Isolation and Characterization of Factors that Interact with Eukaryotic Transcriptional Enhancers

Citation

Harshman, Keith Duncan (1989) Isolation and Characterization of Factors that Interact with Eukaryotic Transcriptional Enhancers. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/5cem-2h95. https://resolver.caltech.edu/CaltechETD:etd-05222007-152402

Abstract

The regulatory region controlling the transcription of the SV40 early genes provides a useful system in which to study the mechanisms of eukaryotic gene expression. The enhancer element of this region is composed of sequence motifs that can function independently or in combination to potentiate transcription in a wide range of cellular contexts. This thesis describes the purification and characterization of two proteins that specifically bind separate motifs.

An enhancer binding protein was identified in bovine thymus extracts and purified to homogeneity. This factor, designated EP2, was shown by chemical and enzymatic footprinting techniques to bind the core as well as two pseudo-core sequences. In a DNase I footprinting experiment, EP2 was unable to bind to a mutated core sequence that is incapable of activating transcription in vivo. EP-2 was shown to consist of a group of polypeptides, ranging in molecular weight from 34 kd to 43 kd, each of which has the ability to bind to the core and two pseudo-core sequences.

It was shown that the sequence bound by the mammalian transcription factor AP-1 -- the AP-1 recognition element (ARE) -- was capable of activating transcription in yeast. The ARE is very similar in sequence to the GCN4 recognition element (GCRE), yet the ARE was shown to activate transcription in a gcn4 yeast strain. A protein present in yeast extracts, designated yAP-l, was shown to bind to the ARE and was purified to near homogeneity based on this ability. yAP-1 and AP-1 display remarkably similar biochemical and DNA binding characteristics. It was shown in vitro that yAP-1 can discriminate between the ARE and the GCRE, while GCN4 exhibits approximately equal affinities for the two elements.

The structural gene encoding yAP-1 was isolated and characterized. The DNA binding domain of the protein was localized to a sequence of 93 amino-acids in the amino-terminus. This sequence was shown to have significant homology with domains in cJUN and GCN4, which have been ascribed the ability to bind DNA. The YAP1 gene was shown to be non-essential by gene disruption. DNA-affinity blot experiments performed using extracts from YAP1 and yap1 strains suggest the existence of a family of yeast genes encoding proteins that recognize the ARE.

Thesis Files