Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase

Abstract

Group II intron-encoded proteins (IEPs) have both reverse transcriptase (RT) activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active RNA structure. The LtrA protein encoded by the Lactococcus lactis Ll.LtrB group II intron contains an N-terminal RT domain, with conserved sequence motifs RT1 to 7 found in the fingers and palm of retroviral RTs; domain X, associated with maturase activity; and C-terminal DNA-binding and DNA endonuclease domains. Here, partial proteolysis of LtrA with trypsin and Arg-C shows major cleavage sites in RT1, and between the RT and X domains. Group II intron and related non-LTR retroelement RTs contain an N-terminal extension and several insertions relative to retroviral RTs, some with conserved features implying functional importance. Sequence alignments, secondary-structure predictions, and hydrophobicity profiles suggest that domain X is related structurally to the thumb of retroviral RTs. Three-dimensional models of LtrA constructed by “threading” the aligned sequence on X-ray crystal structures of HIV-1 RT (1) account for the proteolytic cleavage sites; (2) suggest a template–primer binding track analogous to that of HIV-1 RT; and (3) show that conserved regions in splicing-competent LtrA variants include regions of the RT and X (thumb) domains in and around the template–primer binding track, distal regions of the fingers, and patches on the protein’s back surface. These regions potentially comprise an extended RNA-binding surface that interacts with different regions of the intron for RNA splicing and reverse transcription.

Keywords

• HIV-1
• retrotransposon
• retrovirus
• ribozyme
• RNA–protein interaction
• RNA splicing
• D domain, DNA-binding domain
• En domain, DNA en-donuclease domain
• IEP, intron-encoded protein
• LTR, long terminal repeat
• RNP, ribonucleoprotein
• RT, reverse transcriptase