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Specific phosphorothioate substitutions probe the active site of Bacillus subtilis ribonuclease P

Published online by Cambridge University Press:  20 August 2002

SHARON M. CRARY ,
JEFFREY C. KURZ  and
CAROL A. FIERKE
SHARON M. CRARY
Affiliation:
Biochemistry Department, Duke University Medical Center, Durham, North Carolina 27710, USA Present address: Special Pathogens Branch, Centers for Disease Control and Prevention, 1600 Clifton Road, NE, Mailstop G14, Atlanta, Georgia 30333, USA.
JEFFREY C. KURZ
Affiliation:
Biochemistry Department, Duke University Medical Center, Durham, North Carolina 27710, USA Present address: Archemix Corporation, One Hampshire Street, Fifth Floor, Cambridge, Massachusetts 02139, USA.
CAROL A. FIERKE
Affiliation:
Biochemistry Department, Duke University Medical Center, Durham, North Carolina 27710, USA
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Abstract

Ribonuclease P (RNase P) is a ribonucleoprotein that requires magnesium ions to catalyze the 5′ maturation of transfer RNA. To identify interactions essential for catalysis, the properties of RNase P containing single sulfur substitutions for nonbridging phosphodiester oxygens in helix P4 of Bacillus subtilis RNase P were analyzed using transient kinetic experiments. Sulfur substitution at the nonbridging oxygens of the phosphodiester bond of nucleotide U51 only modestly affects catalysis. However, phosphorothioate substitutions at A49 and G50 decrease the cleavage rate constant enormously (300–4,000-fold for P RNA and 500–15,000-fold for RNase P holoenzyme) in magnesium without affecting the affinity of pre-tRNAAsp, highlighting the importance of this region for catalysis. Furthermore, addition of manganese enhances pre-tRNA cleavage catalyzed by B. subtilis RNase P RNA containing an SP phosphorothioate modification at A49, as observed for Escherichia coli P RNA [Christian et al., RNA, 2000, 6:511–519], suggesting that an essential metal ion may be coordinated at this site. In contrast, no manganese rescue is observed for the A49 Sp phosphorothioate modification in RNase P holoenzyme. These differential manganese rescue effects, along with affinity cleavage, suggest that the protein component may interact with a metal ion bound near A49 in helix P4 of P RNA.

Keywords

magnesiumribozymeRNA ligationRNase Ptransient kinetics
Type
Research Article
Information
RNA , Volume 8 , Issue 7 , July 2002 , pp. 933 - 947
Copyright
2002 RNA Society

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