Biomedical subjects

Jimin Wang

Publications and source records attributed to Jimin Wang.

At least 19 recordsLinked to original sources

PubMed2006

The phi29 DNA polymerase:protein-primer structure suggests a model for the initiation to elongation transition.

The absolute requirement for primers in the initiation of DNA synthesis poses a problem for replicating the ends of linear chromosomes. The DNA polymerase of bacteriophage phi29 solves this problem by using a serine hydroxyl of terminal protein to prime replication. The 3.0 A resolution structure shows one domain of terminal protein making no interactions, a second binding the polymerase and a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation. Thus, the progressively elongating DNA duplex product must displace this priming domain. Further, this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome. We propose a model for the transition from the initiation to the elongation phases in which the priming domain of terminal protein moves out of the active site as polymerase elongates the primer strand. The model indicates that terminal protein should dissociate from polymerase after the incorporation of approximately six nucleotides.

Bacillus Phages↗

PubMed2006

The L561A substitution in the nascent base-pair binding pocket of RB69 DNA polymerase reduces base discrimination.

Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to noncomplementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease-deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k(pol) and K(D) values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k(pol)/K(D)) for mispaired bases relative to the wild-type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 x 10(-)(5) microM(-)(1) s(-)(1) for dCMP opposite templating C to 2 x 10(-)(3) microM(-)(1) s(-)(1) for dAMP opposite templating C. These k(pol)/K(D) values are 3-60-fold greater than those observed with the wild-type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting Escherichia coli harboring a plasmid encoding the L561A variant of RB69 pol with T4 phage bearing a mutant rII locus, and the rates of reversions to rII(+) were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild-type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo(-) mutator activities.

Alanine↗

PubMed2005

Hydrodynamic characterization of the DEAD-box RNA helicase DbpA.

The Escherichia coli DEAD-box protein A (DbpA) belongs to the highly conserved superfamily-II of nucleic acid helicases that play key roles in RNA metabolism. A central question regarding helicase activity is whether the process of coupling ATP hydrolysis to nucleic acid unwinding requires an oligomeric form of the enzyme. We have investigated the structural and functional properties of DbpA by multi-angle laser light-scattering, size-exclusion chromatography, analytical ultracentrifugation, chemical cross-linking and hydrodynamic modeling. DbpA is monomeric in solution up to a concentration of 25 microM and over the temperature range of 4 degrees C to 22 degrees C. Binding of neither nucleotide (ATP or ADP) nor peptidyl transferase center (PTC) RNA, the presumed physiological RNA substrate, favor oligomerization. The hydrodynamic parameters were used together with hydrodynamic bead modeling and structural homology in conjunction with ab initio structure prediction methods to define plausible shapes of DbpA. Collectively, the results favor models where DbpA functions as an active monomer that possesses two distinct RNA binding sites, one in the helicase core domain and the other in the carboxyl-terminal domain that recognizes 23S rRNA and interacts specifically with hairpin 92 of the PTC.

Amino Acid Motifs↗

PubMed2005

DNA polymerases: Hoogsteen base-pairing in DNA replication?

Human polymerase-iota belongs to the error-prone Y family of polymerases, which frequently incorporate incorrect nucleotides during DNA replication but can efficiently bypass DNA lesions. On the basis of X-ray diffraction data, Nair et al. propose that Hoogsteen base-pairing is adopted by DNA during its replication by this enzyme. Here I re-examine their X-ray data and find that the electron density is very weak for a Hoogsteen base pair formed between a template adenine deoxyribonucleotide in the syn conformation and a deoxythymidine 5'-triphosphate (dTTP), and that the fit is better for a normal Watson-Crick base pair. As a guanine-cytosine (G-C) base pair has no potential to form a Hoogsteen base pair at physiological pH, Hoogsteen base-pairing is unlikely to be used in replication by this polymerase.

Base Pairing↗

PubMed2005

Correction of X-ray intensities from an HslV-HslU co-crystal containing lattice-translocation defects.

Because of lattice-translocation defects, two identical but translated lattices can coexist as a single coherent mosaic block in a crystal. The observed structure in such cases is a weighted sum of two identical but translated structures, one from each lattice; the observed structure factors are a weighted vector sum of the structure factors with identical unit amplitudes but shifted phases. The correction of X-ray intensities from a single crystal containing these defects of the hybrid HslV-HslU complex, which consists of Escherichia coli HslU and Bacillus subtilis HslV (also known as CodW), is reported. When intensities are not corrected, a biologically irrelevant complex (with CodW from one lattice and HslU from another) is implied to exist. Only upon correction does a biologically functional CodW-HslU complex structure emerge.

Crystallization↗

PubMed2005

Role of the GYVG pore motif of HslU ATPase in protein unfolding and translocation for degradation by HslV peptidase.

