Transcript Mapping

The sequence of the 1173 nt 12S kinetoplast ribosomal RHA from TiPJ sfimarpa tarentolae was determined from the maxicircle DNA sequence, and the 5* and 3' ends localized by primer runoff and SI nuclease protection experiments. The gene was shown to be free of introns by SI nuclease analysis. A partial secondary structure model of the 12S RHA molecule is presented which is equivalent in certain respects to the corresponding portions of the Escherichia coli 23S ribosomal RHA model. Domain II of the E. coli model is completely missing in the kinetoplast model with the exception of several phylogenetically conserved stems and one loop. There is a striking conservation of the functionally important peptidyltransferase region except for the deletion of a few stems and loops. The 12S RHA is the smallest large subunit ribosomal RHA described to date. INTRODUCTION The 9S and 12S RNA species are the major stable non-polyadenylated RNAs transcribed from the maxicircle DNA in the single mitochondrion of the kinetoplastid protozoa (for reviews see 1-5). These RNAs have been proposed to be unusually small mitochondrial ribosomal RNAs (rRNAs) (2, 6-8). However, mitochondrial ribosomes have not been isolated from kinetoplastid protozoa, and the unusual properties of small size and order of transcription (large to small rRNA instead of small to large rRNA) have raised questions as to the ribosomal nature of these RNAs. We have recently presented a complete secondary structure model for the kinetoplast 9S RNA (9), based on the Rscherichia coli 16S rRNA model (10, 11), which clearly establishes the 9S RNA as the small subunit mitochondrial rRNA of the kinetoplastid protozoa. In this paper we present the sequence of the 12S mitochondrial RNA from Leishmania tarentolae and a partial secondary structure model that conforms well to portions of the IL .coll 23S rRNA © IRL Press Limited, Oxford, England. 2 3 3 7 Nucleic Acids Research model (12). The secondary structure model includes the £^ coli equivalents of domains II and V, and portions of domains IV and VI. The model is supported by several compensatory base pair changes in the Trvpanosoma brucei 12S RNA sequence which preserves the base pairing of the proposed helices. A complete secondary structure model for this unusually small large subunit rENA must await additional cross-species and cross-strain comparisons, as the regions not presented appear to be extremely diverged from the comparable regions of the £^ coli model. MATERIALS AND METHODS Cloning Cloning of the 120 DNA fragment, which contains the 12S rRNA gene, and DNA isolation procedures have been described elsewhere (13-15). Subcloning of the insert into M13 mp7, mp8, or mp9 was carried out using standard procedures (16-20). All restriction and DNA modification enzymes were purchased from Bethesda Research Laboratories (BRL), and New England Biolabs. Enzyme reaction conditions were as recommended by the manufacturer. DNA Sequencing The entire 120 DNA insert was sequenced (the region encompassing nt 1582-2415 was presented in ref. 9, and the region encompassing nt 2213-6561 was presented in ref. 21) using the dideoxy chain termination method (22-24) with modifications of the gel technology as described by Garoff and Ansorge (25) and de la Cruz et al (21). Universal sequencing primers were used for al l clones (New England Biolabs 15-mer). Klenow fragment of DNA polymerase was obtained from Boehringer Mannheim or BRL. Sequence data was compiled using the DB data base management system of Staden (26, 27). The sequence presented here was determined in both orientations and across a l l restriction enzyme sites, with a minimum of three individual clones for each nt position. BSh isolation T. brucei strain 366D ce l l s were grown as the procyclic forms as described (28). Total ce l l RNA from these ce l l s was isolated according to Chirgwin et al (29) and treated as described (30) with RNase-free DNase I (31). Iu_ tarentolae kinetoplast RNA (kRNA) was isolated as described (30).