The pre-tRNA nucleotide base and 2'-hydroxyl at N(-1) contribute to fidelity in tRNA processing by RNase P

Fidelity in tRNA processing by the RNase P RNA from Escherichia coli depends, in part, on interactions with the nucleobase and 2' hydroxyl group of N(-1), the nucleotide immediately upstream of the site of RNA strand cleavage. Here, we report a series of biochemical and structure-function studies designed to address how these interactions contribute to cleavage site selection. We find that simultaneous disruption of cleavage site nucleobase and 2' hydroxyl interactions results in parallel reactions leading to correct cleavage and mis-cleavage one nucleotide upstream (5') of the correct site. Changes in Mg(2+) concentration and pH can influence the fraction of product that is incorrectly processed, with pH effects attributable to differences in the rate-limiting steps for the correct and mis-cleavage reaction pathways. Additionally, we provide evidence that interactions with the 2' hydroxyl group adjacent to the reactive phosphate group also contribute to catalysis at the mis-cleavage site. Finally, disruption of the adjacent 2'-hydroxyl contact has a greater effect on catalysis when pairing between the ribozyme and N(-1) is also disrupted, and the effects of simultaneously disrupting these contacts on binding are also non-additive. One implication of these results is that mis-cleavage will result from any combination of active site modifications that decrease the rate of correct cleavage beyond a certain threshold. Indeed, we find that inhibition of correct cleavage and corresponding mis-cleavage also results from disruption of any combination of active site contacts including metal ion interactions and conserved pairing interactions with the 3' RCCA sequence. Such redundancy in interactions needed for maintaining fidelity may reflect the necessity for multiple substrate recognition in vivo. These studies provide a framework for interpreting effects of substrate modifications on RNase P cleavage fidelity and provide evidence for interactions with the nucleobase and 2' hydroxyl group adjacent to the reactive phosphate group in the transition state.     

The role of protein surface charge in catalytic activity and chloroplast membrane association of the pea NADPH: protochlorophyllide oxidoreductase (POR) as revealed by alanine scanning mutagenesis

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (pchlide) to chlorophyllide (chlide) in the biosynthesis of chlorophyll. POR is a peripheral membrane protein that accumulates to high levels in the prolamellar bodies of vascular plant etioplasts and is present at low levels in the thylakoid membranes of developing and mature plastids. Clustered charged-to-alanine scanning mutagenesis of the pea (Pisum sativum L.) POR was carried out and the resulting mutant enzymes analyzed for their ability to catalyze pchlide photoconversion in vivo and to associate properly with thylakoid membrane preparations in vitro. Of 37 mutant enzymes examined, 5 retained wild-type levels of activity, 14 were catalytically inactive, and the remaining 18 exhibited altered levels of function. Several of the mutant enzymes showed temperature- dependent enzymatic activity, being inactive at 32°C, but partially active at 24°C. Mutations in predicted - helical regions of the protein showed the least effect on enzyme activity, whereas mutations in predicted -sheet regions of the protein showed a consistent adverse affect on enzyme function. In the absence of added NADPH, neither wild-type POR nor any of the mutant PORs resisted proteolysis by thermolysin following assembly onto the thylakoid membranes. In contrast, when NADPH was present in the assay mixture, 13 of the 37 mutant PORs examined were found to be resistant to thermolysin upon treatment, suggesting that the mutations did not affect their ability to be properly attached to the thylakoid membrane. In general, the replacement of charged amino acids by alanine in the most N- and C-terminal regions of the mature protein did not significantly affect POR assembly, whereas mutations within the central core of the protein (between residues 86 and 342) were incapable of proper attachment to the thylakoid. Failure to properly associate with the thylakoid membrane in a protease resistant manner was only weakly correlated to loss of catalytic function. These studies are a first step towards defining structural determinants crucial to POR function and intraorganellar localization.     

The C609T inborn polymorphism in NAD(P)H:quinone oxidoreductase 1 is associated with susceptibility to multiple sclerosis and affects the risk of development of the primary progressive form of the disease

Oxidative stress plays a pivotal role in the pathogenesis of multiple sclerosis (MS). Inactivating polymorphisms of genes encoding detoxification enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), could influence susceptibility to MS. To test this hypothesis we performed a case-control study in which we compared the distribution of NQO1 genotypes between 231 MS patients and 380 controls, using both PCR-RFLP and real-time PCR assays. Correlations with MS clinical subtype classification and gender were also evaluated. A significantly higher frequency of the homozygous (T/T) and heterozygous (C/T) NQO1 C⁶⁰⁹T variant genotypes was observed among MS patients compared to controls (P = 0.01), with MS patients showing a 1.5-fold increased risk of carrying at least one variant T allele (P = 0.009). Interestingly, patients belonging to the primary progressive subgroup exhibited a significantly higher incidence of the heterozygous C/T variant genotype, compared to the other forms of MS (P = 0.019). There was no correlation of the NQO1 polymorphism with gender. These results provide the first evidence for a pathogenetic role for the NQO1 C⁶⁰⁹T polymorphism in MS susceptibility and suggest a possible role for the NQO1 genetic background in the development of primary progressive MS.