Quantitative characterization of the interactions among c-myc transcriptional regulators FUSE, FBP, and FIR

Biochemistry. 2010 Jun 8;49(22):4620-34. doi: 10.1021/bi9021445.

Abstract

Human c-myc is critical for cell homeostasis and growth but is a potent oncogenic factor if improperly regulated. The c-myc far-upstream element (FUSE) melts into single-stranded DNA upon active transcription, and the noncoding strand FUSE recruits an activator [the FUSE-binding protein (FBP)] and a repressor [the FBP-interacting repressor (FIR)] to fine-tune c-myc transcription in a real-time manner. Despite detailed biological experiments describing this unique mode of transcriptional regulation, quantitative measurements of the physical constants regulating the protein-DNA interactions remain lacking. Here, we first demonstrate that the two FUSE strands adopt different conformations upon melting, with the noncoding strand DNA in an extended, linear form. FBP binds to the linear noncoding FUSE with a dissociation constant in the nanomolar range. FIR binds to FUSE more weakly, having its modest dissociation constants in the low micromolar range. FIR is monomeric under near-physiological conditions but upon binding of FUSE dimerizes into a 2:1 FIR(2)-FUSE complex mediated by the RRMs. In the tripartite interaction, our analysis suggests a stepwise addition of FIR onto an activating FBP-FUSE complex to form a quaternary FIR(2)-FBP-FUSE inhibitory complex. Our quantitative characterization enhances understanding of DNA strand preference and the mechanism of the stepwise complex formation in the FUSE-FBP-FIR regulatory system.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Carrier Proteins / chemistry
  • Carrier Proteins / metabolism*
  • Carrier Proteins / physiology
  • DNA Helicases / chemistry
  • DNA Helicases / metabolism*
  • DNA Helicases / physiology
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / metabolism*
  • DNA-Binding Proteins / physiology
  • Dimerization
  • Guanine Nucleotide Exchange Factors / chemistry
  • Guanine Nucleotide Exchange Factors / metabolism*
  • Guanine Nucleotide Exchange Factors / physiology
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Protein Binding
  • Proto-Oncogene Proteins c-myc / chemistry
  • Proto-Oncogene Proteins c-myc / metabolism*
  • Proto-Oncogene Proteins c-myc / physiology
  • RNA-Binding Proteins
  • Repressor Proteins / chemistry
  • Repressor Proteins / metabolism*
  • Repressor Proteins / physiology
  • Rho Guanine Nucleotide Exchange Factors
  • Solutions
  • Trans-Activators / chemistry
  • Trans-Activators / metabolism*
  • Trans-Activators / physiology

Substances

  • Carrier Proteins
  • DNA-Binding Proteins
  • FARP2 protein, human
  • FUBP1 protein, human
  • Guanine Nucleotide Exchange Factors
  • MYC protein, human
  • Proto-Oncogene Proteins c-myc
  • RNA-Binding Proteins
  • Repressor Proteins
  • Rho Guanine Nucleotide Exchange Factors
  • Solutions
  • Trans-Activators
  • poly-U binding splicing factor 60KDa
  • DNA Helicases