Protein Engineering of Multi-Modular Transcription Factor Alcohol Dehydrogenase Repressor 1 (Adr1p), a Tool for Dissecting In Vitro Transcription Activation

Biomolecules. 2019 Sep 17;9(9):497. doi: 10.3390/biom9090497.

Abstract

Studying transcription machinery assembly in vitro is challenging because of long intrinsically disordered regions present within the multi-modular transcription factors. One example is alcohol dehydrogenase repressor 1 (Adr1p) from fermenting yeast, responsible for the metabolic switch from glucose to ethanol. The role of each individual transcription activation domain (TAD) has been previously studied, but their interplay and their roles in enhancing the stability of the protein is not known. In this work, we designed five unique miniAdr1 constructs containing either TADs I-II-III or TAD I and III, connected by linkers of different sizes and compositions. We demonstrated that miniAdr1-BL, containing only PAR-TAD I+III with a basic linker (BL), binds the cognate DNA sequence, located in the promoter of the ADH2 (alcohol dehydrogenase 2) gene, and is necessary to stabilize the heterologous expression. In fact, we found that the sequence of the linker between TAD I and III affected the solubility of free miniAdr1 proteins, as well as the stability of their complexes with DNA. miniAdr1-BL is the stable unit able to recognize ADH2 in vitro, and hence it is a promising tool for future studies on nucleosomal DNA binding and transcription machinery assembly in vitro.

Keywords: Adr1p; heterologous expression; mini-gene design; multi-modular proteins; structural bioinformatics; transcription activation domains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Dehydrogenase / chemistry
  • Alcohol Dehydrogenase / genetics*
  • Alcohol Dehydrogenase / metabolism
  • Binding Sites
  • Cloning, Molecular
  • DNA, Fungal / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Pichia / genetics
  • Pichia / metabolism
  • Protein Binding
  • Protein Domains
  • Protein Engineering / methods*
  • Protein Stability
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Transcription Factors / chemistry*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Transcriptional Activation

Substances

  • ADR1 protein, S cerevisiae
  • DNA, Fungal
  • DNA-Binding Proteins
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • ADH2 protein, S cerevisiae
  • Alcohol Dehydrogenase