Monomeric solution structure of the helicase-binding domain of Escherichia coli DnaG primase

DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB. The C-terminal helix hairpin present in the DnaG C-terminal domain, however, is either less stable or absent in DnaB, as evidenced by high mobility of the C-terminal 35 residues in a construct comprising residues 1-171. The present structure identifies the previous crystal structure of the E. coli DnaG C-terminal domain as a domain-swapped dimer. It is also significantly different from the NMR structure reported for the corresponding domain of DnaG from the thermophile Bacillus stearothermophilus. NMR experiments showed that the DnaG C-terminal domain does not bind to residues 1-171 of the E. coli DnaB helicase with significant affinity.     

Phosphorylation of p68 RNA helicase regulates RNA binding by the C-terminal domain of the protein

We previously reported ATPase, RNA unwinding, and RNA-binding activities of recombinant p68 RNA helicase that was expressed in Escherichia coli. Huang et al. The recombinant protein bound both single-stranded (ss) and double-stranded (ds) RNAs. To further characterize the substrate RNA binding by p68 RNA helicase, we expressed and purified the recombinant N-terminal and C-terminal domains of the protein. RNA-binding property and protein phosphorylation of the recombinant domains of p68 were analyzed. Our data demonstrated that the C-terminal domain of p68 RNA helicase bound ssRNA. More interestingly, the C-terminal domain was a target of protein kinase C (PKC). Phosphorylation of the C-terminal domain of p68 abolished its RNA binding. Based on our observations, we propose that the C-terminal domain is an RNA substrate binding site for p68. The protein phosphorylation by PKC regulates the RNA binding of p68 RNA helicase, which consequently controls the enzymatic activities of the protein.     

In the Bacillus stearothermophilus DnaB-DnaG complex, the activities of the two proteins are modulated by distinct but overlapping networks of residues

We demonstrate the primase activity of Bacillus stearothermophilus DnaG and show that it initiates at 3'-ATC-5' and 3'-ATT-5' sites synthesizing primers that are 22 or 23 nucleotides long. In the presence of the helicase DnaB the size distribution of primers is different, and a range of additional smaller primers are also synthesized. Nine residues from the N- and C-terminal domains of DnaB, as well as its linker region, have been reported previously to affect this interaction. In Bacillus stearothermophilus only three residues from the linker region (I119 and I125) and the N-terminal domain (Y88) of DnaB have been shown previously to have direct structural importance, and I119 and I125 mediate DnaG-induced effects on DnaB activity. The functions of the other residues (L138, T191, E192, R195, and M196) are still a mystery. Here we show that the E15A, Y88A, and E15A Y88A mutants bind DnaG but are not able to modulate primer size, whereas the R195A M196A mutant inhibited the primase activity. Therefore, four of these residues, E15 and Y88 (N-terminal domain) and R195 and M196 (C-terminal domain), mediate DnaB-induced effects on DnaG activity. Overall, the data suggest that the effects of DnaB on DnaG activity and vice versa are mediated by distinct but overlapping networks of residues.     

Characterization and mutational analysis of the RecQ core of the bloom syndrome protein

Bloom syndrome protein forms an oligomeric ring structure and belongs to a group of DNA helicases showing extensive homology to the Escherichia coli DNA helicase RecQ, a suppressor of illegitimate recombination. After over-production in E.coli, we have purified the RecQ core of BLM consisting of the DEAH, RecQ-Ct and HRDC domains (amino acid residues 642-1290). The BLM(642-1290) fragment could function as a DNA-stimulated ATPase and as a DNA helicase, displaying the same substrate specificity as the full-size protein. Gel-filtration experiments revealed that BLM(642-1290) exists as a monomer both in solution and in its single-stranded DNA-bound form, even in the presence of Mg(2+) and ATPgammaS. Rates of ATP hydrolysis and DNA unwinding by BLM(642-1290) showed a hyperbolic dependence on ATP concentration, excluding a co-operative interaction between ATP-binding sites. Using a lambda Spi(-) assay, we have found that the BLM(642-1290) fragment is able to partially substitute for the RecQ helicase in suppressing illegitimate recombination in E.coli. A deletion of 182 C-terminal amino acid residues of BLM(642-1290), including the HRDC domain, resulted in helicase and single-stranded DNA-binding defects, whereas kinetic parameters for ATP hydrolysis of this mutant were close to the BLM(642-1290) values. This confirms the prediction that the HRDC domain serves as an auxiliary DNA-binding domain. Mutations at several conserved residues within the RecQ-Ct domain of BLM reduced ATPase and helicase activities severely as well as single-stranded DNA-binding of the enzyme. Together, these data define a minimal helicase domain of BLM and demonstrate its ability to act as a suppressor of illegitimate recombination.     

