Primary sequence and post‐transcriptional modification pattern of an unusual mitochondrial tRNA^Met from Tetrahymena pyriformis

Abstract

We have now isolated and characterized the smallest T. pyriformis mitochondrial tRNA and we show here that it has the primary sequence that we had predicted from the trnM sequence. The transcript also contains a Y-terminal CCA sequence, not encoded in the mtDNA, as well as several modified nucleosides characteristic of known functional tRNAs. Materials and methodsT. pyriformis, amicronucleate strain ST (obtained from Y. Suyama, Department of Biology, University of Pennsylvania), was grown at 28°C with constant shaking in 500 ml NeWs medium [9]. The culture was chilled on ice for 10 15 min, following which cells were collected by centrifugation at 2000 rpm for 5 min, resuspended, and centrifuged at 2000 rpm for 5 min through a layer of ice-cold homogenizing medium (0.35 M sucrose, 10 mM Tris-HC1, pH 7.2, 2 mM EDTA) [10]. Resuspended cells in 50 ml homogenizing medium were disrupted by three passages through a hand emulsifier (nozzle unscrewed 2.5 turns) [11]. Unbroken cells and debris were removed by centrifugation at 3000 rpm (IEC) for 5 min. A mitochondrial pellet was recovered from the resulting supernatant by centrifugation at 8000 rpm for 5 min and washed twice by resuspension in 50 ml homogenizing medium followed by centrifugation (5 min, 8000 rpm). The final mitochondrial pellet was resuspended in 10 ml 0.15 M NaC1, 0.1 M EDTA (pH 9.0) [12] at room temperature. Ten ml of 4% SDS in the same buffer were added and the mitochondrial lysate was then extracted three times with phenol/cresol [13] at room temperature. Nucleic acids were precipitated by addition of 2 vols. of 95% ethanol. For preparation of a post-ribosomal supernatant [14], purified mitochondria were resuspended in buffer (100 mM KC1, 10 mM MgCI 2, 10 mM Tris-HCl, pH 7.4) and lysed at a final concentration of 2% Triton X-100. After removal of membrane fragments by centrifugation at 10,000 rpm for 10 rain (IEC), the clarified mitochondrial lysate was centrifuged at 42,000 rpm for 90 min in a Ti50 rotor to sediment ribosomes. Transfer RNA was isolated from the supernatant by detergent phenol/cresol extraction.Nucleic acid samples were either used directly for 3'-end-labeling or, alternatively, the RNA samples (5 ~tg post-ribosomal supernatant RNA or 20/lg total nucleic acid) were dissolved in 7.5/11 NMF/urea loading buffer [15] containing 0.1% (w/v) xylene cyanol or Bromophenol blue and heated to 65°C for 5 min. After electrophoresis in a thin (0.5 ram) 6% polyacrylamide gel containing 7 M urea, the smallest tRNA was visualized by ethidium bromide staining and eluted [16] in the presence of 20 gtg linear polyacrylamide carrier (prepared as in [17], extracted with phenol, precipitated with ethanol and re-dissolved at 2.5 flg//.tl). The unlabeled tRNA was either re-purified by gel-electrophoresis and then subjected to modified nucleotide analysis [16] or used directly for end-labeling. 3'-End-labeled tRNA [18] was purified by gel-electrophoresis and used for chemical sequence analysis [18]; both 3'-and 5'-endlabeled tRNAs were subjected to enzymatic s...

The Oxytricha trifallax Mitochondrial Genome

“…In Oxytricha , we think it is unlikely that RNA editing removes all the stops necessary to join the nad2 ORFs (at least two stop codons would need to be eliminated or read through). Instead, it appears that, like nad1 (Seilhamer, Gutell, et al 1984; Seilhamer, Olsen, et al 1984; Heinonen et al 1987; Pritchard et al 1990; Schnare et al 1995; Burger et al 2000), this gene is not transspliced (supported by cDNA PCR results, data not shown), and therefore, the gene pieces are translated as separate subunits that require co-assembly to form the functional protein structure.…”

“…Ciliate mitochondrial genomes are both gene-rich and relatively large (20–60 kb) (Gray et al 1998), though many mitochondrial genes remain unclassified (Pritchard et al 1990; Burger et al 2000; Brunk et al 2003; Moradian et al 2007), in part due to their extreme divergences from other eukaryotic mitochondrial genomes (Pritchard et al 1990; Burger et al 2000; Moradian et al 2007). Split ribosomal RNA and nad1 genes (Seilhamer, Gutell, et al 1984; Seilhamer, Olsen, et al 1984; Schnare et al 1986, 1995; Heinonen et al 1987; Pritchard et al 1990; Burger et al 2000) were also discovered in ciliate mitochondria. Some anaerobic ciliates contain hydrogen-producing organelles, or hydrogenosomes, that may derive from mitochondria, and the ciliate Nyctotherus ( Armophorea ) has a partially sequenced hydrogenosome genome (Akhmanova et al 1998; Boxma et al 2005).…”

Complete Modification Maps for the Cytosolic Small and Large Subunit rRNAs of Euglena gracilis: Functional and Evolutionary Implications of Contrasting Patterns between the Two rRNA Components

“…We previously obtained similar results when sequencing the D loops of tRNAs. 35,36 The sequencing results for LSU rRNA position 3540 are diagnostic of 3-methyluridine (m 3 U), 37 which gives a normal band in alkali ladders (Fig. 7e) but yields very intense bands in the U and C lanes of chemical sequencing gels (Fig.…”

Structural rules and conformational compensations in the tRNA L-form 1 1Edited by D. E. Draper

“…The sequence of the mature tRNA corresponding to the tRNA gene sequence DX3800, published recently (Schnare et al, 1995), is incorporated in the Table, but has not yet received its ID number in the tRNA Compilation.…”