Cloning, purification and characterization of the protein subunit of ribonuclease P from the cyanobacterium Synechocystis sp. PCC 6803.
The rnpA gene from the cyanobacterium Synechocystis sp. PCC 6803, which codes for the protein subunit of ribonuclease P (RNase P), has been cloned by functional complementation of an Escherichia coli mutant. This protein had previously been characterized only in proteobacteria and gram-positive bacteria. rnpA and the closely linked rpmH gene, which code for the large subunit ribosomal protein L34, have been sequenced. The Synechocystis 6803 L34 protein is more similar to the homologous protein from some non-green chloroplasts than to the L34 protein from other bacteria. The protein subunit of RNase P from Synechocystis 6803 has been overexpressed in E. coli and purified to homogeneity. Antibodies raised against the Synechocystis 6803 RNase P protein did not recognize the homologous protein from E. coli (C5 protein). Similarly, antibodies raised against the E. coli C5 protein did not recognize significantly the Synechocystis 6803 protein. In spite of the lack of immunological cross-reactivity and the low level of sequence identity, the E. coli and Synechocystis 6803 proteins are functionally interchangeable. In enzymatic assays using either an E. coli precursor tRNA(Tyr) or a Synechocystis 6803 precursor tRNA(Gln) as substrates, we have detected RNase P activity with holoenzymes reconstituted with the RNA subunit from E. coli and the protein subunit from Synechocystis 6803 or with the RNA subunit from Synechocystis 6803 and the protein subunit from E. coli. The relative efficiency of cleavage of the different substrates is dependent on the origin of the protein subunit used to reconstitute the holoenzyme.
Pascual
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Instituto de Bioquímica Vegetal y Fotosíntesis, Facultad de Biología, Universidad de Sevilla-CSIC, Spain.
Vioque
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D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D000458:Cyanobacteria / Q000737:chemistry / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012351:RNA, Transfer, Gln / Q000235:genetics; D012365:RNA, Transfer, Tyr / Q000235:genetics; D043262:Ribonuclease P; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis
D000906:Antibodies; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012322:RNA Precursors; D016337:RNA, Catalytic; D012351:RNA, Transfer, Gln; D012365:RNA, Transfer, Tyr; D012269:Ribosomal Proteins; C041672:ribosomal protein L34; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli