Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference

J Mol Biol. 2001 Jun 1;309(2):361-86. doi: 10.1006/jmbi.2001.4658.

Abstract

Group II intron mobility occurs by a target DNA-primed reverse transcription mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer to reverse transcribe the inserted intron RNA. The group II intron endonuclease, which mediates this process, is an RNP particle that contains the intron-encoded protein and the excised intron RNA and uses both cooperatively to recognize DNA target sequences. Here, we analyzed the interaction of the Lactococcus lactis Ll.LtrB group II intron endonuclease with its DNA target site by DNA footprinting and modification-interference approaches. In agreement with previous mutagenesis experiments showing a relatively large target site, DNase I protection extends from position -25 to +19 from the intron-insertion site on the top strand and from -28 to +16 on the bottom strand. Our results suggest that the protein first recognizes a small number of specific bases in the distal 5'-exon region of the DNA target site via major-groove interactions. These base interactions together with additional phosphodiester-backbone interactions along one face of the helix promote DNA unwinding, enabling the intron RNA to base-pair to DNA top-strand positions -12 to +3 for reverse splicing. Notably, DNA unwinding extends to at least position +6, somewhat beyond the region that base-pairs with the intron RNA, but is not dependent on interaction of the conserved endonuclease domain with the 3' exon. Bottom-strand cleavage occurs after reverse splicing and requires recognition of a small number of additional bases in the 3' exon, the most critical being T+5 in the now single-stranded downstream region of the target site. Our results provide the first detailed view of the interaction of a group II intron endonuclease with its DNA target site.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenine / metabolism
  • Base Pairing
  • Base Sequence
  • Binding Sites
  • DNA / chemistry
  • DNA / genetics
  • DNA / metabolism*
  • DNA Footprinting*
  • DNA Methylation
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Deoxyribonuclease I / metabolism
  • Deoxyuridine / metabolism
  • Endonucleases / chemistry
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Exons / genetics
  • Introns / genetics*
  • Kinetics
  • Lactococcus lactis / enzymology*
  • Lactococcus lactis / genetics*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation / genetics
  • Nucleic Acid Denaturation
  • Potassium Permanganate / metabolism
  • Protein Structure, Tertiary
  • RNA Splicing / genetics
  • Ribonucleoproteins / chemistry
  • Ribonucleoproteins / genetics
  • Ribonucleoproteins / metabolism*
  • Substrate Specificity

Substances

  • DNA-Binding Proteins
  • Ribonucleoproteins
  • Potassium Permanganate
  • DNA
  • Endonucleases
  • Deoxyribonuclease I
  • Adenine
  • Deoxyuridine