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At least 1,063 records · Page 59Linked to original sources
PubMed2003

Trans-acting hepatitis delta virus ribozyme: catalytic core and global structure are dependent on the 5' substrate sequence.

The hepatitis delta virus (HDV), an infectious human pathogen affecting millions of people worldwide, leads to intensified disease symptoms, including progression to liver cirrhosis upon coinfection with its helper virus, HBV. Both the circular RNA genome of HDV and its complementary antigenome contain a common cis-cleaving catalytic RNA motif, the HDV ribozyme, which plays a crucial role in viral replication. Previously, the crystal structure of the product form of the cis-acting genomic HDV ribozyme has been determined, and the precursor form has been suggested to be structurally similar. In contrast, solution studies by fluorescence resonance energy transfer (FRET) on a trans-cleaving form of the ribozyme have shown significant global conformational changes upon catalysis, while 2-aminopurine (AP) fluorescence assays have detected concomitant local conformational changes in the catalytic core. Here, we augment these studies by using terbium(III) to probe the structure of the trans-acting HDV ribozyme at nucleotide resolution. We observe significant structural differences between the precursor and product forms, especially in the P1.1 helix and the trefoil turn in the single-stranded region connecting P4 and P2 (termed J4/2) of the catalytic core. We show, using terbium(III) footprinting and sensitized luminescence spectroscopy as well as steady-state, time-resolved, and gel-mobility FRET assays on a systematic set of substrates, that the substrate sequence immediately 5' to the cleavage site significantly modulates these local as well as resultant global structural differences. Our results suggest a structural basis for the previously observed impact of the 5' substrate sequence on catalytic activity.

Fluorescence Resonance Energy Transfer↗
PubMed2003

Simultaneous DNA binding and bending by EcoRV endonuclease observed by real-time fluorescence.

The complete catalytic cycle of EcoRV endonuclease has been observed by combining fluorescence anisotropy with fluorescence resonance energy transfer (FRET) measurements. Binding, bending, and cleavage of substrate oligonucleotides were monitored in real time by rhodamine-x anisotropy and by FRET between rhodamine and fluorescein dyes attached to opposite ends of a 14-mer DNA duplex. For the cognate GATATC site binding and bending are found to be nearly simultaneous, with association and bending rate constants of (1.45-1.6) x 10(8) M(-1) s(-1). On the basis of the measurement of k(off) by a substrate-trapping approach, the equilibrium dissociation constant of the enzyme-DNA complex in the presence of inhibitory calcium ions was calculated as 3.7 x 10(-12) M from the kinetic constants. Further, the entire DNA cleavage reaction can be observed in the presence of catalytic Mg(2+) ions. These measurements reveal that the binding and bending steps occur at equivalent rates in the presence of either Mg(2+) or Ca(2+), while a slow decrease in fluorescence intensity following bending corresponds to k(cat), which is limited by the cleavage and product dissociation steps. Measurement of k(on) and k(off) in the absence of divalent metals shows that the DNA binding affinity is decreased by 5000-fold to 1.4 x 10(-8) M, and no bending could be detected in this case. Together with crystallographic studies, these data suggest a model for the induced-fit conformational change in which the role of divalent metal ions is to stabilize the sharply bent DNA in an orientation suitable for accessing the catalytic transition state.

Calcium↗
PubMed2004

Binding of the b-subunit in the ATP synthase from Escherichia coli.

