title: The 5' leader of precursor tRNAAsp bound to the Bacillus subtilis RNase P holoenzyme has an extended conformation.
abstract: RNase P catalyzes the 5' maturation of transfer RNA (tRNA). RNase P from Bacillus subtilis comprises a large RNA component (130 kDa, P RNA) and a small protein subunit (14 kDa, P protein). Although P RNA alone can efficiently catalyze the maturation reaction in vitro, P protein is strictly required under physiological conditions. We have used time-resolved fluorescence resonance energy transfer on a series of donor-labeled substrates and two acceptor-labeled P proteins to determine the conformation of the pre-tRNA 5' leader relative to the protein in the holoenzyme-pre-tRNA complex. The resulting distance distribution measurements indicate that the leader binds to the holoenzyme in an extended conformation between nucleotides 3 and 7. The conformational mobility of nucleotides 5-8 in the leader is reduced, providing further evidence that these nucleotides interact with the holoenzyme. The increased fluorescence intensity and lifetime of the 5'-fluorescein label of these leaders indicate a more hydrophobic environment, consistent with the notion that such interactions occur with the central cleft of the P protein. Taken together, our data support a model where the P protein binds to the 5' leader between the fourth and seventh nucleotides upstream of the cleavage site, extending the leader and decreasing its structural dynamics. Thus, P protein acts as a wedge to separate the 5' from the 3' terminus of the pre-tRNA and to position the cleavage site in the catalytic core. These results reveal a structural basis for the P protein dependent discrimination between precursor and mature tRNAs.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: David
authors.initials: D
authors.lastname: Rueda
authors.forename: John
authors.initials: J
authors.lastname: Hsieh
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Day-Storms
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
authors.forename: Nils G
authors.initials: NG
authors.lastname: Walter
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012349:RNA, Transfer, Asp / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D043262:Ribonuclease P / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012349:RNA, Transfer, Asp; D043262:Ribonuclease P