Structural and Functional Characterization of an RNase HI Domain From the Bifunctional Protein Rv2228c From Mycobacterium Tuberculosis

J Bacteriol. 2010 Jun;192(11):2878-86. doi: 10.1128/JB.01615-09. Epub 2010 Apr 2.

Abstract

The open reading frame Rv2228c from Mycobacterium tuberculosis is predicted to encode a protein composed of two domains, each with individual functions, annotated through sequence similarity searches. The N-terminal domain is homologous with prokaryotic and eukaryotic RNase H domains and the C-terminal domain with alpha-ribazole phosphatase (CobC). The N-terminal domain of Rv2228c (Rv2228c/N) and the full-length protein were expressed as fusions with maltose binding protein (MBP). Rv2228c/N was shown to have RNase H activity with a hybrid RNA/DNA substrate as well as double-stranded RNase activity. The full-length protein was shown to have additional CobC activity. The crystal structure of the MBP-Rv2228c/N fusion protein was solved by molecular replacement and refined at 2.25-A resolution (R = 0.182; R(free) = 0.238). The protein is monomeric in solution but associates in the crystal to form a dimer. The Rv2228c/N domain has the classic RNase H fold and catalytic machinery but lacks several surface features that play important roles in the cleavage of RNA/DNA hybrids by other RNases H. The absence of either the basic protrusion of some RNases H or the hybrid binding domain of others appears to be compensated by the C-terminal CobC domain in full-length Rv2228c. The double-stranded-RNase activity of Rv2228c/N contrasts with classical RNases H and is attributed to the absence in Rv2228c/N of a key phosphate binding pocket.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Catalytic Domain / genetics
  • Catalytic Domain / physiology
  • Crystallography, X-Ray
  • Kinetics
  • Maltose-Binding Proteins
  • Molecular Sequence Data
  • Mycobacterium tuberculosis / genetics
  • Mycobacterium tuberculosis / metabolism*
  • Periplasmic Binding Proteins / genetics
  • Periplasmic Binding Proteins / metabolism
  • Protein Structure, Secondary
  • Protein Structure, Tertiary / genetics
  • Protein Structure, Tertiary / physiology*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Ribonuclease H / chemistry*
  • Ribonuclease H / genetics
  • Ribonuclease H / metabolism*
  • Sequence Homology, Amino Acid
  • Transaminases / genetics
  • Transaminases / metabolism

Substances

  • Bacterial Proteins
  • Maltose-Binding Proteins
  • Periplasmic Binding Proteins
  • Recombinant Fusion Proteins
  • CobC protein, Bacteria
  • Transaminases
  • Ribonuclease H
  • ribonuclease HI

Associated data

  • PDB/3HST