Upstream activating sequence

About: Upstream activating sequence is a research topic. Over the lifetime, 1633 publications have been published within this topic receiving 100112 citations.

Specific protein binding to far upstream activating sequences in polymerase II promoters

Abstract: A binding activity specific for the upstream activating sequence of the GAL1-GAL10 promoter of Saccharomyces cerevisiae has been purified 220-fold on the basis of a nitrocellulose filter-binding assay. The binding activity is enriched in a nuclear preparation and is likely to be the GAL4 gene product. DNase I-protection mapping patterns reveal binding to two 30-base-pair regions at the boundaries of the sequence. A nearly identical mapping pattern is obtained with the coordinately regulated GAL7 promoter. The four 30-base-pair regions of binding in the two promoters are closely homologous, with a core consensus sequence of C-G-CG-TG-C-A-A-C-A-G-T-G-C-T-C-C-G-A-A- GC-G-A-T. A synthetic oligonucleotide with such a sequence competes with the upstream activating sequence in the binding reaction.

Escherichia coli Promoters with UP Elements of Different Strengths: Modular Structure of Bacterial Promoters

Abstract: Promoter sequences involved in recognition by Escherichia coli RNA polymerase (RNAP) were identified from comparisons of a large number of known promoters and from mutational analyses (28, 29, 38, 62). These sequences, the −10 and −35 hexamers (5′ TATAAT 3′ and 5′TTGACA 3′, respectively), are recognized by the ς70 subunit of RNAP (11). The strength of a promoter correlates generally with its degree of identity to these sequences and with the length of the spacer between them (the homology score [42]), although exceptions to this rule have been described (7, 26). It was proposed more than 10 years ago that optimal transcription activity could be achieved by different combinations of promoter elements, including not only the −10 and −35 hexamers, but also upstream and downstream regions (7). In accord with this suggestion, RNAP protects regions both upstream and downstream of the −10 and −35 hexamers in footprints (8, 45, 47, 56), and A+T-rich sequences upstream of the −35 hexamer in several E. coli or Bacillus subtilis promoters were found to increase transcription in vitro in the absence of accessory proteins (3, 19, 31, 37, 39, 50, 54). Phased A-tracts inserted upstream of the −35 region in various promoter constructs were also shown to increase transcription (6, 12, 24). The A+T-rich region upstream of −40 in the rRNA promoter rrnB P1, the UP element, increases transcription 30- to 70-fold by binding the RNAP α subunit (13, 50, 53). A consensus UP element sequence was determined by using in vitro selection for upstream sequences that promote rapid RNAP binding to the rrnB P1 promoter, followed by in vivo screening for high promoter activity. The consensus UP element consists of alternating A- and T-tracts (13). UP elements matching the consensus increased promoter activity as much as 326-fold, about 5-fold more than the wild-type rrnB P1 UP element. UP elements were also identified in other promoters, for example, the flagellin (hag) promoter of B. subtilis (18), the PL2 promoter of phage lambda (25), and the Pe promoter of phage Mu (61), although the effects of these elements were not as large as that of rrnB P1. UP elements also function in promoters recognized by RNAP holoenzymes with alternate ς factors (18). UP elements are not as highly conserved as the −10 and −35 elements and were not described in studies comparing the large sets of E. coli promoters used to define the consensus hexamers (28, 29, 38). However, A+T-rich sequences were identified as a prominent feature of a subset of E. coli promoters (the −44 motif [23]), and a recent E. coli promoter analysis (48) identified two A+T-rich regions at upstream positions corresponding to those crucial for UP element function (14). A+T-rich upstream sequences were also identified in compilations of B. subtilis and Clostridium promoters (27, 30). We have proposed that UP elements may be a recognition feature in many bacterial promoters (13, 53), but in most promoters, the role of upstream sequences has not been evaluated experimentally. Therefore, in this paper, we have examined the role of upstream sequences from six promoters (rrnB P2, rrnD P1, RNA II, merT, lac, and λ pR). We find that several of the sequences function as UP elements and that their effects on promoter activity differ, correlating generally with the degree of similarity to the UP element consensus sequence. These results support the model that bacterial promoters consist of at least three modules, not just −10 and −35 elements. We also show that upstream protection in footprints is not a reliable indicator of UP element function.

cis and trans activation of globin gene transcription in transient assays.

