The non-LTR (long terminal repeat) retrotransposon L1Tc from Trypanosoma cruzi codes for a protein with RNase H activity.
The deduced amino acid sequence of the region downstream of the reverse transcriptase (RT) motif of the Trypanosoma cruzi L1Tc non-LTR retrotransposon shows a significant homology with the sequence coding for proteins with RNase H activity from different organisms and retroelements. The 25-kDa His(6)-tagged recombinant protein bearing only the L1Tc RNase H domain, named RHL1Tc, exhibits RNase H activity as measured on the [(3)H]poly(rA)/poly(dT) hybrid used as substrate as well as on specific homologous and heterologous [(32)P]RNA/DNA hybrids. The mutation of the conserved aspartic acid at position 39 of the enzyme catalytic site, but not of the serine at position 56 (non-conservative amino acid), abolishes protein RNase H activity. The RNase H activity of the RHL1Tc protein is Mg(2+)-dependent, and it is also active in the presence of the Mn(2+) ion. The optimal condition of RNase H activity is found at pH 8 and 37 degrees C, although it also has significant enzymatic activity at 19 degrees C and pH 6. However, it cannot be excluded that the RNase H activity level and its optimal conditions may be different from that of a protein containing both RT and RNase H domains.
Olivares
Mónica
M
Departamento de Biología Molecular, Instituto de Parasitología y Biomedicina López Neyra, Consejo Superior de Investigaciones Cientificas Calle Ventanilla, Granada, Spain.
García-Pérez
José Luis
JL
Thomas
M Carmen
MC
Heras
Sara R
SR
López
Manuel C
MC
D000595:Amino Acid Sequence; D000818:Animals; D016384:Consensus Sequence; D016054:DNA, Protozoan / Q000235:genetics; D007700:Kinetics; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016053:RNA, Protozoan / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D018626:Retroelements / Q000235:genetics*; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D014349:Trypanosoma cruzi / Q000235:genetics*
D016054:DNA, Protozoan; D016053:RNA, Protozoan; D018626:Retroelements; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H