Limited proteolysis unmasks specific DNA-binding of the murine RNA polymerase I-specific transcription termination factor TTFI

J Mol Biol. 1992 Oct 5;227(3):635-47. doi: 10.1016/0022-2836(92)90213-4.

Abstract

Previously we have shown that nuclear extracts from mouse cells contain a heterogeneous group of polypeptides (p65, p80, p90, p100) which form distinct DNA-protein complexes on the 18 base-pair sequence element (termed Sal-box), which constitutes the murine rDNA transcription termination signal. These distinct proteins mediate cessation of RNA polymerase I (pol I) transcription elongation and release of the nascent RNA chains, indicating that they function as termination factor(s). Here, we report the biochemical analysis of the pol I-specific transcription termination factor TTFI. We show that the heterogeneity of TTFI is due to limited proteolysis of a larger, 130 kDa precursor protein (p130). The DNA-binding activity of p130 is strongly reduced as compared to the proteolytic derivatives, indicating that the DNA-binding domain is repressed within the full-length molecule. We have used limited proteolysis to purify and functionally characterize a TTFI core polypeptide (p50) which still specifically binds to the Sal-box target sequence and directs rDNA transcription termination. The equilibrium constant of purified p50 to bind specifically to DNA is 9 x 10(9) M-1. Additionally, we demonstrate that TTFI binds to DNA as a monomer and that binding induces DNA bending. This observation suggests that not only specific DNA-protein and protein-protein interactions but also conformational alterations of DNA may play a role in the termination process.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites / genetics
  • Cells, Cultured
  • Chromatography, Gel
  • DNA, Ribosomal / genetics
  • DNA, Ribosomal / metabolism
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism*
  • Methylation
  • Mice
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Oligodeoxyribonucleotides / metabolism*
  • RNA Polymerase I / metabolism*
  • Transcription Factors / isolation & purification
  • Transcription Factors / metabolism*
  • Transcription, Genetic / genetics*

Substances

  • DNA, Ribosomal
  • DNA-Binding Proteins
  • Oligodeoxyribonucleotides
  • Transcription Factors
  • RNA Polymerase I