1H, 15N and 13C NMR assignments of the 434 repressor fragments 1-63 and 44-63 unfolded in 7 M urea.

An E. coli overexpression system for the N-terminal domain of the 434 repressor with residues 1-63 (434 repressor(1-63)) was constructed and used to produce this polypeptide with uniform 15N-labeling, and with 13C-labeling of the methyl groups of valine and leucine. Using these protein preparations almost complete sequence-specific resonance assignments were obtained for the urea-unfolded form of the 434 repressor(1-63). In addition, the isotope-labeled tryptic peptide, 44-63, was produced by enzymatic cleavage of the recombinant 434 repressor(1-63), and its NMR spectrum was assigned. Corresponding residues in 434 repressor(1-63) and 434 repressor(44-63) in 7 M urea were found to have nearly identical chemical shifts, and in both species similar deviations from 1H random coil shifts were found as previously in 434 repressor(1-69). These indicate the presence of residual non-random structure in the polypeptide segment 50-60. The present NMR assignments, which include stereospecific assignments for the diastereotopic methyl groups of Val and Leu, are the basis for detailed studies of this residual structure in the urea-unfolded form of the 434 repressor.     

Determination of the nuclear magnetic resonance solution structure of the DNA-binding domain (residues 1 to 69) of the 434 repressor and comparison with the X-ray crystal structure.

The DNA-binding domain of the phage 434 repressor consisting of N-terminal residues 1 to 69 (434 repressor(1-69)), was expressed in Escherichia coli with natural isotope abundance, uniform 15N-labeling and biosynthetically directed fractional 13C-labeling in extent of about 10%. With these protein preparations the three-dimensional structure was determined in solution. The techniques used were nuclear magnetic resonance (n.m.r.) spectroscopy for the collection of conformational constraints, calculation of the protein structure from the n.m.r. data with the program DIANA and structure refinements by restrained energy minimization with a modified version of the program AMBER. A group of 20 conformers characterizes a well-defined structure for residues 1 to 63, with an average of 0-6 A for the root-mean-square deviations (RMSD) calculated for the backbone atoms of the individual conformers relative to the mean co-ordinates. The spatial structure of C-terminal residues 64 to 69 is not defined by the n.m.r. data. The molecular architecture of the 434 repressor(1-69) in solution includes five alpha-helices extending from residues 2 to 13, 17 to 24, 28 to 35, 45 to 52 and 56 to 60, which enclose a well-defined hydrophobic core. The n.m.r. structure is closely similar to the reported crystal structure of the 434 repressor(1-69), with an RMSD value of 1.1 A for the backbone atoms of residues 1 to 63. Small differences between the two structures in regions of the first helix and the loop between helices 3 and 4 were analyzed relative to possible correlations with protein-protein contacts in the crystal lattice and the different milieus of pH and ionic strength in the crystals and n.m.r. samples. Further systematic comparisons of local conformational features indicated that there are correlations between amino acid types, local precision of the structure determination by both techniques and local differences between the structures in the crystals and in solution. Overall, hydrophobic residues are most precisely characterized and agree most closely in the two environments.     

Expression, purification and structural characterization of the scaffoldin hydrophilic X-module from the cellulosome of Clostridium thermocellum

The cellulosome is a membrane-bound, extracellular multi-subunit complex responsible for the degradation of crystalline cellulose by a number of organisms including anaerobic bacteria and fungi. The hydrophilic X-module (CipA-X) from the modular scaffoldin subunit of Clostridium thermocellum cellulosome has been proposed to play various roles in cellulosomal function, including thermal and structural stability. Towards elucidating the function of CipA-X using structural and biophysical studies, the region comprising residues 1692-1785 from the C. thermocellum CipA cDNA encoding CipA-X was cloned into a pET21b expression vector. When expressed in Escherichia coli, the C-terminal His-tagged protein accumulated in the insoluble fraction. Cell fractionation experiments showed that the recombinant protein was localized to inclusion bodies. Refolding and purification involved denaturation of the whole cell lysate by addition of urea, followed by a nickel-Sepharose chromatography step and dialysis into native conditions (25 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 10 mM EDTA). A final gel filtration step purified the protein to homogeneity, yielding 40 mg/L. The two-dimensional 1H-15N correlation spectrum of uniformly 15N-labelled CipA-X showed the characteristics of a well-folded protein comprising significant beta-structure, which is in agreement with the circular dichroism data.     

