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.

RNA Post-transcriptional Modifications of an Early-Stage Large-Subunit Ribosomal Intermediate.

Protein production by ribosomes is fundamental to life, and proper assembly of the ribosome is required for protein production. The RNA, which is post-transcriptionally modified, provides the platform for ribosome assembly. Thus, a complete understanding of ribosome assembly requires the determination of the RNA post-transcriptional modifications in all of the ribosome assembly intermediates and on each pathway. There are 26 RNA post-transcriptional modifications in 23S RNA of the mature Escherichia coli (E. coli) large ribosomal subunit. The levels of these modifications have been investigated extensively only for a small number of large subunit intermediates and under a limited number of cellular and environmental conditions. In this study, we determined the level of incorporations of 2-methyl adenosine, 3-methyl pseudouridine, 5-hydroxycytosine, and seven pseudouridines in an early-stage E. coli large-subunit assembly intermediate with a sedimentation coefficient of 27S. The 27S intermediate is one of three large subunit intermediates accumulated in E. coli cells lacking the DEAD-box RNA helicase DbpA and expressing the helicase inactive R331A DbpA construct. The majority of the investigated modifications are incorporated into the 27S large subunit intermediate to similar levels to those in the mature 50S large subunit, indicating that these early modifications or the enzymes that incorporate them play important roles in the initial events of large subunit ribosome assembly.

RNA Post-Transcriptional Modifications in Two Large Subunit Intermediates Populated in E. coli Cells Expressing Helicase Inactive R331A DbpA.

23S ribosomal RNA (rRNA) of Escherichia coli 50S large ribosome subunit contains 26 post-transcriptionally modified nucleosides. Here, we determine the extent of modifications in the 35S and 45S large subunit intermediates, accumulating in cells expressing the helicase inactive DbpA protein, R331A, and the native 50S large subunit. The modifications we characterized are 3-methylpseudouridine, 2-methyladenine, 5-hydroxycytidine, and nine pseudouridines. These modifications were detected using 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT) treatment followed by alkaline treatment. In addition, KMnO4 treatment of 23S rRNA was employed to detect 5-hydroxycytidine modification. CMCT and KMnO4 treatments produce chemical changes in modified nucleotides that cause reverse transcriptase misincorporations and deletions, which were detected employing next-generation sequencing. Our results show that the 2-methyladenine modification and seven uridines to pseudouridine isomerizations are present in both the 35S and 45S to similar extents as in the 50S. Hence, the enzymes that perform these modifications, namely, RluA, RluB, RluC, RluE, RluF, and RlmN, have already acted in the intermediates. Two uridines to pseudouridine isomerizations, the 3-methylpseudouridine and 5-hydroxycytidine modifications, are significantly less present in the 35S and 45S, as compared to the 50S. Therefore, the enzymes that incorporate these modifications, RluD, RlmH, and RlhA, are in the process of modifying the 35S and 45S or will incorporate these modifications during the later stages of ribosome assembly. Our study employs a novel high throughput and single nucleotide resolution technique for the detection of 2-methyladenine and two novel high throughput and single nucleotide resolution techniques for the detection of 5-hydroxycytidine.

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.

Retardation of Folding Rates of Substrate Proteins in the Nanocage of GroEL.

The Escherichia coli ATP-consuming chaperonin machinery, a complex between GroEL and GroES, has evolved to facilitate folding of substrate proteins (SPs) that cannot do so spontaneously. A series of kinetic experiments show that the SPs are encapsulated in the GroEL/ES nanocage for a short duration. If confinement of the SPs is the mechanism by which GroEL/ES facilitates folding, it follows that the assisted folding rate, relative to the bulk value, should always be enhanced. Here, we show that this is not the case for the folding of rhodanese in the presence of the full machinery of GroEL/ES and ATP. The assisted folding rate of rhodanese decreases. On the basis of our finding and those reported in other studies, we suggest that the ATP-consuming chaperonin machinery has evolved to optimize the product of the folding rate and the yield of the folded SPs on the biological time scale. Neither the rate nor the yield is separately maximized.

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.

Selective nano-sensing approach for the determination of inorganic phosphate in human urine samples.

