Substrate Specificities of DDX1: A Human DEAD-box protein.

DDX1 is a human protein which belongs to the DEAD-box protein family of enzymes and is involved in various stages of RNA metabolism from transcription to decay. Many members of the DEAD-box family of enzymes use the energy of ATP binding and hydrolysis to perform their cellular functions. On the other hand, a few members of the DEAD-box family of enzymes bind and/or hydrolyze other nucleotides in addition to ATP. Furthermore, the ATPase activity of DEAD-box family members is stimulated differently by nucleic acids of various structures. The identity of the nucleotides that the DDX1 hydrolyzes and the structure of the nucleic acids upon which it acts in the cell remain largely unknown. Identifying the DDX1 protein's in vitro substrates is important for deciphering the molecular roles of DDX1 in cells. Here we identify the nucleic acid sequences and structures supporting the nucleotide hydrolysis activity of DDX1 and its nucleotide specificity. Our data demonstrate that the DDX1 protein hydrolyzes only ATP and deoxy-ATP in the presence of RNA. The ATP hydrolysis activity of DDX1 is stimulated by multiple molecules: single-stranded RNA molecules as short as ten nucleotides, a blunt-ended double-stranded RNA molecule, a hybrid of a double-stranded DNA-RNA molecule, and a single-stranded DNA molecule. Under our experimental conditions, the single-stranded DNA molecule stimulates the ATPase activity of DDX1 at a significantly reduced extent when compared to the other investigated RNA constructs or the hybrid double-stranded DNA/RNA molecule.

Interactions of the C-Terminal Truncated DEAD-Box Protein DDX3X With RNA and Nucleotide Substrates.

DDX3X is a human DEAD-box RNA helicase implicated in many important cellular processes. In addition to the RecA-like catalytic core, DDX3X contains N- and C-terminal domains. The ancillary domains of DEAD-box RNA helicases have been shown to modulate their interactions with RNA and nucleotide substrates. Here, with the goal of understanding the role of N- and C-terminal domains of DDX3X on the DDX3X catalytic activity, we examined the interactions of RNA substrates and nucleotides with a DDX3X construct possessing the entire N-terminal domain and the catalytic core but lacking 80 residues from its C-terminal domain. Next, we compared our results with previously investigated DDX3X constructs. Our data show that the C-terminal truncated DDX3X does not bind to a blunt-ended double-helix RNA. This conclusion agrees with the data obtained on the wild-type LAF-1 protein, the DDX3X ortholog in Caenorhabditis elegans, and disagrees with the data obtained on the minimally active DDX3X construct, which misses 131 residues from its N-terminal domain and 80 residues from its C-terminal domain. The minimally active DDX3X construct was able to bind to the blunt-ended RNA construct. Combined, the previous studies and our results indicate that the N-terminal of DDX3X modulates the choice of DDX3X-RNA substrates. Furthermore, a previous study showed that the wild-type DDX3X construct hydrolyzes all four nucleotides and deoxynucleotides, both in the presence and absence of RNA. The C-terminal truncated DDX3X investigated here hydrolyzes only cytidine triphosphate (CTP) in the absence of RNA and CTP, adenosine triphosphate (ATP), and deoxyribose adenosine triphosphate (dATP) in the presence of RNA. Hence, the C-terminal truncated DDX3X has a more stringent nucleotide specificity than wild-type DDX3X.

Kinetics and Thermodynamics of DbpA Protein's C-Terminal Domain Interaction with RNA.

DbpA is an Escherichia coli DEAD-box RNA helicase implicated in RNA structural isomerization in the peptide bond formation site. In addition to the RecA-like catalytic core conserved in all of the members of DEAD-box family, DbpA contains a structured C-terminal domain, which is responsible for anchoring DbpA to hairpin 92 of 23S ribosomal RNA during the ribosome assembly process. Here, surface plasmon resonance was used to determine the equilibrium dissociation constant and the microscopic rate constants of the DbpA C-terminal domain association and dissociation to a fragment of 23S ribosomal RNA containing hairpin 92. Our results show that the DbpA protein's residence time on the RNA is 10 times longer than the time DbpA requires to hydrolyze one ATP. Thus, our data suggest that once bound to the intermediate ribosomal particles via its RNA-binding domain, DbpA could unwind a number of double-helix substrates before its dissociation from the ribosomal particles.

DbpA is a region-specific RNA helicase.