The rotary mechanism of ATP synthase requires a strong binding within stator subunits. In this work we studied the binding affinity of the b-subunit to F(1)-ATPase of Escherichia coli. The dimerization of the truncated b-subunit without amino acids 1-33, b(34-156)T62C, was investigated by analytical ultracentrifugation, resulting in a dissociation constant of 1.8 microM. The binding of b-subunit monomeric and dimeric forms to the isolated F(1) part was investigated by fluorescence correlation spectroscopy and steady-state fluorescence. The mutants b(34-156)T62C and EF(1)-gammaT106C were labeled with several fluorophores. Fluorescence correlation spectroscopy was used to measure translational diffusion times of the labeled b-subunit, labeled F(1), and a mixture of the labeled b-subunit with unlabeled F(1). Data analysis revealed a dissociation constant of 0.2 nM of the F(1)b(2) complex, yielding a Gibbs free energy of binding of DeltaG(o)= -55 kJ mol(-1). In steady-state fluorescence resonance energy transfer (FRET) measurements it was found that binding of the b-subunit to EF(1)-gammaT106C-Alexa488 resulted in a fluorescence decrease of one-third of the initial FRET donor fluorescence intensity. The decrease of fluorescence was measured as a function of b-concentration, and data were described by a model including equilibria for dimerization of the b-subunit and binding of b and b(2) to F(1). For a quantitative description of fluorescence decrease we used two different models: the binding of the first and the second b-subunit causes the same fluorescence decrease (model 1) or only the binding of the first b-subunit causes fluorescence decrease (model 2). Data evaluation revealed a dissociation constant for the F(1)b(2) complex of 0.6 nM (model 1) or 14 nM (model 2), giving DeltaG(o)= -52 kJ mol(-1) and DeltaG(o)= -45 kJ mol(-1), respectively. The maximal DeltaG observed for ATP synthesis in cells is approximately DeltaG= 55 kJ mol(-1). Therefore, the binding energy of the b-subunit seems to be too low for models in which the free energy for ATP synthesis is accumulated in the elastic strain between rotor and stator subunits and then transduced to the catalytic site in one single step. Models in which energy transduction takes place in at least two steps are favored.

Bacterial Proton-Translocating ATPases↗
PubMed2005

Reversible photobleaching of enhanced green fluorescent proteins.

Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25-58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based FRET determinations, and FRAP assays.

Bacterial Proteins↗
PubMed2004

On the solution structure of the T4 sliding clamp (gp45).

Examination by time-resolved fluorescence spectroscopy of the trimeric bacteriophage T4 clamp protein labeled across its three subunit interfaces with a fluorescence resonance energy transfer (FRET) pair indicates that the clamp exists in just one state in solution, with one open and two closed interfaces. This is in contrast to what is observed in the X-ray crystal structure. The population distribution of the trFRET distance is bimodal, giving 67% as 17 A and 33% as 42 A. This leads to the conclusion that gp45 exists in an asymmetric open state in solution. The further increase in the separation of the FRET pair in the presence of the clamp loader and ATP may be ascribed to either further opening of the open interface or the opening of a closed interface. The ramifications for replisome remodeling by this pathway are discussed.

Bacteriophage T4↗
PubMed2004

Dynamics inherent in helix 27 from Escherichia coli 16S ribosomal RNA.

The original interpretation of a series of genetic studies suggested that the highly conserved Escherichia coli 16S ribosomal RNA helix 27 (H27) adopts two alternative secondary structure motifs, the 885 and 888 conformations, during each cycle of amino acid incorporation. Recent crystallographic and genetic evidence has called this hypothesis into question. To ask whether a slippery sequence such as that of H27 may harbor inherent conformational dynamics, we have designed a series of model RNAs based on E. coli H27 for in vitro physicochemical studies. One-dimensional (1)H NMR spectroscopy demonstrates that both the 885 and 888 conformations are occupied to approximately the same extent (f(888) = 0.427 +/- 0.04) in the native H27 sequence at low pH (6.4) and low ionic strength (50 mM NaCl). UV irradiation assays conducted under conditions analogous to those used for assays of ribosomal function (pH 7.5 and 20 mM MgCl(2)) suggest that nucleotides 892 and 905, which are too far apart in the known 885 crystal structures, can approach each other closely enough to form an efficient cross-link. The use of a fluorescence resonance energy transfer (FRET)-labeled RNA together with a partially complementary DNA oligonucleotide that induces a shift to the 888 conformation shows that H27 interchanges between the 885 and 888 conformations on the millisecond time scale, with an equilibrium constant of 0.33 +/-0.12. FRET assays also show that tetracycline interferes with the induced shift to the 888 conformation, a finding that is consistent with crystallographic localization of tetracycline bound to the 885 conformation of H27 in the 30S ribosomal subunit. Taken together, our data demonstrate the innate tendency of an isolated H27 to exist in a dynamic equilibrium between the 885 and 888 conformations. This begs the question of how these inherent structural dynamics are suppressed within the context of the ribosome.

