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.

A protein-binding site in the c-myc promoter functions as a terminator of RNA polymerase II transcription.

Abstract: Termination of transcription not only allows polymerases that have completed RNA synthesis to recycle, but it also has important functions in transcriptional regulation and in preventing promoter interference. The molecular basis for termination by RNA polymerase II (pol II} is unclear, however. We have identified a termination site in the promoter region of the c-myc gene, whose function correlates with DNA binding by a nuclear factor. When the c-myc gene was transcribed in injected Xenopus oocytes or a HeLa nuclear extract, a fraction of RNA initiated at the first promoter, P1, terminated at two positions, T1A and TIB, which flank the TATA box of the second promoter, P2. T1B is a T-rich sequence that resembles previously identified attenuation sites, but TIA appears to represent a different class of termination site. TIA is situated -10 bases upstream of an element that overlaps the P2 TATA box. Mutagenesis of this element affected both the efficiency and the position at which termination occurred. A 28-base sequence including this element caused a low level of termination when inserted into the ~-globin gene in either orientation. This sequence bound a factor called TBF I (terminator-binding factorl, whose binding specificity correlated with T1A terminator function. We suggest that TBF I may function as a pol II termination factor.

The upstream factor-binding site is not essential for activation of transcription from the adenovirus major late promoter.

Abstract: An adenovirus major late promoter (MLP) has been constructed with a 4-bp alteration in the sequence which binds the transcription factor known as USF or MLTF. This upstream element has often been considered necessary and sufficient for maximal transcription of the MLP. A duplex oligonucleotide containing the mutant sequence was not capable of binding specific proteins in a band shift assay, nor was it capable of inhibiting such binding by the wild-type sequence. In an in vitro assay, the mutant sequence was incapable of inhibiting transcription from a duplex sequence containing the MLP, whereas the wild-type sequence could. These two pieces of evidence suggest that the sequence is functionally impaired. Surprisingly, a virus containing the mutant MLP had a normal replication phenotype. On more detailed examination however, we show that the mutant viral MLP was deficient in transcription at 9 h postinfection but that the rate of transcription was close to normal by 20 h postinfection. An inverted CAAT box located immediately upstream of the USF-binding element was not previously thought to be of importance to the functioning of the MLP. However, a single point mutation in the CAAT box, placed in the USF mutant background, had a marked effect upon transcription from the MLP. This result suggests that the MLP may exhibit functional redundancy in which either the USF-binding site or the CAAT box can serve as an upstream promoter element. Neither of the mutant viruses displayed any change in the levels of the divergent IVa2 transcription unit, suggesting that the levels of divergent transcription are not determined by competition for limiting transcription factors.

Phosphorylation influences the binding of the yeast RAP1 protein to the upstream activating sequence of the PGK gene.

Abstract: Yeast repressor activator protein 1 (RAP1) binds in vitro to specific DNA sequences that are found in diverse genetic elements. Expression of the yeast phosphoglycerate kinase gene (PGK) requires the binding of RAP1 to the activator core sequence within the upstream activating sequence (UAS) of PGK. A DNA fragment Z+ which contains the activator core sequence of the PGK(UAS) has been shown to bind RAP1. Here we report that phosphatase treatment of RAP1 affected its binding to the PGK(UAS) but that this depended on the nature of the sequence flanking the 5' end of the activator core sequence. When the sequence flanking the 5' end of the activator core sequence was different from the PGK RAP1-binding site, phosphatase treatment of RAP1 decreased its binding to the DNA. When the 5' end of the binding site was a match to the PGK RAP1-binding site dephosphorylation of RAP1 increased RAP1 binding to the DNA. These observations were reproduced when the minimal functional DNA-binding domain of the RAP1 protein was used, implicating a phosphorylation-dependent binding of RAP1. This is the first evidence for phosphorylation-dependent binding of RAP1.

Demarcation of a sequence involved in mediating catabolite repression of the gene for the 11 kDa subunit VIII of ubiquinol-cytochrome c oxidoreductase in Saccharomyces cerevisiae

Abstract: A regulatory element has been identified in the promoter region of the gene encoding the 11 kDa subunit VIII of the ubiquinol-cytochrome c oxidoreductase in Saccharomyces cerevisiae. The element, which is approximately 40 bp long and situated 185 bp upstream of the initiator ATG, is essential for induction of gene expression during growth in the presence of non-fermentable carbon sources. This is shown by the regulated synthesis of beta-galactosidase in yeast cells harbouring a CYC1-lacZ fusion gene, in which the CYC1 UAS's had been replaced by a 43 bp subunit VIII gene promoter fragment. In addition two DNA-binding activities, which may represent either separate factors or different forms of a single factor, have been detected. Both factors are abundant and they bind in a mutually exclusive fashion to a DNA sequence just upstream of the regulatory element. Although it is unlikely that these factors are directly involved in the response of the subunit VIII gene to catabolite repression, the position of their binding sites relative to the UAS and to the 3'-terminus of a gene located only 361 bp upstream suggest that they are important in modulating transcriptional activity of this region.