruses show that the onset, peak, and decline oftranscripts from these early regions is temporal and initiated with the expression of ElA (5-8). The two RNAs derived from ElA share the saime overlapping 5' and 3' termini but differ in length by the amount of intervening sequence removed (4). Adenovirus S (AdS) hostrange (Ad5hr) mutants defective within region ElA transcribe only the two cytoplasmic RNAs that originate from this region (5, 6). Nuclease S1 mapping shows that these ElA RNAs are indistinguishable from those of wild-type virus (5). These data imply that either the ElA RNAs or their encoded polypeptides (9) are required for the accumulation ofcytoplasmic transcripts from other early regions.In this paper, we analyze how the defect within E1A of each
Uracil-DNA glycosylases (UDGs) are evolutionarily conserved DNA repair enzymes that initiate the base excision repair pathway and remove uracil from DNA. The UDG superfamily is classified into six families based on their substrate specificity. This review focuses on the family I enzymes since these are the most extensively studied members of the superfamily. The structural basis for substrate specificity and base recognition as well as for DNA binding, nucleotide flipping and catalytic mechanism is discussed in detail. Other topics include the mechanism of lesion search and molecular mimicry through interaction with uracil-DNA glycosylase inhibitors. The latest studies and findings detailing structure and function in the UDG superfamily are presented.
We describe a simple procedure for isolating specific mRNAs and for mapping them to the regions of the DNA from which they originate. The method involves hybridization of total cytoplasmic RNA to restriction fragments of DNA that have been fractionated in agarose gels and immobilized on nitrocellulose filters. The hybridization-selected RNAs are eluted and translated in a cell-free system in order to identify their encoded polypeptides. Optimal hybridization and filter washing conditions are given for selection of mRNAs from adenovirus 2-infected cells and transformed cells.Synthesis of individual polypeptides directed by purified mRNAs in a cell-free translation system provides compelling evidence for the expression of specific genes. The ability to purify functional mRNAs is a prerequisite for understanding some of the detailed mechanisms involved in gene regulation. Characterization of purified mRNAs by cell-free translation, in combination with other techniques, can yield information about the spatial and topological arrangement of transcripts along the genome, the location of protein coding regions within the mRNA, mRNA structure, and mRNA abundance.Several methods for isolating specific mRNAs have been developed. DNA, either in liquid or bound to immobilized supports, has been used to hybridize and thereby sequester specific mRNAs from the plethora of cellular RNA species. These methods have included isolation of RNA-DNA hybrids by: selective binding to hydroxylapatite (1); selective exclusion through agarose (2) or Sepharose 4B (3); the use of DNA covalently bound to cellulose (4, 5) or Sepharose (6); DNA bound directly to nitrocellulose (7-9); DNA enzymatically synthesized and covalently bound to oligo(dT)-cellulose (10).Here we describe an efficient method by which specific mRNAs can be purified and used to determine the location of these mRNAs with respect to their DNA coding regions. This method relies on hybridization of total cytoplasmic RNA to restriction fragments of DNA which have been immobilized on nitrocellulose filters. The hybridization-selected mRNAs are eluted from the DNA and identified by the polypeptide products that are synthesized in a reticulocyte cell-free system. The map positions of the mRNA transcripts on the DNA can be determined directly because the genomic coordinates of the DNA restriction fragments are known.This procedure has several advantages. Most importantly, purification of the DNA restriction fragments is not required. Rather, DNA restriction fragments are fractionated by electrophoresis in agarose gels and then directly transferred to the nitrocellulose filter membrane by the method of Southern (11). In this way, many different DNA fragments may be easily handled in a single experiment. In addition, the nitrocellulose filters that contain the bound DNA fragments can be used several times. By this procedure, it is possible to identify the translation product of a specific mRNA which is otherwise obscured by the large number of polypeptides that are synthesized...