A novel sensing approach is presented for the analysis of phosphate ions in human urine samples. The new sensor is based on the fluorescence energy transfer between a luminescent probe ([Tb-EDTA]-1) and gold nanoparticles capped with a cetyltrimethylammonium bromide (Au NPs-CTAB). The strong affinity between CTAB receptors and phosphate ions results in a highly selective assay with minimum sample preparation steps. Possible chemical interference is monitored on real-time basis via luminescence lifetime analysis. The simplicity of analysis and the competitive limit of detection in the micro-molar concentration range provide a well-suited approach for routine monitoring of phosphate ions in numerous urine samples.

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.

Time course of large ribosomal subunit assembly in E. coli cells overexpressing a helicase inactive DbpA protein.

DbpA is a DEAD-box RNA helicase implicated in Escherichia coli large ribosomal subunit assembly. Previous studies have shown that when the ATPase and helicase inactive DbpA construct, R331A, is expressed in E. coli cells, a large ribosomal subunit intermediate accumulates. The large subunit intermediate migrates as a 45S particle in a sucrose gradient. Here, using a number of structural and fluorescent assays, we investigate the ribosome profiles of cells lacking wild-type DbpA and overexpressing the R331A DbpA construct. Our data show that in addition to the 45S particle previously described, 27S and 35S particles are also present in the ribosome profiles of cells overexpressing R331A DbpA. The 27S, 35S, and 45S independently convert to the 50S subunit, suggesting that ribosome assembly in the presence of R331A and the absence of wild-type DbpA occurs via multiple pathways.

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.

Folding path of P5abc RNA involves direct coupling of secondary and tertiary structures.

Folding mechanisms in which secondary structures are stabilized through the formation of tertiary interactions are well documented in protein folding but challenge the folding hierarchy normally assumed for RNA. However, it is increasingly clear that RNA could fold by a similar mechanism. P5abc, a small independently folding tertiary domain of the Tetrahymena thermophila group I ribozyme, is known to fold by a secondary structure rearrangement involving helix P5c. However, the extent of this rearrangement and the precise stage of folding that triggers it are unknown. We use experiments and simulations to show that the P5c helix switches to the native secondary structure late in the folding pathway and is directly coupled to the formation of tertiary interactions in the A-rich bulge. P5c mutations show that the switch in P5c is not rate-determining and suggest that non-native interactions in P5c aid folding rather than impede it. Our study illustrates that despite significant differences in the building blocks of proteins and RNA, there may be common ways in which they self-assemble.

Nuclear magnetic resonance spectroscopy with the stringent substrate rhodanese bound to the single-ring variant SR1 of the E. coli chaperonin GroEL.

Nuclear magnetic resonance (NMR) observation of the uniformly (2) H,(15) N-labeled stringent 33-kDa substrate protein rhodanese in a productive complex with the uniformly (14) N-labeled 400 kDa single-ring version of the E. coli chaperonin GroEL, SR1, was achieved with the use of transverse relaxation-optimized spectroscopy, cross-correlated relaxation-induced polarization transfer, and cross-correlated relaxation-enhanced polarization transfer. To characterize the NMR-observable parts of the bound rhodanese, coherence buildup rates by different magnetization transfer mechanisms were measured, and effects of covalent crosslinking of the rhodanese to the apical binding surface of SR1 were investigated. The results indicate that the NMR-observable parts of the SR1-bound rhodanese are involved in intracomplex rate processes, which are not related to binding and release of the substrate protein from the SR1 binding surface. Rather, they correspond to mobility of the stably bound substrate, which thus appears to include flexibly disordered polypeptide segments devoid of long-lived secondary structures or tertiary folds, as was previously observed also with the smaller substrate human dihydrofolate reductase.

Analysis of RNA folding by native polyacrylamide gel electrophoresis.

Polyacrylamide gel electrophoresis under native conditions (native PAGE) is a well-established and versatile method for probing nucleic acid conformation and nucleic acid-protein interactions. Native PAGE has been used to measure RNA folding equilibria and kinetics under a wide variety of conditions. Advantages of this method are its adaptability, absolute determination of reaction endpoints, and direct analysis of conformational hetereogeneity within a sample. Native PAGE is also useful for resolving ligand-induced structural changes.

