TY - JOUR
T1 - The bacterial ribonuclease P holoenzyme requires specific, conserved residues for efficient catalysis and substrate positioning
AU - Reiter, Nicholas J.
AU - Osterman, Amy K.
AU - Mondragón, Alfonso
N1 - Funding Information:
N.J.R. was an National Institutes of Health National Service Award postdoctoral fellow [5F32GM087055]. National Institutes of Health (NIH) [5R01GM058443 to A.M.]. Funding for open access charge: NIH.
PY - 2012/11
Y1 - 2012/11
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/gks744
DO - 10.1093/nar/gks744
M3 - Article
C2 - 22904083
AN - SCOPUS:84869034853
VL - 40
SP - 10384
EP - 10393
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 20
ER -