INTRODUCTIONIt is appropriate that at a meeting dedicated to H. G. Wittmann we should emphasize comparative studies of the three-dimensional structure of the ribosome since he and his collaborators have made such important contributions to this field. In this paper we present data detailing the first isolation of small mitochondrial ribosomal subunits from ihe hemoflagellate Leishmania tarentolae. Th^ir struc;iure is interesting because these are the smallest ribosomes yet found (their small subunit rRNA sediments at 9S and is only 610 nucleotides long). We also show that particles similar in structure to these small subunits can be reconstituted from in vitro transcribed mitochondrial 9S rRNA and E. coli proteins.The 98 and 12S ribosomal RNAs (rRNAs) are the major RNA components of the mitochondrion (kinetoplast) of Leishnumia tarentolae (Simpson and Simpson, 1978). It has been proposed that the 9S RNA is the small kinetoplastid ribosomal subunit rRNA (de la Cruz et al., 198Sa) and the 12S RNA is the large kinetoplastid ribosomal subunit rRNA (de la Cruz et al., 1985b). These assignments were based on the strong similarities between the secondary structures of these RNAs with the consensus small and large subunit rRNA secondary structures and on the occurrence of essential conserved sequences, such as the 520, 720 and 1400 regions (£. coli numbering system). However, the existence of kinetoplast ribosomes has not been directly demonstrated.To test whether 9S RNA can form small subunits, we attempted to reconstitute the small ribosomal subunit (Traub et al., 1971) from heterologous components, Leishmania tarentolae 9S RNA and E. coli small subunit ribosomal proteins. Heterologous reconstitutipns have been previously achieved when the rRNA and ribosomal proteins are from difierent bact»ia (Nomura et al., 1968; Higo et al., 1973; Goldberg and Steitz, 1974; Held et al., 1974). Recently, in vitro T7 transcripts of wild type and mutant £. coli 16S rRNA have also been reconstituted with E. coli ribosomal proteins (Krzyzosiak et al., 1987; Melancon et al., 1987; Scheinman, 1989).We show in this paper that hybrid subunits can be reconstituted using in vitro MATERIALS AND METHODS In Sucrose Gradient Analyses and Electron MicroscopyReconstituted subunits were concentrated by ultracentrifugation in a Beckman SW50.1 rotor at 41,000 RPM (157,000 x g) for 3.5 hrs at 4'*C, resuspended in 200 microliters of 10 mM Tris-HCl (pH 7.6), 6 mM MgCl,, 50 mM NH4CI, 1 mM DTT, 0.5 mM EDTA and loaded onto sucrose gradients that were 20-30% sucrose in the same buffer. These gradients were centrifuged for 40 minutes at 50,000 RPM (220,000 x g) at 4°C in a Beckman VTi65 rotor, after which they were fractionated and monitored through an ISCO UA-5 continuous absorbance monitor. Subunits were collected and concentrated by ultracentrifugation, resuspended to a final concentration of 0.2 AJM units/ml, negatively stained by the double-layer carbon method (Lake, 1979), and visualized with a Philips 400 electron microscope. To character^ the 20S...
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