The bacterial ribonuclease P holoenzyme requires specific, conserved residues for efficient catalysis and substrate positioning.
RNase P is an RNA-based enzyme primarily responsible for 5'-end pre-tRNA processing. A structure of the bacterial RNase P holoenzyme in complex with tRNAPhe revealed the structural basis for substrate recognition, identified the active site location, and showed how the protein component increases functionality. The active site includes at least two metal ions, a universal uridine (U52), and P RNA backbone moieties, but it is unclear whether an adjacent, bacterially conserved protein loop (residues 52-57) participates in catalysis. Here, mutagenesis combined with single-turnover reaction kinetics demonstrate that point mutations in this loop have either no or modest effects on catalytic efficiency. Similarly, amino acid changes in the 'RNR' region, which represent the most conserved region of bacterial RNase P proteins, exhibit negligible changes in catalytic efficiency. However, U52 and two bacterially conserved protein residues (F17 and R89) are essential for efficient Thermotoga maritima RNase P activity. The U52 nucleotide binds a metal ion at the active site, whereas F17 and R89 are positioned >20 Å from the cleavage site, probably making contacts with N(-4) and N(-5) nucleotides of the pre-tRNA 5'-leader. This suggests a synergistic coupling between transition state formation and substrate positioning via interactions with the leader.
Department of Molecular Biosciences, Northwestern University, 2205 Tech Dr., Evanston, IL 60208, USA. nick.reiter@vanderbilt.edu
Nicholas J
NJ
Reiter
Amy K
AK
Osterman
Alfonso
A
Mondragón
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D043262:Ribonuclease P / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020124:Thermotoga maritima / Q000201:enzymology
D001426:Bacterial Proteins; D012322:RNA Precursors; D012343:RNA, Transfer; D043262:Ribonuclease P