Structural requirements for processing of synthetic tRNAHis precursors by the catalytic RNA component of RNase P.
Experiments were conducted to investigate structural features of the aminoacyl stem region of precursor histidine tRNA critical for the proper cleavage by the catalytic RNA component of RNase P that is responsible for 5' maturation. Histidine tRNA was chosen for study because tRNAHis has an 8 base pair instead of the typical 7-base pair aminoacyl stem. The importance of the 3' proximal CCA sequence in the 5'-processing reaction was also investigated. Our results show that the tRNAHis precursor patterned after the natural Bacillus subtilis gene is cleaved by catalytic RNAs from B. subtilis or Escherichia coli, leaving an extra G residue at the 5'-end of the aminoacyl stem. Replacing the 3' proximal CCA sequence in the substrate still allowed the catalytic RNA to cleave at the proper position, but it increased the Km of the reaction. Changing the sequence of the 3' leader region to increase the length of the aminoacyl stem did not alter the cleavage site but reduced the reaction rate. However, replacing the G residue at the expected 5' mature end by an A changed the processing site, resulting in the creation of a 7-base pair aminoacyl stem. The Km of this reaction was not substantially altered. These experiments indicate that the extra 5' G residue in B. subtilis tRNAHis is left on by RNase P processing because of the precursor's structure at the aminoacyl stem and that the cleavage site can be altered by a single base change. We have also shown that the catalytic RNA alone from either B. subtilis or E. coli is capable of cleaving a precursor tRNA in which the 3' proximal CCA sequence is replaced by other nucleotides.
D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000378:metabolism*; D012322:RNA Precursors / Q000138:chemical synthesis*; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012354:RNA, Transfer, His / Q000378:metabolism*; D043262:Ribonuclease P; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D012322:RNA Precursors; D012345:RNA, Transfer, Amino Acid-Specific; D012354:RNA, Transfer, His; D012313:RNA; D004722:Endoribonucleases; D043262:Ribonuclease P; C470865:ribonuclease P, E coli