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Query: UNIPROT:P06889 (Mol)
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The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed.
J Mol Biol 2000 Sep 08
PMID:Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1. 1096 61

Pseudouridine (5-beta-D-ribofuranosyluracil, Psi) is the most commonly found modified base in RNA. Conversion of uridine to Psi is performed enzymatically in both prokaryotes and eukaryotes by pseudouridine synthases (EC 4.2.1.70). The Escherichia coli Psi-synthase RluD modifies uridine to Psi at positions 1911, 1915 and 1917 within 23S rRNA. RluD also possesses a second function related to proper assembly of the 50S ribosomal subunit that is independent of Psi-synthesis. Here, we report the crystal structure of the catalytic module of RluD (residues 68-326; DeltaRluD) refined at 1.8A to a final R-factor of 21.8% (R(free)=24.3%). DeltaRluD is a monomeric enzyme having an overall mixed alpha/beta fold. The DeltaRluD molecule consists of two subdomains, a catalytic subdomain and C-terminal subdomain with the RNA-binding cleft formed by loops extending from the catalytic sub-domain. The catalytic sub-domain of DeltaRluD has a similar fold as in TruA, TruB and RsuA, with the location of the RNA-binding cleft, active-site and conserved, catalytic Asp residue superposing in all four structures. Superposition of the crystal structure of TruB bound to a T-stem loop with RluD reveals that similar RNA-protein interactions for the flipped-out uridine base would exist in both structures, implying that base-flipping is necessary for catalysis. This observation also implies that the specificity determinants for site-specific RNA-binding and recognition likely reside in parts of RluD beyond the active site.
J Mol Biol 2004 Jan 02
PMID:Crystal structure of the RluD pseudouridine synthase catalytic module, an enzyme that modifies 23S rRNA and is essential for normal cell growth of Escherichia coli. 1465 42

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.
Plant Mol Biol 1984 Mar
PMID:Accurate processing and pseudouridylation of chloroplast transfer RNA in a chloroplast transcription system. 2431 Mar 5