Molecular cloning and characterization of the ribonucleases H of Trypanosoma brucei

Ribonucleases H (RNases H) are nucleic acid metabolic enzymes best known for their ability to degrade the RNA strand of RNA:DNA heteroduplexes. Using the protozoal model, Trypanosoma brucei, studies were undertaken to define the relationship between the structure and function of the enzymes. In eukaryotes, RNases H appear to be specifically sorted to nucleic acid containing organelles where they execute their functions. The type 1 RNase H of T. brucei, TbRNHI, was previously shown to contain a nuclear localization signal within amino acids 1--46. Here we show that TbbRNHI also localizes to the mitochondrion and that the N-terminal pentatpeptide sequence, MGKKR, is necessary and sufficient for this targeting. Amino acids 6--46 encode a highly conserved functional double-stranded RNA binding domain. Surprisingly, amino acid substitutions of this domain do not abolish nuclear accumulation. Antibodies to recombinant TbRNHI localize endogenous TbRNHI to the nuclear periphery in a distinct punctate pattern. Co-localization studies using antisera to TbRNHI and antisera to U2 snRNP 40K protein shows that the distribution of both proteins overlap in the nucleus.; This thesis also reports the cloning of the type 2 RNase of T. brucei. This gene encodes a 477 amino acid open reading frame (ORF) containing two kinetoplastid specific motifs. Antibodies to the enzyme recognize a protein of {dollar}43kDa in total parasite extracts which is significantly smaller than the 54kDa protein predicted by the entire ORF. A short form of the protein localizes to the parasite nucleus and a 16 amino acid sequence, containing a bi-partite nuclear localization signal, is sufficient to encode nuclear accumulation of reporter fusion protein. The results of these studies define the organellar localization signals of the type 1 and 2 RNases H of T. brucei. By defining the sequence responsible for enzyme localization, we have begun to define the structure-function relationship of the different domains of these enzymes. The highly conserved properties of these enzymes allow us to apply what we have learned about the T. brucei enzymes to the RNases H of higher eukaryotes.