Interaction of N-terminal domain of U1A protein with an RNA stem/loop.

The U1A protein is a sequence-specific RNA binding protein found in the U1 snRNP particle where it binds to stem/loop II of U1 snRNA. U1A contains two 'RNP' or 'RRM' (RNA Recognition Motif) domains, which are common to many RNA-binding proteins. The N-terminal RRM has been shown to bind specifically to the U1 RNA stem/loop, while the RNA target of the C-terminal domain is unknown. Here, we describe experiments using a 102 amino acid N-terminal RRM of U1A (102A) and a 25-nucleotide RNA stem/loop to measure the binding constants and thermodynamic parameters of this RNA:protein complex. Using nitrocellulose filter binding, we measure a dissociation constant KD = 2 x 10(-11) M in 250 mM NaCl, 2 mM MgC2, and 10 mM sodium cacodylate, pH 6 at room temperature, and a half-life for the complex of 5 minutes. The free energy of association (delta G degrees) of this complex is about -14 kcal/mol in these conditions. Determination of the salt dependence of the binding suggests that at least 8 ion-pairs are formed upon complex formation. A mutation in the RNA loop sequence reduces the affinity 10 x, or about 10% of the total free energy.     

Recognition of 16S rRNA by ribosomal protein S4 from Bacillus stearothermophilus

Protein S4 is essential for bacterial small ribosomal subunit assembly and recognizes the 5' domain (approximately 500 nt) of small subunit rRNA. This study characterizes the thermodynamics of forming the S4-5' domain rRNA complex from a thermophile, Bacillus stearothermophilus, and points out unexpected differences from the homologous Escherichia coli complex. Upon incubation of the protein and RNA at temperatures between 35 and 50 degrees C under ribosome reconstitution conditions [350 mM KCl, 8 mM MgCl2, and 30 mM Tris (pH 7.5)], a complex with an association constant of > or = 10(9) M(-1) was observed, more than an order of magnitude tighter than previously found for the homologous E. coli complex under similar conditions. This high-affinity complex was shown to be stoichiometric, in equilibrium, and formed at rates on the order of magnitude expected for diffusion-controlled reactions ( approximately 10(7) M(-1) x s(-1)), though at low temperatures the complex became kinetically trapped. Heterologous binding experiments with E. coli S4 and 5' domain RNA suggest that it is the B. stearothermophilus S4, not the rRNA, that is activated by higher temperatures; the E. coli S4 is able to bind 5' domain rRNA equally well at 0 and 37 degrees C. Tight complex formation requires a low Mg ion concentration (1-2 mM) and is very sensitive to KCl concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 9.3]. The protein has an unusually strong nonspecific binding affinity of 3-5 x 10(6) M(-1), detected as a binding of one or two additional proteins to the target 5' domain RNA or two to three proteins binding a noncognate 23S rRNA fragment of the approximately same size. This binding is not as sensitive to monovalent ion concentration [- partial differential[log(K)]/partial differential(log[KCl]) = 6.3] as specific binding and does not require Mg ion. These findings are consistent with S4 stabilizing a compact form of the rRNA 5' domain.     

Structure of raw starch-digesting Bacillus cereus beta-amylase complexed with maltose.

The crystals of beta-amylase from Bacillus cereus belong to space group P21 with the following cell dimensions: a = 57.70 A, b = 92.87 A, c = 65.93 A, and beta =101.95 degrees. The structures of free and maltose-bound beta-amylases were determined by X-ray crystallography at 2.1 and 2.5 A with R-factors of 0.170 and 0.164, respectively. The final model of the maltose-bound form comprises 516 amino acid residues, four maltose molecules, 275 water molecules, one Ca2+, one acetate, and one sulfate ion. The enzyme consists of a core (beta/alpha)8-barrel domain (residues 5-434) and a C-terminal starch-binding domain (residues 435-613). Besides the active site in the core where two maltose molecules are bound in tandem, two novel maltose-binding sites were found in the core L4 region and in the C-terminal domain. The structure of the core domain is similar to that of soybean beta-amylase except for the L4 maltose-binding site, whereas the C-terminal domain has the same secondary structure as domain E of cyclodextrin glucosyltransferase. These two maltose-binding sites are 32-36 A apart from the active site. These results indicate that the ability of B. cereus beta-amylase to digest raw starch can be attributed to the additional two maltose-binding sites.     