Escherichia coli Proteins↗
PubMed2004

Magnesium dependence of the amplified conformational switch in the trans-acting hepatitis delta virus ribozyme.

The ability of divalent metal ions to participate in both structure formation and catalytic chemistry of RNA enzymes (ribozymes) has made it difficult to separate their cause and effect in ribozyme function. For example, the recently solved crystal structures of precursor and product forms of the cis-cleaving genomic hepatitis delta virus (HDV) ribozyme show a divalent metal ion bound in the active site that is released upon catalysis due to an RNA conformational change. This conformational switch is associated with a repositioning of the catalytically involved base C75 in the active-site cleft, thus controlling catalysis. These findings confirm previous data from fluorescence resonance energy transfer (FRET) on a trans-acting form of the HDV ribozyme that found a global conformational change to accompany catalysis. Here, we further test the conformational switch model by measuring the Mg(2+) dependence of the global conformational change of the trans-acting HDV ribozyme, using circular dichroism and time-resolved FRET as complementary probes of secondary and tertiary structure formation, respectively. We observe significant differences in both structure and Mg(2+) affinity of the precursor and product forms, in the presence and absence of 300 mM Na(+) background. The precursor shortens while the product extends with increasing Mg(2+) concentration, essentially amplifying the structural differences observed in the crystal structures. In addition, the precursor has an approximately 2-fold and approximately 13-fold lower Mg(2+) affinity than the product in secondary and tertiary structure formation, respectively. We also have compared the C75 wild-type with the catalytically inactive C75U mutant and find significant differences in global structure and Mg(2+) affinity for both their precursor and product forms. Significantly, the Mg(2+) affinity of the C75 wild-type is 1.7-2.1-fold lower than that of the C75U mutant, in accord with the notion that C75 is essential for a catalytic conformational change that leads to a decrease in the local divalent metal ion affinity and release of a catalytic metal. Thus, a consistent picture emerges in which divalent metal ions and RNA functional groups are intimately intertwined in affecting structural dynamics and catalysis in the HDV ribozyme.

Catalysis↗
PubMed2004

Direct detection of phospholamban and sarcoplasmic reticulum Ca-ATPase interaction in membranes using fluorescence resonance energy transfer.

We used fluorescence resonance energy transfer (FRET) to detect and quantitate the interaction of the sarcoplasmic reticulum Ca-ATPase (SERCA) with phospholamban (PLB) in membranes. PLB inhibits SERCA only at submicromolar Ca. It has been proposed that relief of inhibition at micromolar Ca is due to dissociation of the inhibitory complex. To test this hypothesis, we co-reconstituted donor-labeled SERCA and acceptor-labeled I40A-PLB (superinhibitory, monomeric PLB mutant) in membranes of defined lipid and protein composition, with full retention of Ca-dependent ATPase activity and inhibitory regulation by PLB. FRET from SERCA to PLB was measured as a function of membrane concentrations of PLB and SERCA, and functional activity was measured on the same samples. The data revealed clearly that the stoichiometry of binding is one PLB per SERCA, and that binding is a strict function of the ratio of total PLB to SERCA in the membrane. We conclude that the dissociation constant of PLB binding to SERCA is far less than physiological PLB membrane concentrations. Binding at low Ca (pCa 6.5), where I40A-PLB inhibits SERCA, was virtually identical to that at high Ca (pCa 5.0), where no inhibition was observed. However, the limiting energy transfer at saturating PLB was less at high Ca, indicating a greater donor-acceptor distance. We conclude that (a) the affinity of PLB for SERCA is so great that PLB is essentially a SERCA subunit under physiological conditions and (b) relief of inhibition at micromolar Ca is due to a structural rearrangement within the SERCA-PLB complex, rather than dissociation.

