Sequence-dependent cleavage site selection by RNase Z from the cyanobacterium Synechocystis sp. PCC 6803.
Biosynthesis of transfer RNA requires processing from longer precursors at the 5'- and 3'-ends. In eukaryotes, in archaea, and in those bacteria where the 3'-terminal CCA sequence is not encoded, 3' processing is carried out by the endonuclease RNase Z, which cleaves after the discriminator nucleotide to generate a mature 3'-end ready for the addition of the CCA sequence. We have identified and cloned the gene coding for RNase Z in the cyanobacterium Synechocystis sp. PCC 6803. The gene has been expressed in Escherichia coli, and the recombinant protein was purified. The enzymatic activity of RNase Z from Synechocystis has been studied in vitro with a variety of substrates. The presence of C or CC after the discriminator nucleotide modifies the cleavage site of RNase Z so that it is displaced by one and two nucleotides to the 3'-side, respectively. The presence of the complete 3'-terminal CCA sequence in the precursor of the tRNA completely inhibits RNase Z activity. The inactive CCA-containing precursor binds to Synechocystis RNase Z with similar affinity than the mature tRNA. The properties of the enzyme described here could be related with the mechanism by which CCA is added in this organism, with the participation of two separate nucleotidyl transferases, one specific for the addition of C and another for the addition of A. This work is the first characterization of RNase Z from a cyanobacterium, and the first from an organism with two separate nucleotidyl transferases.
Ceballos-Chávez
María
M
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Américo Vespucio 49, E-41092 Sevilla, Spain.
Vioque
Agustín
A
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D000458:Cyanobacteria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004722:Endoribonucleases / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010727:Phosphoric Diester Hydrolases / Q000032:analysis / Q000378:metabolism; D010802:Phylogeny; D012322:RNA Precursors / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D012322:RNA Precursors; D011994:Recombinant Proteins; D012343:RNA, Transfer; D004722:Endoribonucleases; C110250:RNase Z; D010727:Phosphoric Diester Hydrolases