An in vitro study of the DNA and protein-binding properties of basic helix-loop-helix (bHLH) and associated proteins

Robert Quoc To, Purdue University

Abstract

The activation or repression of genes is a highly complex process that involves proteins called transcription factors. A typical transcription factor contains a sequence-specific DNA binding domain, a multimerization domain, and an activation domain. These factors elicit their effects on gene expression by two general methods. First, they participate in molecular associations with proteins (protein:protein interactions) which help to form a biologically functional complex. Second, they undergo molecular associations with DNA (DNA:protein interactions) at specific recognition sites to activate or to repress the transcriptional activity of a particular gene. In this study, those two general methods will be discussed as they relate to the regulation of the muscle-specific gene, troponin I (TnI), to the characterization of Mist1, a novel basic helix-loop-helix (bHLH) protein, and to the characterization of 9-1, a protein partner to Mist1. The TnI gene is controlled by the muscle regulatory factors (MRFs) which include MyoD, myogenin, Myf-5, and MRF4. The MRFs are bHLH proteins which bind to a DNA recognition sequence of -CANNTG-, referred to as an E-box, and participate in protein:protein interactions with other bHLH proteins, E12 and E47, to activate the TnI gene and to initiate the myogenic program. In addition to the MRFs, two ubiquitous proteins, the Site I and the Site II binding factors, also regulate the TnI gene with evidence suggesting that the Site I protein is a transcription factor while the Site II protein is unknown. Unlike the MRFs, Mist1 is a bHLH protein that inhibits muscle differentiation by titrating E12 and E47 away from the MRFs. Furthermore, Mist1 interacts with 9-1, a novel non-bHLH protein, to activate transcription in yeast. The 9-1 partial cDNA encodes a 258 amino acid protein that possesses an acidic rich region and a serine/threonine rich domain that interacts specifically with Mist1. In contrast to Mist1, 9-1 can independently activate transcription in yeast. The muscle regulatory factors, Mist1, and 9-1 all exemplify the importance of DNA:protein and protein:protein interactions in regulating the biological process of gene expression.

Degree

Ph.D.

Advisors

Konieczny, Purdue University.

Subject Area

Molecular biology

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