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Sequence analysis of 5'[32p] labeled mRNA and tRNA using polyacrylamide gel electrophoresis ABSTRACT Sequence analysis of 5'-[32P]labeled tRNA and eukaryotic mRNA using an adaptation of a method recently described by Donis-Keller, Maxam and Gilbert for mapping guanines, adenines and pyrimidines from the 5'-end of an RNA is described. In addition, a(More)
We report the sequences of Neurospora crassa mitochondrial alanine, leucine(1), leucine(2), threonine, tryptophan, and valine tRNAs. On the basis of the anticodon sequences of these tRNAs and of a glutamine tRNA, whose sequence analysis is nearly complete, we infer the following: (i) The N. crassa mitochondrial tRNA species for alanine, leucine(2),(More)
The lysine isoacceptor tRNAs differ in two aspects from the majority of the other mammalian tRNA species: they do not contain ribosylthymine (T) in loop IV, and a 'new' lysine tRNA, which is practically absent in non-dividing tissue, appears at elevated levels in proliferating cells. We have therefore purified the three major isoaccepting lysine tRNAs from(More)
Unfractionated tRNA, isolated from maize mitochondria, has been specifically labeled at the -CCA end and used to recover a tRNA gene-bearing fragment from a clone bank of maize mitochondrial DNA. This gene has been mapped, sequenced and found to carry the anticodon for histidine. The sequence of the gene and that of bases in its near vicinity are identical(More)
We have isolated and characterized a Neurospora crassa gene homologous to the yeast CYH2 gene encoding L29, a cycloheximide sensitivity-conferring protein of the cytoplasmic ribosome. The cloned Neurospora gene was isolated by cross-hybridization to CYH2. It was sequenced from both cDNA and genomic clones. The coding region is interrupted by seven(More)
The mitochondrial tyrosine tRNA from Neurospora crassa has been sequenced and found to have several interesting features: (i) It resembles prokaryotic rather than eukaryotic tyrosine tRNAs in that it possesses a large variable loop (loop III); moreover, it can be quantitatively aminoacylated by Escherichia coli tyrosyl-tRNA synthetase but not by yeast(More)
The hairpin ribozyme is a 50-nucleotide RNA enzyme of unknown three-dimensional structure. Here, we, demonstrate that interdomain interactions are required for catalytic function by reconstitution of activity following separation of an essential, independently folding domain (loop B) from the substrate binding strand at a helical junction. The resulting(More)
The catalytic determinants for the cleavage and ligation reactions mediated by the hairpin ribozyme are integral to the polyribonucleotide chain. We describe experiments that place G8, a critical guanosine, at the active site, and point to an essential role in catalysis. Cross-linking and modeling show that formation of a catalytic complex is accompanied by(More)
To form a catalytically active complex, the essential nucleotides of the hairpin ribozyme, embedded within the internal loops of the two domains, must interact with one another. Little is known about the nature of these essential interdomain interactions. In the work presented here, we have used recent topographical constraints and other biochemical data in(More)
The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that(More)