Protein Component of Bacillus subtilis RNase P Specifically Enhances the Affinity for Precursor-tRNA^Asp

Abstract

Ribonuclease P (RNase P) is an endonuclease that cleaves precursor tRNA to form the 5'-end of mature tRNA and is composed of a catalytic RNA subunit and a small protein subunit. The function of the protein component of Bacillus subtilis RNase P in catalysis of B. subtilis precursor tRNAAsp cleavage has been elucidated using steady-state kinetics, transient kinetics, and ligand affinity measurements to compare the functional properties of RNase P holoenzyme to RNase P RNA in 10 mM MgCl2, 100 mM NH4Cl. The protein component modestly affects several steps including </=10-fold increases in the rate constant for tRNA dissociation, the affinity of tRNA, and the rate constant for phosphodiester bond cleavage. However, the protein principally affects substrate binding, increasing the affinity of RNase P for pre-tRNAAsp by a factor of 10(4) as determined from both the ratio of the pre-tRNAAsp dissociation and association rate constants measured in 10 mM MgCl2 and a binding isotherm measured in 10 mM CaCl2 using gel filtration to separate enzyme-bound and free pre-tRNAAsp. Therefore, the main role of the protein component in RNase P is to facilitate recognition of pre-tRNA by enhancing the interaction between the enzyme and the 5'-precursor segment of the substrate, rather than stabilizing the tertiary structure of the folded RNA as has been observed for protein-facilitated group I intron self-splicing. Furthermore, the protein component maximizes the efficiency of RNase P under physiological conditions and minimizes product inhibition.

The RNR motif of B. subtilis RNase P protein interacts with both PRNA and pre-tRNA to stabilize an active conformer

“…Although the RNA component alone is capable of catalyzing pre-tRNA cleavage in high salt in vitro, the protein is essential for catalytic activity in vivo (Hsieh et al 2004;Evans et al 2006;Gossringer et al 2006;Smith et al 2007). In vitro studies indicate that the P protein subunit contributes to numerous facets of RNase P catalysis including substrate and metal affinity and cleavage efficiency (Reich et al 1988;Crary et al 1998;Kurz et al 1998;Sun et al 2006). For example, P protein enhances k cat /K m for Bacillus subtilis RNase P-catalyzed cleavage by 2000-fold and substrate affinity by 10 4 -fold (Kurz et al 1998), while the homologous protein subunit of Escherichia coli RNase P increases the cleavage rate constants for some noncanonical pre-tRNA substrates by >900-fold (Sun et al 2006).…”

“…In vitro studies indicate that the P protein subunit contributes to numerous facets of RNase P catalysis including substrate and metal affinity and cleavage efficiency (Reich et al 1988;Crary et al 1998;Kurz et al 1998;Sun et al 2006). For example, P protein enhances k cat /K m for Bacillus subtilis RNase P-catalyzed cleavage by 2000-fold and substrate affinity by 10 4 -fold (Kurz et al 1998), while the homologous protein subunit of Escherichia coli RNase P increases the cleavage rate constants for some noncanonical pre-tRNA substrates by >900-fold (Sun et al 2006). Both E. coli and B. subtilis proteins also decrease the concentration of divalent metal ions required for catalysis by RNase P (Gardiner et al 1985;Kurz et al 1998;Sun and Harris 2007), perhaps by stabilizing an essential metaldependent conformational change (Hsieh et al 2010).…”

The Bacillus subtilis RNase P holoenzyme contains two RNase P RNA and two RNase P protein subunits

“…The holoenzyme of a bacterial RNase P is a ribonucleoprotein complex containing an RNA component (P RNA) of ;330-420 nt and a protein component (P protein) of ;120 amino acids (Frank & Pace, 1998;Altman & Kirsebom, 1999)+ The functional role of the protein component in the RNase P holoenzyme has been investigated extensively (Guerrier-Takada et al+, 1983, 1984Gardiner et al+, 1985;McClain et al+, 1987;Reich et al+, 1988;Peck-Miller & Altman, 1991;Svard & Kirsebom, 1992;Tallsjo & Kirsebom, 1993;Liu & Altman, 1994;Crary et al+, 1998;Kurz et al+, 1998;Loria et al+, 1998;Niranjanakumari et al+, 1998aNiranjanakumari et al+, , 1998bLoria & Pan, 1999)+ The Bacillus subtilis RNase P holoenzyme is remarkably efficient in the catalysis of precursor tRNA substrates with a k cat /K m near the diffusion limit (Kurz et al+, 1998;Reich et al+, 1988)+ In the absence of the protein component, k cat /K m decreases by 10 4 -fold under physiological conditions (Kurz et al+, 1998)+ A principal effect of the P protein function has been postulated to be the enhancement of substrate binding under physiological conditions (Crary et al+, 1998;Kurz et al+, 1998;Niranjanakumari et al+, 1998b)+ The physical state of the RNase P holoenzyme has received less attention+ Previous work by the Altman group conclusively showed that the Escherichia coli holoenzyme has an equal molar amount of RNA and protein (Talbot & Altman, 1994a)+ The affinity of the P protein binding to P RNA has been estimated to be ;0+5 nM, assuming a simple two-component binding isotherm (Talbot & Altman, 1994b)+ Functional studies by the Fierke group showed that the RNA-protein stoichiometry of the B. subtilis holoenzyme is also 1:1 (Niranjanakumari et al+, 1998a)+ Most studies in this area have focused on the details of P RNA-P protein interactions using chemical modification (Vioque et al+, 1988;Talbot & Altman, 1994b;Loria et al+, 1998;Biswas et al+, 20...…”

Chloroplast ribonuclease P does not utilize the ribozyme-type pre-tRNA cleavage mechanism

“…Transfer RNA (tRNA) molecules are synthesized as precursors possessing both 59-leader and 39-trailer extensions+ Ribonuclease P (RNase P) is a ubiquitous enzyme required for maturation of precursor tRNA (pre-tRNA) by endonucleolytic cleavage between the 59-leader and the mature tRNA domain+ In all varieties of RNase P studied previously, the enzyme is composed of an RNA subunit and a protein subunit+ In bacteria, it is the RNA component alone that is responsible for catalysis (Guerrier-Takada et al+, 1983), whereas in eukaryal and archaeal varieties of RNase P, both subunits are required for activity (for review, see Pace & Brown, 1995)+ The bacterial RNase P protein subunit contains an average of 120 amino acids and the RNA subunit is 350-400 nt long (Brown & Pace, 1992)+ For example, the Escherichia coli RNase P protein has a chain length of 119 nt (13+8 kDa) whereas the RNA subunit is 377 nt (146 kDa)+ The role of the protein subunit is to facilitate binding of the anionic substrate and enzyme in vivo; and to alter substrate versus product-binding kinetics to permit enzyme discrimination between substrate and product (Reich et al+, 1988;Tallsjö & Kirsebom, 1993;Kurz et al+, 1998)+ Bacterial RNase P is a metalloenzyme, requiring divalent metal cations for activity+ The divalent ion functions directly in catalysis, as the RNA subunit can bind substrate in the presence of monovalent ions alone, and displays a burst of activity once a divalent species is added (Smith et al+, 1992)+ Three Mg 2ϩ ions participate in catalysis (Hardt et al+, 1993;Smith & Pace, 1993;Beebe et al+, 1996;Chen et al+, 1997)+ Magnesium(II) is thought to promote catalysis in two ways; by coordinating phosphate oxygen molecules and polarizing the phosphorus center, and by activating the nucleophilic water molecule that attacks the scissile bond (Smith & Pace, 1993;Fersht, 1985)+…”