DbpA is a DEAD-box RNA helicase implicated in RNA structural rearrangements in the peptidyl transferase center. DbpA contains an RNA binding domain, responsible for tight binding of DbpA to hairpin 92 of 23S ribosomal RNA, and a RecA-like catalytic core responsible for double-helix unwinding. It is not known if DbpA unwinds only the RNA helices that are part of a specific RNA structure, or if DbpA unwinds any RNA helices within the catalytic core's grasp. In other words, it is not known if DbpA is a site-specific enzyme or region-specific enzyme. In this study, we used protein and RNA engineering to investigate if DbpA is a region-specific or a site-specific enzyme. Our data suggest that DbpA is a region-specific enzyme. This conclusion has an important implication for the physiological role of DbpA. It suggests that during ribosome assembly, DbpA could bind with its C-terminal RNA binding domain to hairpin 92, while its catalytic core may unwind any double-helices in its vicinity. The only requirement for a double-helix to serve as a DbpA substrate is for the double-helix to be positioned within the catalytic core's grasp.

The DbpA catalytic core unwinds double-helix substrates by directly loading on them.

DbpA is a DEAD-box RNA helicase implicated in the assembly of the large ribosomal subunit. Similar to all the members of the DEAD-box family, the DbpA protein has two N-terminal RecA-like domains, which perform the RNA unwinding. However, unlike other members of this family, the DbpA protein also possesses a structured C-terminal RNA-binding domain that mediates specific tethering of DbpA to hairpin 92 of the Escherichia coli 23S ribosomal RNA. Previous studies using model RNA molecules containing hairpin 92 show that the RNA molecules support the DbpA protein's double-helix unwinding activity, provided that the double helix has a 3' single-stranded region. The 3' single-stranded region was suggested to be the start site of the DbpA protein's catalytic unwinding activity. The data presented here demonstrate that the single-stranded region 3' of the double-helix substrate is not required for the DbpA protein's unwinding activity and the DbpA protein unwinds the double-helix substrates by directly loading on them.

Determination of high-molecular weight polycyclic aromatic hydrocarbons in high performance liquid chromatography fractions of coal tar standard reference material 1597a via solid-phase nanoextraction and laser-excited time-resolved Shpol'skii spectroscopy.

This article presents an alternative approach for the analysis of high molecular weight - polycyclic aromatic hydrocarbons (HMW-PAHs) with molecular mass 302 Da in complex environmental samples. This is not a trivial task due to the large number of molecular mass 302 Da isomers with very similar chromatographic elution times and similar, possibly even virtually identical, mass fragmentation patterns. The method presented here is based on 4.2K laser-excited time-resolved Shpol'skii spectroscopy, a high resolution spectroscopic technique with the appropriate selectivity for the unambiguous determination of PAHs with the same molecular mass. The potential of this approach is demonstrated here with the analysis of a coal tar standard reference material (SRM) 1597a. Liquid chromatography fractions were submitted to the spectroscopic analysis of five targeted isomers, namely dibenzo[a,l]pyrene, dibenzo[a,e]pyrene, dibenzo[a,i]pyrene, naphtho[2,3-a]pyrene and dibenzo[a,h]pyrene. Prior to analyte determination, the liquid chromatographic fractions were pre-concentrated with gold nanoparticles. Complete analysis was possible with microliters of chromatographic fractions and organic solvents. The limits of detection varied from 0.05 (dibenzo[a,l]pyrene) to 0.24 µg L(-1) (dibenzo[a,e]pyrene). The excellent analytical figures of merit associated to its non-destructive nature, which provides ample opportunity for further analysis with other instrumental methods, makes this approach an attractive alternative for the determination of PAH isomers in complex environmental samples.

Parallel Factor Analysis of 4.2 K Excitation-Emission Matrices for the Direct Determination of Dibenzopyrene Isomers in Coal-Tar Samples with a Cryogenic Fiber-Optic Probe Coupled to a Commercial Spectrofluorimeter.

Several studies have shown high concentrations of polycyclic aromatic hydrocarbons (PAHs) in living spaces and soil adjacent to parking lots sealed with coal-tar-based products. Recent attention has been paid to the presence of seven PAHs in coal-tar samples, namely, benz[a]anthracene, benzo[k]-fluoranthene, benzo[b]fluoranthene, benzo[a]pyrene, chrysene, dibenz[a,h]anthracene, and indeno[1,2,3-cd]pyrene, and their association to significant increases in estimated excess lifetime cancer risk for nearby residents. Herein, we present an analytical approach to screen the presence of five highly toxic, high-molecular weight PAHs (HMW-PAHs) in coal-tar samples. These include dibenzo[a,l]pyrene, dibenzo[a,i]pyrene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, and naphtho[2,3-a]pyrene. Their direct analysis, without chromatographic separation, in a reference coal-tar sample is made possible with the combination of excitation-emission matrices (EEMs) and parallel factor analysis (PARAFAC). EEMs are recorded at 4.2 K with the aid of a cryogenic fiber-optic probe and a commercial spectrofluorimeter. The simplicity of the experimental procedure and the excellent analytical figures of merit demonstrate the screening potential of this environmentally friendly approach for the routine analysis of numerous coal-tar samples.