Calcium↗
PubMed2004

Structural dynamics of actin during active interaction with myosin: different effects of weakly and strongly bound myosin heads.

We have used optical spectroscopy (transient phosphorescence anisotropy, TPA, and fluorescence resonance energy transfer, FRET) to detect the effects of weakly bound myosin S1 on actin during the actomyosin ATPase cycle. The changes in actin were reported by (a) a phosphorescent probe (ErIA) attached to Cys 374 and (b) a FRET donor-acceptor pair, IAEDANS attached to Cys 374 and a nucleotide analogue (TNPADP) in the nucleotide-binding cleft. Strong interactions were detected in the absence of ATP, and weak interactions were detected in the presence of ATP or its slowly hydrolyzed analogue ATP-gamma-S, under conditions where a significant fraction of weakly bound acto-S1 complex was present and the rate of nucleotide hydrolysis was low enough to enable steady-state measurements. The results show that actin in the weakly bound complex with S1 assumes a new structural state in which (a) the actin filament has microsecond rotational dynamics intermediate between that of free actin and the strongly bound complex and (b) S1-induced changes are not propagated along the actin filament, in contrast to the highly cooperative changes due to the strongly bound complex. We propose that the transition on the acto-myosin interface from weak to strong binding is accompanied by transitions in the structural dynamics of actin parallel to transitions in the dynamics of interacting myosin heads.

Actins↗
PubMed2004

Sry-directed sex reversal in transgenic mice is robust with respect to enhanced DNA bending: comparison of human and murine HMG boxes.

The testis-determining factor SRY contains an HMG box DNA-bending domain. Human and murine factors (hSRY and mSRY, respectively) exhibit marked sequence divergence and are reported to differ markedly in DNA bending properties. Surprisingly, the combined application of time-resolved fluorescence resonance energy transfer (tr-FRET) and permutation gel electrophoresis demonstrates that the hSRY-DNA complex is more sharply bent than the murine complex and not less bent as previously reported. tr-FRET-based analyses of the distribution of end-to-end distances in the bent DNA-protein complexes further suggest that a broader range of DNA bend angles is populated in the murine ensemble than in the human ensemble. The two domains and their respective DNA complexes nevertheless exhibit similar thermodynamic stabilities. (1)H NMR spectra indicate analogous intercalation of distinct "cantilever" side chains (isoleucine or methionine) with subtle differences in induced DNA structure. Interchange of cantilevers does not affect DNA bending. That transgenic expression of either human or murine Sry in XX mice can confer a male somatic phenotype suggests that SRY-directed transcriptional regulation is robust to enhanced DNA bending and to changes in the precision of DNA bending. We propose that male-specific gene regulation requires DNA bending above a critical threshold set by architectural requirements of enhanceosome assembly.

Amino Acid Sequence↗
PubMed2005

Förster resonance energy transfer measurements are consistent with a helical bundle model for lipid-free apolipoprotein A-I.

