Probing the architecture of the B. subtilis RNase P holoenzyme active site by cross-linking and affinity cleavage.

Journal Article (Journal Article)

Bacterial ribonuclease P (RNase P) is a ribonucleoprotein complex composed of one catalytic RNA (PRNA) and one protein subunit (P protein) that together catalyze the 5' maturation of precursor tRNA. High-resolution X-ray crystal structures of the individual P protein and PRNA components from several species have been determined, and structural models of the RNase P holoenzyme have been proposed. However, holoenzyme models have been limited by a lack of distance constraints between P protein and PRNA in the holoenzyme-substrate complex. Here, we report the results of extensive cross-linking and affinity cleavage experiments using single-cysteine P protein variants derivatized with either azidophenacyl bromide or 5-iodoacetamido-1,10-o-phenanthroline to determine distance constraints and to model the Bacillus subtilis holoenzyme-substrate complex. These data indicate that the evolutionarily conserved RNR motif of P protein is located near (<15 Angstroms) the pre-tRNA cleavage site, the base of the pre-tRNA acceptor stem and helix P4 of PRNA, the putative active site of the enzyme. In addition, the metal binding loop and N-terminal region of the P protein are proximal to the P3 stem-loop of PRNA. Studies using heterologous holoenzymes composed of covalently modified B. subtilis P protein and Escherichia coli M1 RNA indicate that P protein binds similarly to both RNAs. Together, these data indicate that P protein is positioned close to the RNase P active site and may play a role in organizing the RNase P active site.

Full Text

Duke Authors

Cited Authors

  • Niranjanakumari, S; Day-Storms, JJ; Ahmed, M; Hsieh, J; Zahler, NH; Venters, RA; Fierke, CA

Published Date

  • April 2007

Published In

Volume / Issue

  • 13 / 4

Start / End Page

  • 521 - 535

PubMed ID

  • 17299131

Pubmed Central ID

  • PMC1831860

International Standard Serial Number (ISSN)

  • 1355-8382

Digital Object Identifier (DOI)

  • 10.1261/rna.308707


  • eng

Conference Location

  • United States