A new alpha-helical extension promotes RNA binding by the dsRBD of Rnt1p RNAse III

EMBO J. 2004 Jul 7;23(13):2468-77. doi: 10.1038/sj.emboj.7600260. Epub 2004 Jun 10.

Abstract

Rnt1 endoribonuclease, the yeast homolog of RNAse III, plays an important role in the maturation of a diverse set of RNAs. The enzymatic activity requires a conserved catalytic domain, while RNA binding requires the double-stranded RNA-binding domain (dsRBD) at the C-terminus of the protein. While bacterial RNAse III enzymes cleave double-stranded RNA, Rnt1p specifically cleaves RNAs that possess short irregular stem-loops containing 12-14 base pairs interrupted by internal loops and bulges and capped by conserved AGNN tetraloops. Consistent with this substrate specificity, the isolated Rnt1p dsRBD and the 30-40 amino acids that follow bind to AGNN-containing stem-loops preferentially in vitro. In order to understand how Rnt1p recognizes its cognate processing sites, we have defined its minimal RNA-binding domain and determined its structure by solution NMR spectroscopy and X-ray crystallography. We observe a new carboxy-terminal helix following a canonical dsRBD structure. Removal of this helix reduces binding to Rnt1p substrates. The results suggest that this helix allows the Rnt1p dsRBD to bind to short RNA stem-loops by modulating the conformation of helix alpha1, a key RNA-recognition element of the dsRBD.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Catalytic Domain
  • Crystallography, X-Ray
  • Fungal Proteins / chemistry*
  • Fungal Proteins / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Nucleic Acid Conformation
  • Protein Conformation
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • RNA, Double-Stranded / metabolism*
  • RNA, Fungal / chemistry
  • RNA, Fungal / metabolism
  • Ribonuclease III / chemistry*
  • Ribonuclease III / metabolism*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Homology, Amino Acid
  • Solutions
  • Substrate Specificity

Substances

  • Fungal Proteins
  • RNA, Double-Stranded
  • RNA, Fungal
  • Saccharomyces cerevisiae Proteins
  • Solutions
  • RNT1 protein, S cerevisiae
  • Ribonuclease III