枯草芽孢杆菌全长引物酶的溶液结构研究

在细菌DNA复制中,DnaG引物酶合成RNA引物,然后合成的引物通过DNA聚合酶进行延伸. DnaG引物酶由3个结构域组成,N端锌结合结构域（zinc-binding domain,ZBD）、RNA聚合酶结构域（RNA polymerase domain,RPD）和C端解旋酶结合结构域（helicase binding domain,HBD）. 在合成引物的过程中,引物酶的3个结构域协同作用,缺一不可. 尽管引物酶3个结构域的结构均已有研究报道,但到目前为止,引物酶的全长结构尚不清楚. 我们在上海光源利用小角X射线散射技术研究了枯草芽孢杆菌全长引物酶的溶液结构,首次构建了全长引物酶结构模型. 我们发现,枯草芽孢杆菌引物酶在溶液中处于伸展状态,且ZBD和HBD结构域相对于RPD结构域呈现出连续的构象变化. 本文研究表明DnaG引物酶中的结构域重排可能有助于其在DNA复制中发挥功能.     

The RNA binding domains of the nuclear poly(A)-binding protein

The nuclear poly(A)-binding protein (PABPN1) is involved in the synthesis of the mRNA poly(A) tails in most eukaryotes. We report that the protein contains two RNA binding domains, a ribonucleoprotein-type RNA binding domain (RNP domain) located approximately in the middle of the protein sequence and an arginine-rich C-terminal domain. The C-terminal domain also promotes self-association of PABPN1 and moderately cooperative binding to RNA. Whereas the isolated RNP domain binds specifically to poly(A), the isolated C-terminal domain binds non-specifically to RNA and other polyanions. Despite this nonspecific RNA binding by the C-terminal domain, selection experiments show that adenosine residues throughout the entire minimal binding site of approximately 11 nucleotides are recognized specifically. UV-induced cross-links with oligo(A) carrying photoactivatable nucleotides at different positions all map to the RNP domain, suggesting that most or all of the base-specific contacts are made by the RNP domain, whereas the C-terminal domain may contribute nonspecific contacts, conceivably to the same nucleotides. Asymmetric dimethylation of 13 arginine residues in the C-terminal domain has no detectable influence on the interaction of the protein with RNA. The N-terminal domain of PABPN1 is not required for RNA binding but is essential for the stimulation of poly(A) polymerase.     

Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus

For the first time, we demonstrate directly a stable complex between a bacterial DnaG (primase) and DnaB (helicase). Utilizing fragments of both proteins, we are able to dissect interactions within this complex and provide direct evidence that it is the C-terminal domain of primase that interacts with DnaB. Furthermore, this C-terminal domain is sufficient to induce maximal stimulation of the helicase and ATPase activities of DnaB. However, the region of DnaB that interacts with the C-terminal domain of primase appears to comprise a surface on DnaB that includes regions from both of the previously identified N- and C-terminal domains. Using a combination of biochemical and physical techniques, we show that the helicase-primase complex comprises one DnaB hexamer and either two or three molecules of DnaG. Our results show that in Bacillus stearothermophilus the helicase-primase interaction at the replication fork may not be transient, as was shown to be the case in Escherichia coli. Instead, primase appears to interact with the helicase forming a tighter complex with enhanced ATPase and helicase activities.     

Homogenous assays for Escherichia coli DnaB-stimulated DnaG primase and DnaB helicase and their use in screening for chemical inhibitors

Escherichia coli DnaG primase is a single-stranded DNA-dependent RNA polymerase. Primase catalyzes the synthesis of a short RNA primer to initiate DNA replication at the origin and to initiate Okazaki fragment synthesis for synthesis of the lagging strand. Primase activity is greatly stimulated through its interaction with DnaB helicase. Here we report a 96-well homogeneous scintillation proximity assay (SPA) for the study of DnaB-stimulated E. coli primase activity and the identification of E. coli primase inhibitors. The assay uses an adaptation of the general priming reaction by employing DnaG primase, DnaB helicase, and ribonucleotidetriphosphates (incorporation of [(3)H]CTP) for in vitro primer synthesis on single-stranded oligonucleotide and M13mp18 DNA templates. The primase product is captured by polyvinyl toluene-polyethyleneimine-coated SPA beads and quantified by counting by beta-scintography. In the absence of helicase as a cofactor, primer synthesis is reduced by 85%. The primase assay was used for screening libraries of compounds previously identified as possessing antimicrobial activities. Primase inhibitory compounds were then classified as direct primase inhibitors or mixed primase/helicase inhibitors by further evaluation in a specific assay for DnaB helicase activity. By this approach, specific primase inhibitors could be identified.     