Apolipoprotein (apo) A-I mutants were constructed for FRET studies to distinguish between two possible lipid-free conformers, a globular helix bundle and an elongated helical hairpin. Mutants containing a single Trp at position 50 were prepared by replacing Trps at positions 8, 72, and 108 with Phe (W@50). Two mutants were constructed from W@50 by incorporating Cys at Arg83 (W@50R83C) or Arg173 (W@50R173C) for attachment of the fluorescent probe AEDANS. Secondary structure of the mutants is very similar to wild type (wt) apo A-I, and fluorescence emission indicates that W50 is protected from solvent. Thermal stabilities of the AEDANS-labeled mutants are also similar to wt. These results indicate that no discernible changes occur in structure or stability as a result of mutations or labeling. The FRET data from W@50 to AEDANS are well-represented by a single distance distribution function with a distance of approximately 22 A for W@50R83C and approximately 19 A for W@50R173C. These distances are consistent with theoretical values calculated from a helical bundle model but not from a helical hairpin. A probability distance distribution function yields significantly small half-width values of 5.6 and 3.7 A, respectively, suggesting low conformational dynamics in both mutants. Differential scanning calorimetry (DSC) was performed on wt and a C-terminal deletion mutant, Delta(187-243), to obtain information on domain architecture. Contrary to expectations, both proteins unfold cooperatively. The results are consistent with the presence of a single folded domain within residues 1-186. These results support the presence of a discrete globular bundle conformation for lipid-free apo A-I.

Apolipoprotein A-I↗
PubMed2006

The DNA-binding domain of the ultraspiracle drives deformation of the response element whereas the DNA-binding domain of the ecdysone receptor is responsible for a slight additional change of the preformed structure.

Ecdysteroids control molting and metamorphosis in insects via a heterodimeric complex of two nuclear receptors, the ecdysone receptor (EcR) and ultraspiracle protein (Usp). We used fluorescence resonance energy transfer (FRET) to study the topology of the natural pseudopalindromic element from the hsp27 gene (hsp27pal) in complex with the DNA-binding domains of Usp and EcR (UspDBD and EcRDBD, respectively). Steady-state data revealed shortening of the end-to-end distance of the hsp27pal-derived probe. For the 70.8 +/- 0.6 A distance obtained for the UspDBD-complexed DNA a bend of about 23.1 +/- 2.9 degrees was measured. Nearly the same value (23.0 +/- 3.4 degrees) was obtained for the DNA complexed with the UspDBD/EcRDBD heterodimer. The respective bend angles estimated using fluorescence decay measurements were 19.0 +/- 2.1 degrees and 20.9 +/- 3.6 degrees . Thus, the FRET data suggest for the first time that the UspDBD defines the architecture of the UspDBD/EcRDBD heterocomplex due to the significant deformation of the hsp27pal. This suggestion has been further reinforced using gel retardation experiments, which, in conjunction with high-resolution DNase I footprinting, indicate that the main contribution to the observed bend is given by the UspDBD itself, while binding of the EcRDBD molecule brings on a slight additional change of the preformed structure.

Animals↗
PubMed2005

Conformation coupled enzyme catalysis: single-molecule and transient kinetics investigation of dihydrofolate reductase.

Ensemble kinetics and single-molecule fluorescence microscopy were used to study conformational transitions associated with enzyme catalysis by dihydrofolate reductase (DHFR). The active site loop of DHFR was labeled with a fluorescence quencher, QSY35, at amino acid position 17, and the fluorescent probe, Alexa555, at amino acid 37, by introducing cysteines at these sites with site-specific mutagenesis. The distance between the probes was such that approximately 50% fluorescence resonance energy transfer (FRET) occurred. The double-labeled enzyme retained essentially full catalytic activity, and stopped-flow studies of both the forward and reverse reactions revealed that the distance between probes increased prior to hydride transfer. A fluctuation in fluorescence intensity of single molecules of DHFR was observed in an equilibrium mixture of substrates but not in their absence. Ensemble rate constants were derived from the distributions of lifetimes observed and attributed to a reversible conformational change. Studies were carried out with both NADPH and NADPD as substrates, with no measurable isotope effect. Similar studies with a G121V mutant DHFR resulted in smaller rate constants. This mutant DHFR has reduced catalytic activity, so that the collective data for the conformational change suggest that the conformational change being observed is associated with catalysis and probably represents a conformational change prior to hydride transfer. If the change in fluorescence is attributed to a change in FRET, the distance change associated with the conformational change is approximately 1-2 A. These results are correlated with other measurements related to conformation coupled catalysis.

