Time-resolved hydroxyl radical footprinting of RNA with X-rays.

RNA tertiary structure and protein interactions can be detected by protection from hydroxyl radical cleavage. Generation of hydroxyl radicals with a synchrotron X-ray beam provides analysis on a short timescale (50 msec to 100 sec), which enables the structures of folding intermediates or other transient conformational states to be determined. This unit provides detailed instructions on the use of the synchrotron beamline for hydroxyl radical footprinting.

Multiple folding pathways for the P4-P6 RNA domain.

We recently described site-specific pyrene labeling of RNA to monitor Mg(2+)-dependent equilibrium formation of tertiary structure. Here we extend these studies to follow the folding kinetics of the 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA, using stopped-flow fluorescence with approximately 1 ms time resolution. Pyrene-labeled P4-P6 was prepared using a new phosphoramidite that allows high-yield automated synthesis of oligoribonucleotides with pyrene incorporated at a specific 2'-amino-2'-deoxyuridine residue. P4-P6 forms its higher-order tertiary structure rapidly, with k(obs) = 15-31 s(-1) (t(1/2) approximately 20-50 ms) at 35 degrees C and [Mg(2+)] approximately 10 mM in Tris-borate (TB) buffer. The folding rate increases strongly with temperature from 4 to 45 degrees C, demonstrating a large activation enthalpy DeltaH(double dagger) approximately 26 kcal/mol; the activation entropy DeltaS(double dagger) is large and positive. In low ionic strength 10 mM sodium cacodylate buffer at 35 degrees C, a slow (t(1/2) approximately 1 s) folding component is also observed. The folding kinetics are both ionic strength- and temperature-dependent; the slow phase vanishes upon increasing [Na(+)] in the cacodylate buffer, and the kinetics switch completely from fast at 30 degrees C to slow at 40 degrees C. Using synchrotron hydroxyl radical footprinting, we confirm that fluorescence monitors the same kinetic events as hydroxyl radical cleavage, and we show that the previously reported slow P4-P6 folding kinetics apply only to low ionic strength conditions. One model to explain the fast and slow folding kinetics postulates that some tertiary interactions are present even without Mg(2+) in the initial state. The fast kinetic phase reflects folding that is facilitated by these interactions, whereas the slow kinetics are observed when these interactions are disrupted at lower ionic strength and higher temperature.

Folding mechanism of the Tetrahymena ribozyme P4-P6 domain.

Synchrotron X-ray-dependent hydroxyl radical footprinting was used to probe the folding kinetics of the P4-P6 domain of the Tetrahymena group I ribozyme, which forms a stable, closely packed tertiary structure. The 160-nt domain folds independently at a similar rate (approximately 2 s(-1)) as it does in the ribozyme, when folding is measured in 10 mM sodium cacodylate and 10 mM MgCl(2). Surprisingly, tertiary interactions around a three-helix junction (P5abc) within the P4-P6 domain fold at least 25 times more rapidly (k >/= 50 s(-1)) in isolation, than when part of the wild-type P4-P6 RNA. This difference implies that long-range interactions in the P4-P6 domain can interfere with folding of P5abc. P4-P6 was observed to fold much faster at higher ionic strength than in 10 mM sodium cacodylate. Analytical centrifugation was used to measure the sedimentation and diffusion coefficients of the unfolded RNA. The hydrodynamic radius of the RNA decreased from 58 to 46 A over the range of 0-100 mM NaCl. We propose that at low ionic strength, the addition of Mg(2+) causes the domain to collapse to a compact intermediate where P5abc is trapped in a non-native structure. At high ionic strength, the RNA rapidly collapses to the native structure. Faster folding most likely results from a different average initial conformation of the RNA in higher salt conditions.

Fast folding of a ribozyme by stabilizing core interactions: evidence for multiple folding pathways in RNA.

Folding of the Tetrahymena ribozyme under physiological conditions in vitro is limited by slow conversion of long-lived intermediates to the active structure. These intermediates arise because the most stable domain of the ribozyme folds 10-50 times more rapidly than the core region containing helix P3. Native gel electrophoresis and time-resolved X-ray-dependent hydroxyl radical cleavage revealed that mutations that weaken peripheral interactions between domains accelerated folding fivefold, while a point mutation that stabilizes P3 enabled 80 % of the mutant RNA to reach the native conformation within 30 seconds at 22 degrees C. The P3 mutation increased the folding rate of the catalytic core as much as 50-fold, so that both domains of the ribozyme were formed at approximately the same rate. The results show that the ribozyme folds rapidly without significantly populating metastable intermediates when native interactions in the ribozyme core are stabilized relative to peripheral structural elements.

N2-hydroxyguanosine 5'-monophosphate is a time-dependent inhibitor of Escherichia coli guanosine monophosphate synthetase.

In contrast to several other glutamine amidotransferases including asparagine synthetase, cytidine 5'-triphosphate (CTP) synthetase, carbamoyl phosphate synthetase, and phosphoribosyl pyrophosphate (PRPP) amidotransferase, guanosine monophosphate synthetase (GMPS) will not utilize hydroxylamine as an alternative nitrogen source. Instead, the enzyme is inhibited by an unknown mechanism. One untested hypothesis was that hydroxylamine serves as a substrate and intercepts a xanthosine 5'-monophosphate- (XMP-) adenylate intermediate in the enzyme active site. The nucleotide product of this substitution reaction would be N2-hydroxyguanosine 5'-monophosphate (N2-OH-GMP, 2). Here we describe the chemoenzymatic preparation of 2, via the nucleotide 2-fluoroinosine 5'-monophosphate (F-IMP, 5), and characterization of both these compounds as inhibitors of Escherichia coli GMPS. F-IMP was conceived as an electronic mimic of a reactive intermediate in the GMPS reaction but was found to bind weakly to the enzyme (IC50 > 2 mM). In contrast, N2-OH-GMP shows time-dependent inhibition and is competitive with respect to XMP (Ki = 92 nM), representing the first example of a compound that displays these kinetic properties with GMPS. The mechanism of inhibition is proposed to occur via formation of a ternary E.ATP.2 complex, followed by a rate-determining isomerization to a higher affinity complex that has a t1/2 =7.5 min. The contrast in inhibitory activity for 2-substituted purines with GMPS formulates a basis for future inhibitor design. In addition, these results complement recent structural studies of GMPS and implicate the formation of the XMP-adenylate intermediate inducing a probable conformational change that stimulates the hydrolysis of glutamine.

The crystal structure of GMP synthetase reveals a novel catalytic triad and is a structural paradigm for two enzyme families.

The crystal structure of GMP synthetase serves as a prototype for two families of metabolic enzymes. The Class I glutamine amidotransferase domain of GMP synthetase is found in related enzymes of the purine, pyrimidine, tryptophan, arginine, histidine and folic acid biosynthetic pathways. This domain includes a conserved Cys-His-Glu triad and is representative of a new family of enzymes that use a catalytic triad for enzymatic hydrolysis. The structure and conserved sequence fingerprint of the nucleotide-binding site in a second domain of GMP synthetase are common to a family of ATP pyrophosphatases, including NAD synthetase, asparagine synthetase and argininosuccinate synthetase.