Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate mechanisms for processing of plant mitochondrial RNAs, we studied the fate of wheat mitochondrial tRNA precursors in a homologous soluble extract. Artificial precursor transcripts were synthesized in vitro using T3 or T7 RNA polymerase and DNA templates containing wheat mitochondrial tRNA genes and flanking sequences. We found that the mitochondrial extract supports processing of precursors containing both native and chloroplast-like (Joyce, P. B. M., and Gray, M. W. (1989) Nucleic Acids Res. 77, 5461-5476) wheat mitochondrial tRNA sequences. Incubation of precursor transcripts with the extract results in processing of tRNAs via precise 5'- and 3'-endonucleolytic cleavages. However, these cleavages are not ordered in vitro because intermediates composed of 5'-leader + tRNA and tRNA + 3'-trailer are present simultaneously throughout the course of the reaction. Sequence analysis of processed products confirmed that endonucleolytic cleavages occur at the expected positions, generating tRNAs with 5'-phosphoryl and 3'-hydroxyl termini. The mitochondrial extract also contains a tRNA nucleotidyltransferase activity that adds -CCAOH termini to the 3'-ends of processed tRNAs. This cell-free RNA processing system provides the basis for biochemical characterization of the various enzymes involved in the production and maturation of plant mitochondrial tRNAs.
...
PMID:Processing of transfer RNA precursors in a wheat mitochondrial extract. 169 57

To study the mechanism involved in the 3'-processing of mitochondrial tRNA precursors, we examined tRNA processing in a reconstituted system with a yeast mitochondrial extract. Two mitochondrial tRNA(Glu) precursors synthesized from SP6 RNA polymerase-directed transcription system were used as substrates. One contained a 214-nucleotide 5' terminus and 115-123-nucleotide 3' trailer. The other had the same sized 3' trailer, but contained a mature 5' terminus. An endonucleolytic activity was identified in a mitochondrial S30 fraction which cleaves the 3' terminus of the latter tRNA precursor precisely at the in vivo CCA addition site. No cleavage of the 5'-extended precursor was observed in vitro. This mitochondrial 3'-processing activity was partially purified using DEAE-CL-6B chromatography. It removes the 3' trailer sequence from the 5'-matured precursor leaving a 3'-hydroxyl group on the processed tRNA and a 5'-phosphate group on the trailer. The resulting tRNA product serves as a substrate for tRNA nucleotidyltransferase which catalyzes the addition of CCA residues to the tRNA to complete its 3' maturation. Thus, yeast mitochondrial 3'-tRNA processing events resemble those found in eucaryotic cytoplasmic/nuclear systems where a single endonucleolytic cleavage is responsible for the formation of the 3' end of the tRNAs. This is in contrast to the multistep 3'-processing events known to occur in procaryotes.
...
PMID:Biosynthesis of tRNA in yeast mitochondria. An endonuclease is responsible for the 3'-processing of tRNA precursors. 284 29

The genome of Geobacter sulfurreducens contains three genes whose sequences are quite similar to sequences encoding known members of an RNA nucleotidyltransferase superfamily that includes tRNA nucleotidyltransferases and poly(A) polymerases. Reverse transcription-PCR using G. sulfurreducens total RNA demonstrated that the genes encoding these three proteins are transcribed. These genes, encoding proteins designated NTSFI, NTSFII, and NTSFIII, were cloned and overexpressed in Escherichia coli. The corresponding enzymes were purified and assayed biochemically, resulting in identification of NTSFI as a poly(A) polymerase, NTSFII as a C-adding tRNA nucleotidyltransferase, and NTSFIII as an A-adding tRNA nucleotidyltransferase. Analysis of G. sulfurreducens rRNAs and mRNAs revealed the presence of heteropolymeric RNA 3' tails. This is the first characterization of a bacterial system that expresses separate C- and A-adding tRNA nucleotidyltransferases and a poly(A) polymerase.
...
PMID:Geobacter sulfurreducens contains separate C- and A-adding tRNA nucleotidyltransferases and a poly(A) polymerase. 1895 95

The trancription of a cloned trnV1-trnN1-trnR1 cluster from Euglena gracilis chloroplast (ct) DNA and the processing of a tRNA(Val)-tRNA(Asn)-tRNA(Arg) polycistronic precursor were studied in a spinach ct transcription extract. A soluble ct RNA polymerase selectively transcribes the trnV1-trnN1-trnR1-trnL1 locus in the EcoG fragment from the Euglena ct genome. Restriction enzyme modified templates and RNA fingerprint analysis were used to confirm that the tRNA genes were correctly transcribed. The tRNA(Val)-tRNA(Asn)-tRNA(Arg) polycistronic precursor transcribed by RNA polymerase III in a HeLa cell extract was used as a substrate to demonstrate that a ct tRNA precursor molecule is correctly processed by the ct tRNA processing enzymes. The oligonucleotide pattern of tRNAs processed in vitro from the tRNA(Val)-tRNA(Asn)-RNA(Arg) polycistronic precursor is indistinguishable from tRNA(Val), tRNA(Asn) and tRNA(Arg) transcribed by the ct RNA polymerase and processed in the ct transcription extract. The 3'-CCAOH is added to the tRNAs by a 3' nucleotidyltransferase after correct processing of the 3' terminus. Correct pseudouridylation was demonstrated for uridine residues in a tRNA(Met) m molecule transcribed from a spinach ct trnM1 locus. Thus, the enzymatic activities involved in tRNA biosynthesis in vitro include DNA-dependent (tDNA) RNA polymerase, a 5'-processing activity (RNase P-like), a 3'-exonuclease, an endoribonuclease involved in 3'-tRNA maturation, a tRNA nucleotidyltransferase, and pseudouridylate synthetase.
...
PMID:Accurate processing and pseudouridylation of chloroplast transfer RNA in a chloroplast transcription system. 2431 Mar 5