The 289R EMA protein of adenovirus transactivates a variety of viral and cellular promoters through protein-protein interactions. In earlier studies, mutational analyses of the ElA transactivating domain identified residues that are critical for transactivation and implied that the zinc finger region of the transactivating domain binds a transcription factor. Also, the ElA activation domain was found to bind to the TATA box binding protein (TBP) in vitro. Here, we tested the significance of the E1A-TBP interaction for ElA transactivation by analyzing the effects of conservative substitutions at each of the 49 residues of the ElA activation domain. Seven of the substitutions significantly diminished TBP binding in vitro. All of these were in the zinc finger region and were defective for transactivation in vivo. The perfect correlation between reduced TBP binding and transactivation argues strongly that a direct interaction between the ElA activation domain and TBP is critical to the mechanism of ElA activation. This genetic analysis leads us to further suggest that another factor, which is limiting, is also necessary for ElA-mediated transactivation.The adenovirus ElA 289R protein is a potent transactivator of a variety of viral and cellular promoters (reviewed in refs. 1 and 2). The absence of a common cis element in E1A-inducible promoters (1) and the weak, nonspecific DNA binding activity of the 289R protein (3) suggests that ElA stimulates transcription through protein-protein interactions with cellular transcription factors.The transactivation function of the 289R protein maps to an internal stretch of 49 amino acids (residues 140-188) (4, 5). The salient feature of the ElA transactivating domain is a metal binding structure that is formed by four cysteine residues, which coordinate a single zinc atom (6, 7). The importance of a structurally intact Cys4 zinc finger is highlighted by the fact that replacing any of the four cysteine residues produces a mutant protein that is incapable of activating transcription.Individually substituting every amino acid in the transactivating domain identified additional residues that are also critical for transactivation (8). From this study, ElA proteins containing mutations in a contiguous stretch of residues C terminal to the zinc finger were found not only to be defective for transactivation but also to display a strong transdominant negative phenotype. Furthermore, this study revealed that the transactivating domain is composed of two functionally distinct regions, a finger region (residues 147-177) and a carboxyl region (residues 183-188), each of which is postulated to bind to a different cellular protein. Indeed, two distinct classes of factors, the TATA box binding protein (TBP) and the activating transcription factor family (ATF), have been recently shown to directly interact with the transactivating domain of ElA (9, 10, 34). Taken together, these studies suggest that ElA transactivates by interacting with sequence-specific DNA binding transcription factor...
The 289R ElA protein of adenovirus stimulates transcription of early viral and certain cellular genes. trans-Activation requires residues 140 to 188, which encompass a zinc finger. Several studies have indicated that trans-activation by ElA is mediated through cellular transcription factors. In particular, the ability of the trans-dominant ElA point mutant hr5 (Ser-185 to Asn) to inhibit wild-type ElA trans-activator (40, 49, 63). The independent nature of the trans-activating domain was revealed by the ability of a synthetic peptide comprising the 49-amino-acid domain to stimulate transcription from an early viral promoter in microinjection experiments (31).The salient structural feature of the trans-activating domain is a C4-type zinc finger sequence defined by C154, C157, C171, and C174 (7). We have shown that the 289R protein binds a single zinc ion. Since ElA does not bind DNA directly, it is different from other regulatory proteins which mediate DNA binding through their zinc fingers, e.g., TFIIIA (16,39), Spl (21), and the glucocorticoid receptor (11). However, the zinc finger of ElA is clearly an essential component of the trans-activating domain, since individually substituting glycine or serine for each of the four cysteine residues destroyed trans-activation (7). Substituting serine for C157, C171, or C174 also destroyed zinc binding, whereas replacing C154 with serine surprisingly had no effect on zinc binding. X-ray absorption fine structure analysis showed that the zinc in the wild-type (wt) 289R protein is indeed coordinated to four cysteine residues; the single amino acid substitution of serine for C154 resulted in the recruitment of two histidines on the left side of the finger to bind zinc in conjunction with C171 and C174 (59). These results argue that for trans-activation to occur, zinc must be bound by ElA in a specific way.Following our report of the identification of an ElA trans-dominant point mutant, hr5 (185SN) (13), mutant forms of several other viral trans-activator proteins which also exhibit trans-dominance have been identified, including Tax of human T-cell lymphotropic virus type 11 (58), VP16 of herpes simplex virus type 1 (57), and Rev and Tat of human 4287
