Lanny I. Hecker

Structure and function of initiator methionine tRNA from the mitochondria of neurospora crassa

Abstract Initiator methionine tRNA from the mitochondria of Neurospora crassa has been purified and sequenced. This mitochondrial tRNA can be aminoacylated and formylated by E. coli enzymes, and is capable of initiating protein synthesis in E. coli extracts. The nucleotide composition of the mitochondrial initiator tRNA (the first mitochondrial tRNA subjected to sequence analysis) is very rich in A + U, like that reported for total mitochondrial tRNA. In two of the unique features which differentiate procaryotic from eucaryotic cytoplasmic initiator tRNAs, the mitochondrial tRNA appears to resemble the eucaryotic initiator tRNAs. Thus unlike procaryotic initiator tRNAs in which the 5′ terminal nucleotide cannot form a Watson-Crick base pair to the fifth nucleotide from the 3′ end, the mitochondrial tRNA can form such a base pair; and like the eucaryotic cytoplasmic initiator tRNAs, the mitochondrial initiator tRNA lacks the sequence -TΨCG(or A) in loop IV. The corresponding sequence in the mitochondrial tRNA, however, is -UGCA- and not -AU(or Ψ)CG-as found in all eucaryotic cytoplasmic initiator tRNAs. In spite of some similarity of the mitochondrial initiator tRNA to both eucaryotic and procaryotic initiator tRNAs, the mitochondrial initiator tRNA is basically different from both these tRNAs. Between these two classes of initiator tRNAs, however, it is more homologous in sequence to procaryotic (56–60%) than to eucaryotic cytoplasmic initiator tRNAs (45–51%).

The first nucleotide sequence of an organelle transfer RNA: Chloroplastic tRNAPhe

The primary sequence of phenylalanine tRNA (tRNAphe) from the chloroplasts of Euglena gracilis has been determined: pG-C-U-G-G-G-A-U-A-G-C-U-C-A-G-D-U-G-Gm-U(U)-A-G-A-G-C-G-G-A-G-G-A-C-U-G-A-A-A-A-PSI-C-C-U-U-G-U-m7G-Py-C-A-C-C-A-G-T-psi-C-A-A-A-U-C-U-G-G-U-U-C-C-U-A-G-C-A-C-C-A. This represents the first nucleotide sequence determined for an organelle tRNA. As do all other tRNAPhes thus far sequenced, chloroplastic tRNAPhe contains 76 nucleotides. Both in the nature of its modified nucleotides and in its sequence (although the sequence of all known tRNAPhes is quite similar), chloroplastic tRNAPhe more closely resembles procaryotic tRNAPhes than it resembles those from the cytoplasm of eucaryotes. There are eight positions in the tRNAPhe molecule where nucleotides are invariant in procaryotes but differ from invariant nucleotides in eucaryotes; at five of these positions, chloroplastic tRNAPhe is similar to procaryotes. The possible evolutionary significance of this intermediate type of structure is discussed.

The Transfer RNAs of Eukaryotic Organelles

Publisher Summary This chapter reviews that organelles contain a complete apparatus for the synthesis of proteins. It is clear that, at least in most cases, organelle tRNAs are transcripts of the organelle genome. The limited number of tRNAs present in organelles in contrast to prokaryotes and the eukaryotic cytoplasm-poses an interesting problem. The chapter discusses the two hypotheses: (1) That organelles evolved via endosymbiosis, and (2) that organelles evolved through invagination and compartmentalization of function. It suggests that either certain codons are not utilized by organelles or that, because of “wobble” at the third position of the anticodon, or a few isoacceptors-all 61 “sense” codons may be translated. As codon recognition by aminoacyl-tRNAs is determined, solely by the tRNA component, these elements of the protein synthetic machinery are in large part, responsible for maintaining the fidelity of the genetic code. The aim of this chapter is to review the development and progress of organelle tRNA research and the aminoacyl-tRNA synthetases.

The nucleotide sequence of Euglena cytoplasmic phenylalanine transfer RNA. Evidence for possible classifications of Euglena among the animal rather than the plant kingdom.

The nucleotide sequence of cytoplasmic phenylalanine tRNA from Euglena gracilis has been elucidated using procedures described previously for the corresponding chloroplastic tRNA [Cell, 9, 717 (1976)]. The sequence is: pG-C-C-G-A-C-U-U-A-m(2)G-C-U-Cm-A-G-D-D-G-G-G-A-G-A-G-C-m(2)2G-psi-psi-A-G-A-Cm -U-Gm-A-A-Y-A-psi-C-U-A-A-A-G-m(7)G-U-C-*C-C-U-G-G-T-psi-C-G-m(1)A-U-C-C-C-G-G- G-A-G-psi-C-G-G-C-A-C-C-A. Like other tRNA Phes thus far sequenced, this tRNA has a chain length of 76 nucleotides. The sequence of E. gracilis cytoplasmic tRNA Phe is quite different (27 nucleotides out of 76 different) from that of the corresponding chloroplastic tRNA but is surprisingly similar (72 out of 76 nucleotides identical) to that of tRNA Phe from mammalian cytoplasm. This extent of sequence homology even exceeds that found between E. gracilis and wheat germ cytoplasmic tRNA Phe. These findings raise interesting questions on the evolution of tRNAs and the taxonomy of Euglena.