Cold-adaptation in sea-water-borne signal proteins: sequence and NMR structure of the pheromone En-6 from the Antarctic ciliate Euplotes nobilii.

Ciliates of Euplotes species constitutively secrete pleiotropic protein pheromones, which are capable to function as prototypic autocrine growth factors as well as paracrine inducers of mating processes. This paper reports the amino acid sequence and the NMR structure of the pheromone En-6 isolated from the antarctic species Euplotes nobilii. The 63-residue En-6 polypeptide chain forms three alpha-helices in positions 18-25, 36-40 and 46-56, which are arranged in an up-down-up three-helix bundle forming the edges of a distorted trigonal pyramid. The base of the pyramid is covered by the N-terminal heptadecapeptide segment, which includes a 3(10)-turn of residues 3-6. This topology is covalently anchored by four long-range disulfide bonds. Comparison with the smaller pheromones of E. raikovi, a closely related species living in temperate waters, shows that the two-pheromone families have the same three-helix bundle architecture. It then appears that cold-adaptation of the En proteins is primarily related to increased lengths of the chain-terminal peptide segments and the surface-exposed loops connecting the regular secondary structures, and to the presence of solvent-exposed clusters of negatively charged side-chains.

Charge density of divalent metal cations determines RNA stability.

RNA molecules are exquisitely sensitive to the properties of counterions. The folding equilibrium of the Tetrahymena ribozyme is measured by nondenaturing gel electrophoresis in the presence of divalent group IIA metal cations. The stability of the folded ribozyme increases with the charge density (zeta) of the cation. Similar scaling is found when the free energy of the RNA folded in small and large metal cations is measured by urea denaturation. Brownian dynamics simulations of a polyelectrolyte show that the experimental observations can be explained by nonspecific ion-RNA interactions in the absence of site-specific metal chelation. The experimental and simulation results establish that RNA stability is largely determined by a combination of counterion charge and the packing efficiency of condensed cations that depends on the excluded volume of the cations.

Counterion charge density determines the position and plasticity of RNA folding transition states.

The self-assembly of RNA structure depends on the interactions of counterions with the RNA and with each other. Comparison of various polyamines showed that the tertiary structure of the Tetrahymena ribozyme is more stable when the counterions are small and highly charged. By monitoring the folding kinetics of the ribozyme as a function of polyamine concentration, we now find that the charge density of the counterions determines the positions of the folding transition states. The transition state ensemble (TSE) between U and N moves away from the native state as the counterion valence and charge density increase, as predicted by the Hammond postulate. The TSE is broader and less structured when the RNA is refolded in polyamines rather than Mg2+. That the charge density of the counterions determines the plasticity of the TSE demonstrates the importance of interactions among condensed counterions for the self-assembly of RNA structures. We propose that the major barrier to RNA folding is dominated by entropy changes when counterion charge density is low and enthalpy differences when it is high.

Folding of the Tetrahymena ribozyme by polyamines: importance of counterion valence and size.

Polyamines are abundant metabolites that directly influence gene expression. Although the role of polyamines in DNA condensation is well known, their role in RNA folding is less understood. Non-denaturing gel electrophoresis was used to monitor the equilibrium folding transitions of the Tetrahymena ribozyme in the presence of polyamines. All of the polyamines tested induce near-native structures that readily convert to the native conformation in Mg(2+). The stability of the folded structure increases with the charge of the polyamine and decreases with the size of the polyamine. When the counterion excluded volume becomes large, the transition to the native state does not go to completion even under favorable folding conditions. Brownian dynamics simulations of a model polyelectrolyte suggest that the kinetics of counterion-mediated collapse and the dimensions of the collapsed RNA chains depend on the structure of the counterion. The results are consistent with delocalized condensation of polyamines around the RNA. However, the effective charge of the counterions is lowered by their excluded volume. The stability of the folded RNA is enhanced when the spacing between amino groups matches the distance between adjacent phosphate groups. These results show how changes in intracellular polyamine concentrations could alter RNA folding pathways.