ERK modulates DNA bending and enhancesome structure by phosphorylating HMG1-boxes 1 and 2 of the RNA polymerase I transcription factor UBF

Biochemistry. 2006 Mar 21;45(11):3626-34. doi: 10.1021/bi051782h.

Abstract

Transcription of the ribosomal RNA genes of mammals by RNA polymerase I is rapidly activated by epidermal growth factor via the MAP-kinase (ERK) signaling cascade. This activation is mediated by direct phosphorylation of the HMG box DNA binding domains of the architectural transcription factor UBF. Mutation of the ERK sites of UBF inhibits its normal function and blocks growth factor activation of ribosomal transcription. UBF has little or no DNA sequence selectivity and binds throughout the ribosomal genes, defining a specialized chromatin. Indeed, the HMG boxes of UBF induce looping of the ribosomal DNA to create the enhancesome, a structure somewhat reminiscent of the nucleosome. Here, we show that both ERK phosphorylation and mutations that simulate this phosphorylation decrease the affinity of the individual HMG boxes of UBF for linear ribosomal DNA but have little or no effect on the capacity of these HMG boxes to bind to pre-bent DNA and do not affect the overall binding constant of UBF for the DNA. Electron spectroscopic imaging showed that ERK site UBF mutants do not induce the characteristic DNA looping of the enhancesome and associate with no more than half of the enhancesomal DNA. The data demonstrate that ERK phosphorylation of UBF prevents DNA bending by its first two HMG boxes, leading to a cooperative unfolding of the enhancesome.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • DNA / metabolism*
  • DNA, Cruciform / metabolism
  • Enhancer Elements, Genetic*
  • HMGB1 Protein / metabolism*
  • HMGB2 Protein / metabolism*
  • Mitogen-Activated Protein Kinase Kinases / genetics
  • Mitogen-Activated Protein Kinase Kinases / metabolism*
  • Models, Biological
  • Molecular Sequence Data
  • Mutation
  • Phosphorylation
  • Pol1 Transcription Initiation Complex Proteins / genetics
  • Pol1 Transcription Initiation Complex Proteins / isolation & purification
  • Pol1 Transcription Initiation Complex Proteins / metabolism*
  • Protein Binding
  • RNA Polymerase I / genetics
  • RNA Polymerase I / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship
  • Transcription, Genetic
  • Xenopus laevis / metabolism

Substances

  • DNA, Cruciform
  • HMGB1 Protein
  • HMGB2 Protein
  • Pol1 Transcription Initiation Complex Proteins
  • Recombinant Proteins
  • transcription factor UBF
  • DNA
  • Mitogen-Activated Protein Kinase Kinases
  • RNA Polymerase I