Probing the architecture of the B. subtilis RNase P holoenzyme active site by cross-linking and affinity cleavage.
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.
Niranjanakumari
Somashekarappa
S
Chemistry Department, University of Michigan, Ann Arbor, MI 48109, USA.
Day-Storms
Jeremy J
JJ
Ahmed
Mahiuddin
M
Hsieh
John
J
Zahler
Nathan H
NH
Venters
Ronald A
RA
Fierke
Carol A
CA
D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D017665:Hydroxyl Radical / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043262:Ribonuclease P / Q000737:chemistry* / Q000378:metabolism*
D003432:Cross-Linking Reagents; D020035:Holoenzymes; D021122:Protein Subunits; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D017665:Hydroxyl Radical; D043262:Ribonuclease P; D003545:Cysteine