The universal 3'-terminal CCA sequence of tRNA is built and/or synthesized by the CCA-adding enzyme, CTP:(ATP) tRNA nucleotidyltransferase. This RNA polymerase has no nucleic acid template, but faithfully synthesizes the defined CCA sequence on the 3'-terminus of tRNA at one time, using CTP and ATP as substrates. The mystery of CCA-addition without a nucleic acid template by unique RNA polymerases has long fascinated researchers in the field of RNA enzymology. In this review, the mechanisms of RNA polymerization by the remarkable CCA-adding enzyme and its related enzymes are presented, based on their structural features.
...
PMID:Molecular mechanisms of template-independent RNA polymerization by tRNA nucleotidyltransferases. 2459 76

Pathways for tolerating and repairing DNA-protein crosslinks (DPCs) are poorly defined. We used transposon mutagenesis and candidate gene approaches to identify DPC-hypersensitive Escherichia coli mutants. DPCs were induced by azacytidine (aza-C) treatment in cells overexpressing cytosine methyltransferase; hypersensitivity was verified to depend on methyltransferase expression. We isolated hypersensitive mutants that were uncovered in previous studies (recA, recBC, recG, and uvrD), hypersensitive mutants that apparently activate phage Mu Gam expression, and novel hypersensitive mutants in genes involved in DNA metabolism, cell division, and tRNA modification (dinG, ftsK, xerD, dnaJ, hflC, miaA, mnmE, mnmG, and ssrA). Inactivation of SbcCD, which can cleave DNA at protein-DNA complexes, did not cause hypersensitivity. We previously showed that tmRNA pathway defects cause aza-C hypersensitivity, implying that DPCs block coupled transcription/translation complexes. Here, we show that mutants in tRNA modification functions miaA, mnmE and mnmG cause defects in aza-C-induced tmRNA tagging, explaining their hypersensitivity. In order for tmRNA to access a stalled ribosome, the mRNA must be cleaved or released from RNA polymerase. Mutational inactivation of functions involved in mRNA processing and RNA polymerase elongation/release (RNase II, RNaseD, RNase PH, RNase LS, Rep, HepA, GreA, GreB) did not cause aza-C hypersensitivity; the mechanism of tmRNA access remains unclear.
...
PMID:Functions that protect Escherichia coli from DNA-protein crosslinks. 2573 40

tRNA nucleotidyltransferase adds the invariant CCA-terminus to the tRNA 3'-end, a central step in tRNA maturation. This CCA-adding enzyme is a specialized RNA polymerase that synthesizes the CCA sequence at high fidelity in all kingdoms of life. Recently, an additional function of this enzyme was identified, where it generates a specific degradation tag on structurally unstable tRNAs. This tag consists of an additional repeat of the CCA triplet, leading to a 3'-terminal CCACCA sequence. In order to explain how the enzyme catalyzes this extended polymerization reaction, Kuhn et al. solved a series of co-crystal structures of the CCA-adding enzyme from Archaeoglobus fulgidus in complex with different tRNA substrates. They show that the enzyme forces a bound unstable tRNA to refold the acceptor stem for a second round of CCA-addition, while stable transcripts are robust enough to resist this isomerization. In this review, we discuss how the CCA-adding enzyme uses a simple yet very elegant way to scrutinize its substrates for sufficient structural stability and, consequently, functionality.
...
PMID:The CCA-adding enzyme: A central scrutinizer in tRNA quality control. 2617 25

tRNAs are important players in the protein synthesis machinery, where they act as adapter molecules for translating the mRNA codons into the corresponding amino acid sequence. In a series of highly conserved maturation steps, the primary transcripts are converted into mature tRNAs. In the amoebozoan Acanthamoeba castellanii, a highly unusual evolution of some of these processing steps was identified that are based on unconventional RNA polymerase activities. In this context, we investigated the synthesis of the 3'-terminal CCA-end that is added posttranscriptionally by a specialized polymerase, the tRNA nucleotidyltransferase (CCA-adding enzyme). The majority of eukaryotic organisms carry only a single gene for a CCA-adding enzyme that acts on both the cytosolic as well as the mitochondrial tRNA pool. In a bioinformatic analysis of the genome of this organism, we identified a surprising multitude of genes for enzymes that contain the active site signature of eukaryotic/eubacterial tRNA nucleotidyltransferases. In vitro activity analyses of these enzymes revealed that two proteins represent bona fide CCA-adding enzymes, one of them carrying an N-terminal sequence corresponding to a putative mitochondrial target signal. The other enzymes have restricted activities and represent CC- and A-adding enzymes, respectively. The A-adding enzyme is of particular interest, as its sequence is closely related to corresponding enzymes from Proteobacteria, indicating a horizontal gene transfer. Interestingly, this unusual diversity of nucleotidyltransferase genes is not restricted to A. castellanii, but is also present in other members of the Acanthamoeba genus, indicating an ancient evolutionary trait.
...
PMID:CCA Addition gone wild: Unusual Occurrence and Phylogeny of four different tRNA Nucleotidyltransferases in Acanthamoeba castellanii. 3309 40

---