Algorithms↗
PubMed2005

Tetraplex DNA transitions within the human c-myc promoter detected by multivariate curve resolution of fluorescence resonance energy transfer.

The nuclease hypersensitive element (NHE) III(I) of the c-myc promoter regulates the expression of oncogene c-myc and hence is an important anti-cancer target. Paranemic secondary structure formation within the promoter has been implicated in mechanistic regulation models. Here, it is shown that two monomeric tetraplexes form within the c-myc promoter, which coexist in solution. The development and application of a new experimental approach for detection of conformation transitions in nucleic acids [which exploits the sensitivity of fluorescence resonance energy transfer (FRET) for theoretical spectral resolution by multivariate curve resolution-alternating least-squares (MCR-ALS) method] has been used for this study. The pK(a) for tetraplex transitions are centered around 5.9 +/- 0.2 (between two intercalation topologies) and 6.8 +/- 0.1 (tetraplex to random coil). The presence of two tetraplexes has been further confirmed by S1 nuclease digestion. Finally, it is established that MCR-ALS analysis of FRET at different temperatures, pH, and salt concentrations allows resolution of pure species. Results are discussed in the light of recent observations implicating paranemic DNA motifs within the c-myc NHE in regulation of the oncogene. This method has several advantages over other methods vis-à-vis, high sensitivity and linear detection over a wide concentration range and, particularly, potential applications in intracellular probing.

Base Sequence↗
PubMed2006

Catalytic core structure of the trans-acting HDV ribozyme is subtly influenced by sequence variation outside the core.

The human pathogenic hepatitis delta virus (HDV) employs a unique self-cleaving catalytic RNA motif, the HDV ribozyme, during double-rolling circle replication. Fluorescence spectroscopy, circular dichroism, terbium(III) footprinting, and X-ray crystallography of precursor and product forms have revealed that a conformational change accompanies catalysis. In addition, fluorescence resonance energy transfer (FRET) has previously been used on a trans-acting HDV ribozyme to demonstrate surprisingly significant catalytic and global conformational effects of substrate analogues with varying 5' sequences, which reside as dangling overhangs outside the catalytic core. Here, we use the fluorescent guanine analogue 2-aminopurine (AP) in nucleotide position 76, immediately downstream of the catalytically involved C75, to monitor the relative structural effects of these substrate analogues on the ribozyme's trefoil turn of the catalytic core. Steady-state and time-resolved AP fluorescence spectroscopies show that the binding of each substrate analogue induces a unique local conformation with a specific AP76 stacking equilibrium. Binding of the 3' product results in a relative increase in AP fluorescence, suggesting that AP76 becomes more unstacked upon catalysis. These local conformational changes are kinetically concomitant with global conformational changes monitored by FRET. Finally, the rate constant of the local conformational change upon 3' product binding is fast and independent of 3' product concentration yet Mg2+ dependent. Our results demonstrate that the trefoil turn of the HDV ribozyme catalytic core is in a state of dynamic equilibrium not captured by static crystal structures and is highly sensitive to the identity of the 5' sequence and Mg2+ ions.

2-Aminopurine↗
PubMed2006

The pleckstrin homology domain of phospholipase Cbeta transmits enzymatic activation through modulation of the membrane-domain orientation.