Comments on the translational and transcriptional origin of Euglena chloroplastic aminoacyl-tRNA synthetases.

A response to: “A consideration of Euglena gracilis W3BUL as a cytoplasmic control for the wild-type phenylalanyl-tRNA synthetase system” and “A reinvestigation of the sites of transcription and translation of Euglena chloroplastic phenylalanyl-tRNA synthetase” by J. L. Lesiewicz and D. S. Herson.

[13] Isolation of Neurospora mitochondrial tRNA

Publisher Summary This chapter discusses the isolation of Neurospora mitochondrial tRNA. Large quantities of highly purified mitochondria are obtained from Neurospora crassa with the use of zonal rotors. The ability to obtain these organelles in high yields has been instrumental in demonstrating the existence of unique species of tRNAs and aminoacyl-tRNA synthetases in mitochondria and, more recently, to isolate and purify initiator methionine tRNA from these organelles. Neither methionine nor phenylalanine tRNAs from mitochondria are detected in total cell preparations from N. crassa. This situation is probably due to the presence of relatively small amounts of mitochondrial tRNAs in Neurospora and/or the lability of these molecules because of their high (A+U) contents. Thus, in order to detect and purify mitochondrial tRNAs, large amounts of N. crassa mitochondria is isolated free of cytoplasmic contamination. The methods of mitochondrial isolation employed are derived from those of Hall and Greenawalt and in some respects are similar to the small-scale isolation of mitochondria.

[35] Isolation of plastid ribosomes from Euglena

Publisher Summary The chapter discusses the isolation of plasmid ribosomes from Euglena. The chloroplastic ribosomes of Euglena are unstable; if exposed to suboptimal ionic conditions, the 68 S monosome is converted to a 53 S particle. Under the ionic conditions used to stabilize the chloroplast monosome, organelles isolated in 12 mM Mg 2+ clump together, making it impossible to free them of cytoplasmic contamination. Chloroplast ribosomes cannot be isolated directly from whole-cell lysates, therefore, a procedure is developed for the large-scale isolation of structurally intact chloroplasts. If the structural integrity of the chloroplast is maintained, the chloroplast ribosomes are protected from the unfavorable ionic conditions of the chloroplast isolation buffers; these chloroplasts are thus suitable starting material for the isolation of chloroplast monosomes. The purity of each chloroplastic ribosome preparation is checked on sucrose gradients or on polyacrylamide gel after extractions of the rRNAs because the quantity of contaminating cytoplasmic ribosomes is variable. Chloroplastic monosomes are separated in pure form by centrifugation on sucrose gradients in tubes or in zonal rotors. Chloroplast ribosomal subunits are obtained by dialyzing the chloroplast ribosomal pellet against low Mg 2+ (1.0 m M ) before separation on sucrose gradients.

[14] Isolation of Euglena chloroplastic tRNA and purification of chloroplasticPhe

Publisher Summary The chapter discusses the isolation of Euglena chloroplastic tRNA and purification of chloroplastic tRNA Phe . Total mitochondrial and chloroplast tRNAs are isolated using zonal rotors, essentially free from contaminating cytoplasmic components for purposes of identification and characterization and use in hybridization studies. Individual species of organelle tRNA are purified for use in structural and hybridization studies. The various purification procedures, particularly those involving large-scale organelle isolations, are the subject of the chapter. The isolated chloroplasts are suspended in breaking buffer (10 mM Tris.HCl, pH 7.5, 0.1 M sodium chloride, 10 mM magnesium acetate, 10 mM 2-mercaptoethanol, and 3 mM sodium azide) plus 1% sodium dodecyl sulfate, and the total tRNA is isolated. After phenol and chloroform extractions, the deproteinized nucleic acids are precipitated with ethanol (2.5 volumes of ethanol) and resuspended in breaking buffer. RNA obtained from DEAE-cellulose are fractionated using benzoylated DEAE-cellulose (BD-cellulose) column chromatography to separate individual isoacceptors, tRNA is first deacylated in 0.5 M Tris HCl buffer, pH 8, for 1-2 hr at room temperature. Chloroplastic tRNAs can easily be identified in whole-cell preparations of Euglena tRNA assayed for amino acid acceptor after chromatography on BD cellulose columns.