The single subunit DNA-dependent RNA polymerase (RNAP) from bacteriophage T7 catalyzes both promoterdependent transcription initiation and promoter-independent elongation. Using a promoter-free substrate, we have dissected the kinetic pathway of single nucleotide incorporation during elongation. We show that T7 RNAP undergoes a slow conformational change (0.01-0.03 s ؊1 ) to form an elongation competent complex with the promoter-free substrate (dissociation constant (K d ) of 96 nM). The complex binds to a correct NTP (K d of 80 M) and incorporates the nucleoside monophosphate (NMP) into RNA primer very efficiently (220 s ؊1 at 25°C). An overall free energy change (؊5.5 kcal/mol) and internal free energy change (؊3.7 kcal/mol) of single NMP incorporation was calculated from the measured equilibrium constants. In the presence of inorganic pyrophosphate (PP i ), the elongation complex catalyzes the reverse pyrophosphorolysis reaction at a maximum rate of 0.8 s ؊1 with PP i K d of 1.2 mM. Several experiments were designed to investigate the rate-limiting step in the pathway of single nucleotide addition. Acid-quench and pulse-chase kinetics indicated that an isomerization step before chemistry is rate-limiting. The very similar rate constants of sequential incorporation of two nucleotides indicated that the steps after chemistry are fast. Based on available data, we propose that the preinsertion to insertion isomerization of NTP observed in the crystallographic studies of T7 RNAP is a likely candidate for the rate-limiting step. The studies here provide a kinetic framework to investigate structure-function and fidelity of RNA synthesis and to further explore the role of the conformational change in nucleotide selection during RNA synthesis.The single subunit bacteriophage T7 RNA polymerase (RNAP) 2 catalyzes each of the stages of transcription including initiation, elongation, and termination without requiring any accessory proteins that are necessary in multisubunit RNA polymerases (1, 2). Structurally, T7 RNAP is similar to the pol I family of DNA-directed DNA polymerases and reverse transcriptases and shows high sequence homology to mitochondrial RNA polymerases (1). Being a single subunit enzyme, T7 RNAP serves as a model RNAP in understanding the mechanism and regulation of transcription initiation, elongation, and termination.The mechanism of transcription initiation by T7 RNAP is relatively well understood. The kinetic pathway of initiation has been dissected and the steps of promoter DNA and initiating NTP binding as well as promoter DNA melting have been quantified (3, 4). Recent studies have also provided a more detailed understanding of the transition process from initiation to elongation in T7 RNAP (5-11). The mechanism of transcription elongation catalyzed by T7 RNAP has not been characterized in detail, partly because elongation is an intermediate phase of transcription that begins only after 9 -12 nt of RNA is made through promoter-specific initiation (8). In the single subunit T7 RNAP, the transitio...

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been identified as major genetic determinants of Parkinson's disease (PD). The most prevalent mutation, G2019S, increases LRRK2's kinase activity, therefore understanding the sites and substrates that LRRK2 phosphorylates is critical to understanding its role in disease aetiology. Since the physiological substrates of this kinase are unknown, we set out to reveal potential targets of LRRK2 G2019S by identifying its favored phosphorylation motif. A non-biased screen of an oriented peptide library elucidated F/Y-x-T-x-R/K as the core dependent substrate sequence. Bioinformatic analysis of the consensus phosphorylation motif identified several novel candidate substrates that potentially function in neuronal pathophysiology. Peptides corresponding to the most PD relevant proteins were efficiently phosphorylated by LRRK2 in vitro. Interestingly, the phosphomotif was also identified within LRRK2 itself. Autophosphorylation was detected by mass spectrometry and biochemical means at the only F-x-T-x-R site (Thr 1410) within LRRK2. The relevance of this site was assessed by measuring effects of mutations on autophosphorylation, kinase activity, GTP binding, GTP hydrolysis, and LRRK2 multimerization. These studies indicate that modification of Thr1410 subtly regulates GTP hydrolysis by LRRK2, but with minimal effects on other parameters measured. Together the identification of LRRK2's phosphorylation consensus motif, and the functional consequences of its phosphorylation, provide insights into downstream LRRK2-signaling pathways.

Leucine‐rich repeat kinase 2 (LRRK2) is a large, complex, multidomain protein containing kinase and GTPase enzymatic activities and multiple protein–protein interaction domains. Mutations linked to autosomal dominant forms of Parkinson’s disease result in amino acid changes throughout the protein and alterations in both its enzymatic properties and interactions. The best characterized mutation to date, G2019S, leads to increased kinase activity, and mutations in the GTPase domain, such as R1441C and R1441G, have also been reported to influence kinase activity. Therefore, an examination of LRRK2’s properties as a kinase is important for understanding the mechanisms underlying the disorder and has the potential to lead to therapeutics. These findings also suggest that there may be complex interplay between the functional domains of LRRK2. Here, we review LRRK2’s biochemical functions based on structural and kinetic studies of the enzymatic domains, its potential substrates and the role of its interactions. Despite the field’s embryonic understanding of the true relevance of these substrates and interactions, initial studies are providing clues with respect to its pathophysiological functions. Together, these findings should increase our understanding of mechanisms underlying Parkinson’s disease and place LRRK2 as a unique molecular target for effective therapeutic development.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder in humans, and has a relatively poorly understood etiology. Linkage analysis studies in families with PD identified several mutations in the leucine-rich repeat kinase 2 gene (LRRK2) [1,2]. Moreover, epidemiological studies have shown that these mutations are the most prevalent cause of the autosomal form of the disorder, with high penetrance of certain mutations [3]. The similarity in age of onset and clinical symptoms between familial and idiopathic forms may also provide insights into the pathways involved in sporadic cases of PD.LRRK2 Mutations in leucine-rich repeat kinase 2 (LRRK2) comprise the leading cause of autosomal dominant Parkinson's disease, with age of onset and symptoms identical to those of idiopathic forms of the disorder. Several of these pathogenic mutations are thought to affect its kinase activity, so understanding the roles of LRRK2, and modulation of its kinase activity, may lead to novel therapeutic strategies for treating Parkinson's disease. In this study, highly purified, baculovirus-expressed proteins have been used, for the first time providing large amounts of protein that enable a thorough enzymatic characterization of the kinase activity of LRRK2. Although LRRK2 undergoes weak autophosphorylation, it exhibits high activity towards the peptidic substrate LRRKtide, suggesting that it is a catalytically efficient kinase. We have also utilized a time-resolved fluorescence resonance energy transfer (TR-FRET) assay format (LanthaScreen TM ) to characterize LRRK2 and test the effects of nonselective kinase inhibitors. Finally, we have used both radiometric and TR-FRET assays to assess the role of clinical mutations affecting LRRK2's kinase activity. Our results suggest that only the most prevalent clinical mutation, G2019S, results in a robust enhancement of kinase activity with LRRKtide as the substrate. This mutation also affects binding of ATP to LRRK2, with wild-type binding being tighter (K m,app of 57 lm) than with the G2019S mutant (K m,app of 134 lm). Overall, these studies delineate the catalytic efficiency of LRRK2 as a kinase and provide strategies by which a therapeutic agent for Parkinson's disease may be identified.Abbreviations COR, C-terminus of Roc; FRET, fluorescence resonance energy transfer; GST, glutathione S-transferase; LRRK2, leucine-rich repeat kinase 2; LRRK2-FL, full-length leucine-rich repeat kinase 2; PD, Parkinson's disease; Roc, Ras of complex; TR-FRET, time-resolved fluorescence resonance energy transfer; 4E-BP, eukaryotic initiation factor 4E-binding protein.

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