Combining cryogenic fiber optic probes with commercial spectrofluorimeters for the synchronous fluorescence Shpol'skii spectroscopy of high molecular weight polycyclic aromatic hydrocarbons.

Cryogenic fiber optic probes are combined for the first time with a commercial spectrofluorometer for Shpol'skii spectroscopy measurements at liquid nitrogen (77 K) and liquid helium (4.2 K) temperatures. Accurate and reproducible acquisition of fluorescence spectra and signal intensities is demonstrated with three well known Shpol'skii systems, namely, anthracene/heptane, pyrene/hexane, and benzo[a]pyrene/octane. The ability to adjust the excitation and emission bandpass of the spectrofluorimeter to reach both site-resolution and analytically valuable signal-to-noise ratios was illustrated with benzo[a]pyrene in n-octane. The analytical potential of 4.2 K synchronous fluorescence Shpol'skii spectroscopy for the analysis of high molecular weight-polycyclic aromatic hydrocarbons was then explored for the first time. The judicious optimization of wavelength offsets permitted the successful determination of dibenzo[a,l]pyrene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, and naphtho[2,3-a]pyrene without previous chromatographic separation from a soil extract with complex matrix composition. The simplicity of the experimental procedure, the competitive analytical figures of merit, and the selectivity of analysis turn 4.2 K synchronous fluorescence Shpol'skii spectroscopy into a valuable alternative for screening isomers of high molecular weight polycyclic aromatic hydrocarbons in environmental samples.

Single fiber identification with nondestructive excitation-emission spectral cluster analysis.

Identification methods for single textile fibers are in demand for forensic applications, and nondestructive methods with minimal pretreatment have the greatest potential for utility. Excitation-emission luminescence data provide a three-dimensional matrix for comparison of single-fiber dyes, and these data are enhanced by principal component analysis and comparison of fibers using a statistical figure of merit. No dye extraction methods are required to sample the spectra from a single fiber. This approach has been applied to the analysis of single fibers to compare closely matched dye pairs, acid blue (AB) 25 and 41 and direct blue (DB) 1 and 53. In all cases, the accuracy of fiber identification was high and no false positive identifications were made.

Four-way modeling of 4.2 K time-resolved excitation emission fluorescence data for the quantitation of polycyclic aromatic hydrocarbons in soil samples.

A screening method for the soil analysis of 15 Environmental Protection Agency-polycyclic aromatic hydrocarbons (EPA-PAHs) is reported. The new method is based on the collection of 4.2 K fluorescence time-resolved excitation-emission cubes (TREECs) via laser-excited time-resolved Shpol'skii spectroscopy. 4.2 K fluorescence TREECs result from the superposition of fluorescence time-resolved excitation emission matrices recorded at different time windows from the laser excitation pulse. Potential interference from unknown sample concomitants is handled by processing the four-way 4.2 K fluorescence TREEC data arrays with either parallel factor analysis (PARAFAC) or unfolded partial least-squares/residual-trilinearization(U-PLS/RTL). The sensitivity of the two approaches makes possible to determine PAHs at the ng g(-1) to pg g(-1) concentration level with no need for sample pre-concentration. Its selectivity eliminates sample clean-up steps and chromatographic separation. These features reduce PAH loss, analysis time and cost. The method is environmentally friendly as the complete screening of the 15 EPA-PAHs takes only 250 µL of organic solvent per sample.

Portable mercury sensor for tap water using surface plasmon resonance of immobilized gold nanorods.

The surface plasmon resonance of surface immobilized gold nanorods (Au NRs) was used to quantify mercury in tap water. Glass substrates were chemically functionalized with (3-mercaptopropyl)trimethoxysilane, which chemically bound the nanorods to produce a portable and sensitive mercury sensor. The analytical capabilities of the sensor were measured using micromolar mercury concentrations. Since the analytical response was dependent upon number of nanorods present, the limit of detection was 2.28×10(-19) M mercury per nanorod. The possibility to using glass substrates with immobilized Au NRs is a significant step towards the analysis of mercury in tap water flows at this low concentration level.