Abstract: We examined the effects of the simian virus 40 enhancer sequence on transcription of cloned human alpha- and beta-globin genes shortly after their introduction into cultured mammalian cells. We find that (i) detectable transcription of the beta-globin gene but not the alpha-globin gene requires linkage to the enhancer; (ii) the enhancer increases the amount of beta-globin RNA at least 100-fold but results in only a 5- to 10-fold increase in the amount of alpha-globin RNA; (iii) plasmid replication does not increase the level of beta-globin RNA, regardless of linkage to the enhancer, but does result in an approximately equal to 50-fold increase in the level of alpha-globin RNA; (iv) the enhancer is not required for and does not increase transcription of either gene in 293 cells, an adenovirus 5-transformed human kidney cell line. We also show that an enhancer sequence is not required for activity of the normally enhancer-dependent simian virus 40 early promoter in 293 cells, indicating that these cells contain a trans-acting factor(s) that circumvents the requirement for the enhancer sequence.

Mammalian p53 can function as a transcription factor in yeast

Abstract: p53 has previously been shown to contain a transactivation domain using GAL4 fusion proteins and to bind specifically to a 33 base pair DNA sequence in immunoprecipitation assays. We show here that mammalian p53 expressed in S. cerevisiae is able to activate transcription of a reporter gene placed under the control of a CYC1 hybrid promoter containing the 33 base pair p53-binding sequence. The activation is dependent on the orientation and number of copies of the binding site. Three p53 mutants commonly found in human tumours, 175H, 248W and 273H, are unable to activate transcription. A fourth human p53 mutant, 285K, is temperature-sensitive for transcriptional activation. Murine p53 activates transcription from the same sequence. The murine 135V mutant, which is temperature-sensitive for mammalian cell transformation, is also temperature-sensitive for transcriptional activation. There is a much better correlation between mutation and transcriptional competence than between mutation and the structure of p53 determined with conformation-sensitive antibodies. We have therefore developed a simple transcription assay for p53 mutation in which yeast are transfected with p53 PCR products and mutation is scored on X-gal plates.

A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation

Abstract: The interleukin 2 (IL-2) gene is subject to two types of regulation: its expression is T-lymphocyte-specific and it is acutely dependent on specific activation signals. The IL-2 transcriptional apparatus integrates multiple types of biochemical information in determining whether or not the gene will be expressed, using multiple diverse transcription factors that are each optimally activated or inhibited by different signaling pathways. When activation of one or two of these factors is blocked IL-2 expression is completely inhibited. The inability of the other, unaffected factors to work is explained by the striking finding that none of the factors interacts stably with its target site in the IL-2 enhancer unless all the factors are present. Coordinate occupancy of all the sites in the minimal enhancer is apparently maintained by continuous assembly and disassembly cycles that respond to the instantaneous levels of each factor in the nuclear compartment. In addition, the minimal enhancer undergoes specific increases in DNase I accessibility, consistent with dramatic changes in chromatin structure upon activation. Still to be resolved is what interaction(s) conveys T-lineage specificity. In the absence of activating signals, the minimal IL-2 enhancer region in mature T cells is apparently unoccupied, exactly as in non-T lineage cells. However, in a conserved but poorly studied upstream region, we have now mapped several novel sites of DNase I hypersensitivity in vivo that constitutively distinguish IL-2 producer type T cells from cell types that cannot express IL-2. Thus a distinct domain of the IL-2 regulatory sequence may contain sites for competence- or lineage-marking protein contacts.