Biochemical and structural characterization of the RT domain of Leishmania sp. telomerase reverse transcriptase.

BACKGROUND: The Leishmania genus comprises parasites that cause leishmaniasis, a neglected disease spread worldwide. Leishmania sp. telomeres are composed of TTAGGG repeats maintained by telomerase. In most eukaryotes, the enzyme minimal complex contains the TER (telomerase RNA) and the TERT (telomerase reverse transcriptase) components. The TERT holds the enzyme catalytic core and is formed by four structural and functional domains (TEN, Telomerase Essential N-terminal; TRBD, Telomerase RNA Binding Domain; RT, the reverse transcriptase domain and CTE, C-Terminal Extension domain). METHODS AND RESULTS: Amino acid sequence alignments, protein structure prediction analysis, and protein: nucleic acid interaction assays were used to show that the Leishmania major RT domain preserves the canonical structural elements found in higher eukaryotes, including the canonical motifs and the aspartic acid residues that stabilize the Mg2+ ion cofactor. Furthermore, amino acid substitutions specific to the Leishmania genus and partial conservation of the residues involved with nucleic acid interactions are shown. The purified recombinant Leishmania RT protein is biochemically active and interacts with the G-rich telomeric strand and the TER template sequence. CONCLUSION: Our results highlight that the telomerase catalysis mechanism is conserved in a pathogen of medical importance despite the structural peculiarities present in the parasite's RT domain.

Behind Base J: The Roles of JBP1 and JBP2 on Trypanosomatids.

ß-D-glucopyranosyloxymethiluracil (Base J) is a modified thymidine base found in kinetoplastids and some related organisms. Interestingly, Base J distribution into the genome can vary depending on the organism and its life stage. Base J is reported to be found mostly at telomeric repeats, on inactive variant surface glycoproteins (VSG's) expression sites (e.g., T. brucei), in RNA polymerase II termination sites and sub-telomeric regions (e.g., Leishmania). This hypermodified nucleotide is synthesized in two steps with the participation of two distinct thymidine hydroxylases, J-binding protein 1 and 2 (JBP1 and JBP2, respectively) and a ß-glucosyl transferase. A third J-binding protein, named JBP3, was recently identified as part of a multimeric complex. Although its structural similarities with JBP1, it seems not to be involved in J biosynthesis but to play roles in gene expression regulation in trypanosomatids. Over the years, with the characterization of JBP1 and JBP2 mutant lines, Base J functions have been targeted and shone a light on that matter, showing genus-specific features. This review aims to explore Base J's reported participation as a regulator of RNA polymerase II transcription termination and to summarize the functional and structural characteristics and similarities of the remarkable JBP proteins in pathogenic trypanosomatids.