Characterisation of the nucleic-acid-binding activity of KH domains. Different properties of different domains

Eur J Biochem. 1996 Oct 15;241(2):425-31. doi: 10.1111/j.1432-1033.1996.00425.x.

Abstract

The KH module is a sequence motif recently identified in a number of diversified RNA-binding proteins and suggested to be the functional element responsible for RNA binding. So far, however, this hypothesis has not received direct experimental support. We have expressed the three KH-domains from heterogeneous nuclear ribonucleoprotein K (hnRNP-K), the poly(C)-binding proteins PCBP-1 and PCBP-2, the first three to four domains from the high-density binding protein HBP, the one and a half domain from the archaeon Halobacterium halobium ORF139 and one and a half domain of the fragile-X protein FMR1 in Escherichia coli and analysed their nucleic-acid-binding properties in vitro. The results showed that the in vitro poly(rC)-binding activity of hnRNP-K can be assigned to KH-domain 3, whereas both domains 1 and 3 in the PCBPs bind poly(rC). In addition, all these domains exhibit binding activity towards other nucleic acids, albeit at a significantly lower level. The first KH domain from the FMR1 protein binds poly(rG) and single-stranded and double-stranded DNA. The N-terminal three or four domains from HBP bind poly(rG) and, at a much lower level, single-stranded and double-stranded DNA. Thus, single KH domains are discrete and independent nucleic-acid-binding units. Moreover, different KH domains bind different nucleic acids, suggesting that KH domains are composed of a conserved, weakly nucleic-acid-binding, structure that is fine tuned, by sequence variation, resulting in sequence-specific nucleic-acid-binding entities.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • DNA, Complementary / genetics
  • DNA-Binding Proteins*
  • Heterogeneous-Nuclear Ribonucleoprotein K
  • Heterogeneous-Nuclear Ribonucleoproteins
  • Humans
  • Molecular Sequence Data
  • Molecular Structure
  • Poly C / metabolism
  • Poly G / metabolism
  • Protein Denaturation
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Ribonucleoproteins / chemistry
  • Ribonucleoproteins / genetics
  • Ribonucleoproteins / metabolism
  • Sequence Homology, Amino Acid
  • Transcription Factors*

Substances

  • DNA, Complementary
  • DNA-Binding Proteins
  • Heterogeneous-Nuclear Ribonucleoprotein K
  • Heterogeneous-Nuclear Ribonucleoproteins
  • PCBP1 protein, human
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Ribonucleoproteins
  • Transcription Factors
  • Poly G
  • Poly C