Student Theses and Dissertations

Date of Award

1991

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Chua Laboratory

Keywords

35S promoter, as-1 element, TGA1a, ASF-1, bZIP transcription factor, root-specific expression

Abstract

The cauliflower mosaic virus 35S promoter is composed of several cis-elements, each of which confers a different expression pattern in transgenic tobacco. One such cis-element, activation sequence (as)-1 (-83 to -63) mediates preferential expression in root. The element contains a tandem repeat of the motif TGACG, that is crucial for as-1 function and binding of a tobacco nuclear factor, activation sequence factor (ASF)-1. Two other TGACG-containing cis-elements from plant promoters were identified by a computer assisted search of a gene bank. These cis-elements, hex-1 from the wheat histone H3 promoter and nos-1 from the nopaline synthase promoter of T-DNA, also bind ASF-1. Another tobacco nuclear factor, designated hex-i-specific binding factor (HSBF), binds only to hex-1, A tobacco cDNA library was screened for DNA-binding proteins using hex-1as a binding probe and two types of clones encoding hex-1-binding proteins were isolated. Based on their binding specificities to as-1, hex-1, and nos-1, the encoded DNA-binding proteins, named TGA1a and TGA1b, appear to correspond to ASF-1 and HSBF, respectively. Both TGA1a and TGA1b are bZIP proteins. The TGA1a mRNA level is much higher in root than in leaf. TGA1a can function as an as-1-specific transcription activator in a HeLa cell in vitro system as well as a wheat germ in vitro system. In both systems, TGA1a stimulates transcription by increasing the number of preinitiation complexes. When microinjected into cotyledon cells of transgenic tobacco plants carrying a promoter linked to as-I, TGA1a can induce the expression of the promoter. Judging from these results, TGA1a is likely to be the factor that is responsible for the as-1 function in vivo. Such a factor is present in leaf cells in a limiting concentration. Although the bZIP domain of TGA1a is sufficient for the specific binding to its target site, another domain (DS domain) increases the apparent DNA-binding affinity by stabilizing the dimeric form of TGA1a. The N-terminal region of TGA1a, which is enriched in acidic residues, appears to be essential for transactivation in vivo. However, when assayed in a HeLa cell in vitro system, most parts of the protein except the bZIP domain seem dispensable for activity.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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