Single amino acid replacements affecting the thermostability of kanamycin nucleotidyltransferase

Summary Amino acid residues of the carboxyl-terminal region of kanamycin nucleotidyltransferase were modified using segment-directed mutagenesis. Six different mutant enzymes with single amino acid replacements were selected out of 59 clones by DNA sequence analyses. The mutant enzymes were purified and it was found that the thermostability of one mutant enzyme was identical to the wild type, whereas the other five were less thermostable at varying degrees. The data suggested that changes in the enzyme thermostability depend not only on the position but also on the species of amino acid residue replaced.     

A possible role for the pcnB gene product of Escherichia coli in modulating RNA: RNA interactions.

Summary The sequence of the PcnB protein of Escherichia coli, a protein required for copy number maintenance of ColE1-related plasmids, was compared with the PIR sequence database. Strong local similarities to the sequence of the E. coli protein tRNA nucleotidyltransferase were found. Since a substrate of the latter protein, tRNA, structurally resembles the RNAs that control ColE1 copy number we believe that we may have identified a region in PcnB that interacts with these RNAs. Consistent with this idea is our observation that PcnB is required for the replication of R1, a plasmid whose replication is also regulated by a small RNA.     

Solution NMR Studies of Chlorella Virus DNA Ligase-adenylate

DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3′-OH/5′-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone ¹⁵N spin relaxation and ¹⁵N,¹H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5′-phosphate.     

tRNA-nucleotidyltransferases: Highly unusual RNA polymerases with vital functions

tRNA-nucleotidyltransferases are fascinating and unusual RNA polymerases responsible for the synthesis of the nucleotide triplet CCA at the 3′-terminus of tRNAs. As this CCA end represents an essential functional element for aminoacylation and translation, these polymerases (CCA-adding enzymes) are of vital importance in all organisms. With a possible origin of ancient telomerase-like activity, the CCA-adding enzymes obviously emerged twice during evolution, leading to structurally different, but functionally identical enzymes. The evolution as well as the unique polymerization features of these interesting proteins will be discussed in this review.     

Mechanism for the definition of elongation and termination by the class II CCA‐adding enzyme

The CCA‐adding enzyme synthesizes the CCA sequence at the 3′ end of tRNA without a nucleic acid template. The crystal structures of class II Thermotoga maritima CCA‐adding enzyme and its complexes with CTP or ATP were determined. The structure‐based replacement of both the catalytic heads and nucleobase‐interacting neck domains of the phylogenetically closely related Aquifex aeolicus A‐adding enzyme by the corresponding domains of the T. maritima CCA‐adding enzyme allowed the A‐adding enzyme to add CCA in vivo and in vitro. However, the replacement of only the catalytic head domain did not allow the A‐adding enzyme to add CCA, and the enzyme exhibited (A, C)‐adding activity. We identified the region in the neck domain that prevents (A, C)‐adding activity and defines the number of nucleotide incorporations and the specificity for correct CCA addition. We also identified the region in the head domain that defines the terminal A addition after CC addition. The results collectively suggest that, in the class II CCA‐adding enzyme, the head and neck domains collaboratively and dynamically define the number of nucleotide additions and the specificity of nucleotide selection.     

Schizosaccharomyces pombe contains separate CC- and A-adding tRNA nucleotidyltransferases

A specific cytidine-cytidine-adenosine (CCA) sequence is required at the 3′-terminus of all functional tRNAs. This sequence is added during tRNA maturation or repair by tRNA nucleotidyltransferase enzymes. While most eukaryotes have a single enzyme responsible for CCA addition, some bacteria have separate CC- and A-adding activities. The fungus, Schizosaccharomyces pombe, has two genes (cca1 and cca2) that are thought, based on predicted amino acid sequences, to encode tRNA nucleotidyltransferases. Here, we show that both genes together are required to complement a Saccharomyces cerevisiae strain bearing a null mutation in the single gene encoding its tRNA nucleotidyltransferase. Using enzyme assays we show further that the purified S. pombe cca1 gene product specifically adds two cytidine residues to a tRNA substrate lacking this sequence while the cca2 gene product specifically adds the terminal adenosine residue thereby completing the CCA sequence. These data indicate that S. pombe represents the first eukaryote known to have separate CC- and A-adding activities for tRNA maturation and repair. In addition, we propose that a novel structural change in a tRNA nucleotidyltransferase is responsible for defining a CC-adding enzyme.     

Structures of the Mitochondrial CDP-DAG Synthase Tam41 Suggest a Potential Lipid Substrate Pathway from Membrane to the Active Site

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The ability of an arginine to tryptophan substitution in Saccharomyces cerevisiae tRNA nucleotidyltransferase to alleviate a temperature-sensitive phenotype suggests a role for motif C in active site organization

We report that the temperature-sensitive (ts) phenotype in Saccharomyces cerevisiae associated with a variant tRNA nucleotidyltransferase containing an amino acid substitution at position 189 results from a reduced ability to incorporate AMP and CMP into tRNAs. We show that this defect can be compensated for by a second-site suppressor converting residue arginine 64 to tryptophan. The R64W substitution does not alter the structure or thermal stability of the enzyme dramatically but restores catalytic activity in vitro and suppresses the ts phenotype in vivo. R64 is found in motif A known to be involved in catalysis and nucleotide triphosphate binding while E189 lies within motif C previously thought only to connect the head and neck domains of the protein. Although mutagenesis experiments indicate that residues R64 and E189 do not interact directly, our data suggest a critical role for residue E189 in enzyme structure and function. Both R64 and E189 may contribute to the organization of the catalytic domain of the enzyme. These results, along with overexpression and deletion analyses, show that the ts phenotype of cca1-E189F does not arise from thermal instability of the variant tRNA nucleotidyltransferase but instead from the inability of a partially active enzyme to support growth only at higher temperatures.