The NF-B p50/p50 homodimer is mainly associated with transcriptional repression. Previously, we demonstrated that phosphorylation of NF-B p50 Ser 337 is critical for DNA binding. Here, we report that p50 Ser 337 is constitutively phosphorylated by the protein kinase A catalytic subunit (PKAc) in three different cell types, which may account for the constant binding of p50/p50 to DNA in unstimulated cells. This was demonstrated first by showing that treatment of cells with PKAc-specific inhibitors blocked p50/p50 DNA binding. Second, phosphorylation of p50 by PKAc was prevented by substitution of Ser 337 to alanine. Third, both p50 and PKAc proteins as well as kinase activity that phosphorylates p50 were found to co-fractionate following gel filtration chromatography. Finally, PKAc and p50 were shown to be able to reciprocally co-immunoprecipitate one another, and their physical association was blocked by a PKA catalytic site inhibitory peptide. This indicates that phosphorylation of p50 Ser 337 involves direct contact with the PKAc catalytic center. In contrast to the dramatic elevation of nuclear p50/p65 heterodimers induced by tumor necrosis factor ␣, DNA binding of p50/ p50 homodimers was not greatly altered. Taken together, these findings reveal for the first time that there is a direct interaction between PKAc and p50 that accounts for constitutive phosphorylation of p50 Ser 337 and the existence of DNA bound p50/p50 in the nuclei of most resting cells. This mechanism of DNA binding by p50/p50 following phosphorylation of Ser 337 by PKAc may represent an important means for maintaining stable negative regulation of NF-B gene expression in the absence of extracellular stimulation.The nuclear factor NF-B is a transcription factor identified by Sen and Baltimore nearly 20 years ago (1). NF-B plays a critical role in transcription regulation of genes involved in immune response, inflammation, cell proliferation, differentiation, apoptosis, and oncogenesis (2-6). In vertebrates, five members of NF-B family have been identified. These include RelA (p65), RelB, c-Rel, NF-B1 (p50/p105), and NF-B2 (p52/ p100). All of these proteins share a highly conserved Rel homology domain termed RHD within the N-terminal 300 amino acid region. The RHD is responsible for DNA binding, dimerization, nuclear translocation, and interaction with IB inhibitory proteins. Although all NF-B family members can bind to DNA, only p65, RelB, and c-Rel contain a transactivation domain in their C-terminal regions.The NF-B family members can form various homodimers and heterodimers among which the p50/p65 heterodimer is the most abundant and studied species. In most resting or unstimulated cells, p50/p65 heterodimers are confined in the cytoplasm in an inactive form by forming a complex with IB proteins. Treatment of cells with NF-B stimuli such as cytokines, mitogens, and bacterial lipopolysaccharide leads to phosphorylation of IB by IB kinase and subsequent ubiquitination and degradation of IB by the 26 S proteasome (7,8). This allows NF-B to ...
It has been demonstrated that phosphorylation of the p50 subunit of NF-B is required for efficient DNA binding, yet the specific phospho-residues of p50 have not been determined. In this study, we substituted all of the serine and conserved threonine residues in the p50 Rel homology domain and identified three serine residues, Ser 65 , Ser 337 , and Ser 342 , as critical for DNA binding without affecting dimerization. Although substitution with negatively charged aspartic acid at each of these positions failed to restore DNA binding, substitution with threonine, a potential phospho-acceptor, retained DNA binding for residues 65 and 337. In particular, Ser 337 , in a consensus site for protein kinase A (PKA) and other kinases, was shown to be phosphorylated both in vitro and in vivo. Importantly, phosphorylation of Ser 337 by PKA in vitro dramatically increased DNA binding of p50. This study shows for the first time that the DNA binding ability of NF-B p50 subunit is regulated through phosphorylation of residue Ser 337 , which has implications for both positive and negative control of NF-B transcription.The transcription factor NF-B acts as a central regulator of inflammatory, immune, and stress responses by controlling gene expression of cytokines, chemokines, immunoreceptors, antigen-presenting proteins, growth factors, transcription factors, cell adhesion molecules, stress response proteins, and apoptotic regulators (1, 2). Members of Rel/NF-B transcription factor family include Dorsal, Relish, and Dif in Drosophila and p65 (RelA), RelB, c-Rel, p50/p105, and p52/p100 in vertebrates. All of these proteins have a highly conserved DNAbinding and dimerization domain called the Rel homology domain. The vertebrate Rel family proteins can form homodimers or heterodimers that bind to 10-basepair B sites in the promoters of NF-B target genes (1, 3, 4). The most common and important NF-B transactivating species is the p50/p65 heterodimer. The p65 subunit, which contains a transactivation domain in the C terminus of the Rel homology domain, is responsible for the ability of NF-B to stimulate transcription. By contrast, p50 subunit, which lacks a transactivation domain, functions mainly in NF-B DNA binding (5-9). Another important dimeric species, the p50/p50 homodimer, serves mainly as a negative regulator of NF-B activity through competing with p50/p65 for NF-B response elements on DNA and through its association with co-repressor histone deacetylase (10, 11). The x-ray crystal structures of p50/p65 heterodimer and p50/p50 and p65/p65 homodimer binding to DNA have revealed a conformation often referred to as a "butterfly" (12-16). The DNA recognition loop (L1) in the N-terminal half of NF-B Rel homology domain mediates base-specific DNA contacts, whereas the C-terminal half is responsible for dimerization and nonspecific DNA contacts (17).NF-B activity is regulated by nuclear translocation. In most cell types, p50/p65 heterodimers exist in the cytoplasm as an inactive form associated with the inhibitor protein, I B. A w...
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