In vivo and in vitro investigation of bacterial type B RNase P interaction with tRNA 3'-CCA.
For catalysis by bacterial type B RNase P, the importance of a specific interaction with p(recursor)tRNA 3'-CCA termini is yet unclear. We show that mutation of one of the two G residues assumed to interact with 3'-CCA in type B RNase P RNAs inhibits cell growth, but cell viability is at least partially restored at increased RNase P levels due to RNase P protein overexpression. The in vivo defects of the mutant enzymes correlated with an enzyme defect at low Mg(2+) in vitro. For Bacillus subtilis RNase P, an isosteric C259-G(74) bp fully and a C258-G(75) bp slightly rescued catalytic proficiency, demonstrating Watson-Crick base pairing to tRNA 3'-CCA but also emphasizing the importance of the base identity of the 5'-proximal G residue (G258). We infer the defect of the mutant enzymes to primarily lie in the recruitment of catalytically relevant Mg(2+), with a possible contribution from altered RNA folding. Although with reduced efficiency, B. subtilis RNase P is able to cleave CCA-less ptRNAs in vitro and in vivo. We conclude that the observed in vivo defects upon disruption of the CCA interaction are either due to a global deceleration in ptRNA maturation or severe inhibition of 5'-maturation for a ptRNA subset.
Wegscheid
Barbara
B
Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, D-35037 Marburg, Germany.
Hartmann
Roland K
RK
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001419:Bacteria / Q000201:enzymology*; D001483:Base Sequence; D002021:Buffers; D004252:DNA Mutational Analysis; D029968:Escherichia coli Proteins / Q000235:genetics; D005816:Genetic Complementation Test; D020035:Holoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics
D002021:Buffers; D029968:Escherichia coli Proteins; D020035:Holoenzymes; D012322:RNA Precursors; D012343:RNA, Transfer; D043262:Ribonuclease P; C470865:ribonuclease P, E coli