Solution structure of the GUCT domain from human RNA helicase II/Gu beta reveals the RRM fold, but implausible RNA interactions

Human RNA helicase II/Gu alpha (RH-II/Gu alpha) and RNA helicase II/Gu beta (RH-II/Gu beta) are paralogues that share the same domain structure, consisting of the DEAD box helicase domain (DEAD), the helicase conserved C-terminal domain (helicase_C), and the GUCT domain. The N-terminal regions of the RH-II/Gu proteins, including the DEAD domain and the helicase_C domain, unwind double-stranded RNAs. The C-terminal tail of RH-II/Gu alpha, which follows the GUCT domain, folds a single RNA strand, while that of RH-II/Gu beta does not, and the GUCT domain is not essential for either the RNA helicase or foldase activity. Thus, little is known about the GUCT domain. In this study, we have determined the solution structure of the RH-II/Gu beta GUCT domain. Structural calculations using NOE-based distance restraints and residual dipolar coupling-based angular restraints yielded a well-defined structure with beta-alpha-alpha-beta-beta-alpha-beta topology in the region for K585-A659, while the Pfam HMM algorithm defined the GUCT domain as G571-E666. This structure-based domain boundary revealed false positives in the sequence homologue search using the HMM definition. A structural homology search revealed that the GUCT domain has the RRM fold, which is typically found in RNA-interacting proteins. However, it lacks the surface-exposed aromatic residues and basic residues on the beta-sheet that are important for the RNA recognition in the canonical RRM domains. In addition, the overall surface of the GUCT domain is fairly acidic, and thus the GUCT domain is unlikely to interact with RNA molecules. Instead, it may interact with proteins via its hydrophobic surface around the surface-exposed tryptophan.     

Domain swapping reveals that the C- and N-terminal domains of DnaG and DnaB, respectively, are functional homologues

The bacterial primase (DnaG)-helicase (DnaB) interaction is mediated by the C-terminal domain of DnaG (p16) and a linker that joins the N- and C-terminal domains (p17 and p33 respectively) of DnaB. The crystal and nuclear magnetic resonance structures of p16 from Escherichia coli and Bacillus stearothermophilus DnaG proteins revealed a unique structural homology with p17, despite the lack of amino acid sequence similarity. The functional significance of this is not clear. Here, we have employed a 'domain swapping' approach to replace p17 with its structural homologue p16 to create chimeras. p33 alone hydrolyses ATP but exhibits no helicase activity. Fusing p16 (p16-p33) or DnaG (G-p33) to the N-terminus of p33 produced chimeras with partially restored helicase activities. Neither chimera interacted with DnaG. The p16-p33 chimera formed hexamers while G-p33 assembled into tetramers. Furthermore, G-p33 and DnaB formed mixed oligomers with ATPase activity better than that of the DnaB/DnaG complex and helicase activity better than the sum of the individual DnaB and G-p33 activities but worse than that of the DnaB/DnaG complex. Our combined data provide direct evidence that p16 and p17 are not only structural but also functional homologues, albeit their amino acid composition differences are likely to influence their precise roles.     

Contributions of basic residues to ribosomal protein L11 recognition of RNA

The C-terminal domain of ribosomal protein L11, L11-C76, binds in the distorted minor groove of a helix within a 58 nucleotide domain of 23 S rRNA. To study the electrostatic component of RNA recognition in this protein, arginine and lysine residues have been individually mutated to alanine or methionine residues at the nine sequence positions that are conserved as basic residues among bacterial L11 homologs. In measurements of the salt dependence of RNA-binding, five of these mutants have a reduced value of - partial differentiallog(K(obs))/ partial differentiallog[KCl] as compared to the parent L11-C76 sequence, indicating that these residues interact with the RNA electrostatic field. These five residues are located at the perimeter of the RNA-binding surface of the protein; all five of them form salt bridges with phosphates in the crystal structure of the complex. A sixth residue, Lys47, was found to make an electrostatic contribution to binding when measurements were made at pH 6.0, but not at pH 7.0; its pK in the free protein must be <6.5. The unusual behavior of Lys47 is explained by its burial in the hydrophobic core of the free protein, and unburial in the RNA-bound protein, where it forms a salt bridge with a phosphate. The contributions of these six residues to the electrostatic component of binding are not additive; thus the magnitude of the salt dependence cannot be used to count the number of ionic interactions in this complex. By interacting with irregular features of the RNA backbone, including an S-turn, these basic residues contribute to the specificity of L11 for its target site.