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.

Efficient transcriptional activation of many simple modular promoters by simian virus 40 large T antigen.

Abstract: Simian virus 40 (SV40) large T antigen is a multifunctional protein which plays central roles during both lytic and transforming infections by SV40. It is a potent transcriptional activator and increases expression from the SV40 late promoter and from several cellular promoters. To understand better the transcriptional activation activity of large T antigen, we examined its ability to transactivate a set of simple modular promoters containing one of four upstream activation sequences coupled with one of three different TATA box sequences originally constructed and studied by Taylor and Kingston (Mol. Cell. Biol. 10:165-175, 1990). Large T antigen activated transcription from all of these simple promoters. The identity of the TATA box was a more important determinant of the final level of gene expression than was the identity of the upstream activating sequence element. We also determined the ability of a set of mutant SV40 large T antigens to activate a subset of these promoters. Several mutant SV40 large T antigens which had reduced ability to activate the complex SV40 late and Rous sarcoma virus long terminal repeat promoters showed reduced transcriptional activation activity on all of the modular promoters tested. We used a set of promoter derivatives of the human U6 small nuclear RNA promoter containing different TATA boxes and found that wild-type large T antigen could activate transcription from all of them, although to widely different levels of expression.

Focusing Transgene Expression in Drosophila by Coupling Gal4 With a Novel Split-LexA Expression System

Abstract: Here we report the development of a ternary version of the LexA::VP16/LexAop system in which the DNA-binding and trans-activating moieties are independently targeted using distinct promoters to achieve highly restricted, intersectional expression patterns. This Split LexA system can be concatenated with the Gal4/upstream activating sequence system to refine the expression patterns of existing Gal4 lines with minimal genetic manipulations.

Interactions of purified transcription factors: binding of yeast MAT alpha 1 and PRTF to cell type-specific, upstream activating sequences.

Abstract: Pheromone receptor transcription factor (PRTF) and MAT alpha 1 are protein transcription factors that are involved in the regulation of the alpha-specific genes in Saccharomyces cerevisiae. We have expressed MAT alpha 1 as a fusion protein in Escherichia coli and purified it from inclusion bodies in milligram quantities. The MAT alpha 1 protein was obtained after specific cleavage of the fusion protein. Quantitative band shift electrophoresis was used to determine the equilibrium dissociation constants that describe the multicomponent binding equilibrium between the PRTF and MAT alpha 1 proteins, and alpha-specific STE3 upstream activating sequence (UAS) DNA. The dissociation constant for the complex of PRTF and the a-specific UAS of STE2 was also measured and found to be 5.9 X 10(-11) M, only three times less than that for the PRTF-STE3 UAS complex. Analyses of these complexes by DNase I footprinting demonstrate that the PRTF binding site is confined to the palindromic P-box sequence in the case of the STE3 UAS, but extends symmetrically from this central region to cover 28 bp for the STE2 UAS. When MAT alpha 1 is bound to the PRTF-STE3 complex, the region of DNA protected is enlarged to that seen for the PRTF-STE2 complex. Our results using these two purified factors in vitro suggest that PRTF has nearly the same affinity for a- and alpha-specific UAS elements and that transcriptional activation requires a particular conformational state for the PRTF-DNA complex which occurs in the PRTF-STE2 and MAT alpha 1-PRTF-STE3 complexes, but not in the PRTF-STE3 complex.

The upstream activation site of a Ty2 element of yeast is necessary but not sufficient to promote maximal transcription of the element.

Abstract: The Ty elements of yeast are a family of retrovirus-like transposons that are highly transcribed, accounting for about 10% of total mRNA. We have mapped two sites to the nontranscribed region of the element upstream of the transcription start site that are required for maximal gene expression and are similar to sites previously defined in other genes. One, the TATA site, is located 74 base pairs upstream of the transcription start site and has the canonical sequence TATAAAA. This site is required for normal rates of initiation; deletion of the site greatly reduces the amount of Ty917 mRNA without changing its 5' end. A second site is located in a region from 140 to 110 base pairs upstream of the start site. Unlike other upstream activation site (UAS) elements previously defined, the Ty917 UAS is not sufficient to promote any transcription in the absence of downstream transcription regulatory sites. Thus the UAS is necessary but not sufficient for maximal transcription. Comparison of constructs lacking either the UAS or the downstream enhancer or both shows evidence of synergistic interaction between the sites since the effect of the sites on the rate of transcription initiation is more than additive.