Phospholipase Cbeta (PLCbeta) enzymes are activated by Galpha q and Gbetagamma subunits and catalyze the hydrolysis of the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Activation of PLCbeta2 by Gbetagamma subunits has been shown to be conferred through its N-terminal pleckstrin homology (PH) domain, although the underlying mechanism is unclear. Also unclear are observations that the extent of Gbetagamma activation differs on different membrane surfaces. In this study, we have identified a unique region of the PH domain of the PLCbeta2 domain (residues 71-88) which, when added to the enzyme as a peptide, causes enzyme activation similar to that with Gbetagamma subunits. This PH domain segment interacts strongly with membranes composed of lipid mixtures but not those containing lipids with electrically neutral zwitterionic headgroups. Also, addition of this segment perturbs interaction of the catalytic domain, but not the PH domain, with membrane surfaces. We monitored the orientation of the PH and catalytic domains of PLC by intermolecular fluorescence resonance energy transfer (FRET) using the Gbetagamma activatable mutant, PLCbeta2/delta1(C193S). We find an increase in the level of FRET with binding to membranes with mixed lipids but not to those containing only lipids with electrically neutral headgroups. These results suggest that enzymatic activation can be conferred through optimal association of the PHbeta71-88 region to specific membrane surfaces. These studies allow us to understand the basis of variations of Gbetagamma activation on different membrane surfaces.

Amino Acid Sequence↗
PubMed2006

An alternative clamp loading pathway via the T4 clamp loader gp44/62-DNA complex.

In bacteriophage T4, a clamp loading pathway that utilizes the T4 clamp loader (gp44/62) and ATP hydrolysis initially to form a complex with the clamp (gp45) has been demonstrated, followed by interaction with DNA and closing of the clamp. However, the recent observation that gp45 exists as an opened form in solution raises the possibility of other pathways for clamp loading. In this study, an alternative clamp loading sequence is evaluated in which gp44/62 first recognizes the DNA substrate and then sequesters the clamp from solution and loads it onto DNA. This pathway differs in terms of the initial formation of a gp44/62-DNA complex that is capable of loading gp45. In this work, we demonstrate ATP-dependent DNA binding by gp44/62. Among various DNA structures that were tested, gp44/62 binds specifically to primer-template DNA but not to single-stranded DNA or blunt-end duplex DNA. By tracing the dynamic clamp closing with pre-steady-state FRET measurements, we show that the clamp loader-DNA complex is functional in clamp loading. Furthermore, pre-steady-state ATP hydrolysis experiments suggest that 1 equiv of ATP is hydrolyzed when gp44/62 binds to DNA, and additional ATP hydrolysis is associated with the completion of the clamp loading process. We also investigated the detailed kinetics of binding of MANT-nucleotide to gp44/62 through stopped-flow FRET and demonstrated a conformational change as the result of ATP, but not ADP binding. The collective kinetic data allowed us to propose and evaluate a sequence of steps describing this alternative pathway for clamp loading and holoenzyme formation.

Adenosine Diphosphate↗
PubMed2006

In situ analysis of single-stranded and duplex siRNA integrity in living cells.

To attain the full therapeutic promise of short interfering RNA (siRNA), it is believed that improvements such as increased biostability are critical. Regrettably, thus far, insufficient in situ data are on hand regarding the intracellular stability of siRNAs. We report on the use of an advanced fluorescence-based method to probe the nucleolytic decay of double labeled siRNAs, which are subject to fluorescence resonance energy transfer (FRET). In vitro measurements with RNAse A and cellular extracts demonstrate that the ratio of acceptor (5'-Cy5) to donor (3'-rhodamine green) fluorescence can be used to study the degradation of the labeled siRNA substrates upon donor excitation. Intracellular FRET analysis showed substantial degradation of single-stranded siRNA, whereas duplex siRNA stayed intact during the measured time period. These data underline the high intrinsic nuclease resistance of unmodified duplex siRNA and prove that cellular persistence is much more critical for the single-stranded structure. For the first time, the stability of siRNA is investigated in real-time inside living cells. The fluorescence-based method presented here is a straightforward technique to gain direct information on siRNA integrity inside living cells and provides a bright outlook to learn more about the intracellular fate of siRNA therapeutics.

Animals↗