HslVU is an ATP-dependent protease consisting of HslU ATPase and HslV peptidase. In an HslVU complex, the central pores of HslU hexamer and HslV dodecamer are aligned and the proteolytic active sites are sequestered in the inner chamber of HslV. Thus, the degradation of natively folded proteins requires unfolding and translocation processes for their access into the proteolytic chamber of HslV. A highly conserved GYVG(93) sequence constitutes the central pore of HslU ATPase. To determine the role of the pore motif on protein unfolding and translocation, we generated various mutations in the motif and examined their effects on the ability of HslU in supporting the proteolytic activity of HslV against three different substrates: SulA as a natively folded protein, casein as an unfolded polypeptide, and a small peptide. Flexibility provided by Gly residues and aromatic ring structures of the 91st amino acid were essential for degradation of SulA. The same structural features of the GYVG motif were highly preferred, although not essential, for degradation of casein. In contrast, none of the features were required for peptide hydrolysis. Mutations in the GYVG motif of HslU also showed marked influence on its ATPase activity, affinity to ADP, and interaction with HslV. These results suggest that the GYVG motif of HslU plays important roles in unfolding of natively folded proteins as well as in translocation of unfolded proteins for degradation by HslV. These results also implicate a role of the pore motif in ATP cleavage and in the assembly of HslVU complex.

Adenosine Triphosphate↗

PubMed2005

A specific subdomain in phi29 DNA polymerase confers both processivity and strand-displacement capacity.

Recent crystallographic studies of phi29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a phi29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of phi29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity.

Amino Acid Sequence↗

PubMed2005

Base selectivity is impaired by mutants that perturb hydrogen bonding networks in the RB69 DNA polymerase active site.

To investigate the molecular basis for the selective utilization of nucleoside triphosphates complementary to templating bases, by RB69 DNA polymerase (RB69 pol), we constructed a set of mutants that we predicted would perturb the "floor" of the nascent base-pairing interface in the enzyme. We then determined the pre-steady-state kinetic parameters for the incorporation of complementary and noncomplementary dNTPs by the exo(-) form of RB69 pol and its mutants. We found that the Y567A mutant had the same K(d) and k(pol) values for incorporation of C versus G as the wild-type exo(-) enzyme; however, the k(pol)/K(d) ratio for G versus G incorporation with the Y567A mutant was 10 times higher than the k(pol)/K(d) efficiency of G versus G incorporation using the exo(-) RB69 pol. The reduced level of discrimination by the Y567A mutant against incorporation of mismatched bases was also seen with the Y391A mutant. Stopped-flow fluorescence was also employed to monitor rates of putative conformational changes with the exo(-) RB69 pol and its mutants using a primer-template complex containing 2-aminopurine. The rates of fluorescence changes were equal to or greater than the rates of the rapid chemical quench, indicating that we were monitoring a process occurring before or during the phosphoryl transfer reaction. We have interpreted our results within the context of the crystal structure of the RB69 pol ternary complex [Franklin, M. C., et al. (2001) Cell 105, 657-667].

Alanine↗

PubMed2005

Recent cyanobacterial Kai protein structures suggest a rotary clock.

The cyanobacterial circadian oscillator consists of three Kai proteins, KaiA, KaiB, and KaiC, in its oscillation feedback loop. Structural comparison reveals that the Kai system resembles the F1-ATPase system in which KaiC is equivalent to alpha(3)beta(3), KaiA to gammadelta, and KaiB to its inhibitory factor. It also suggests that there exists a possible haemagglutinin-like spring-loaded mechanism for the activation of KaiA during the formation of Kai complexes.

Bacterial Proteins↗

PubMed2005

A twisted four-sheeted model for an amyloid fibril.

The formation of amyloid fibers and their deposition in the body is a characteristic of a number of devastating human diseases. Here, we propose a structural model, based on X-ray diffraction data, for the basic structure of an amyloid fibril formed by using the variants of the B1 domain of IgG binding protein G of Streptococcus. The model for the fibril incorporates four beta sheets in a bundle with a diameter of 45 A. Its cross-section, or layer, consists of four strands, one strand from each sheet. Layers stack on top of each other to form the fibril, which has an overall helical twist with a periodicity of about 154 A. Each strand interacts in a parallel fashion with the strands in the layers above and below it, in an infinite beta sheet. Some geometric features of this model and the logic behind it may be applicable for constructing other related cross-beta amyloid fibrils.

Amyloid↗

PubMed2004

Correction of X-ray intensities from single crystals containing lattice-translocation defects.

In 1954, Howells and colleagues described an unusual diffraction pattern from imidazole methemoglobin crystals caused by lattice-translocation defects. In these crystals, two identical lattices coexist as a single coherent mosaic block, but are translated by a fixed vector with respect to each other. The observed structure is a weighted sum of the two identical but translated structures, one from each lattice; the observed structure factors are a weighted vector sum of the two structure factors with identical unit amplitudes but shifted phases. A general procedure is described to obtain the unit amplitudes of observed structure factors from a realigned single lattice through an X-ray intensity correction. An application of this procedure is made to determine the crystal structure of phi29 DNA polymerase at 2.2 A resolution using multiple isomorphous replacement and multiwavelength anomalous dispersion methods.

Bacillus Phages↗

PubMed2004

Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29.

The DNA polymerase from phage phi29 is a B family polymerase that initiates replication using a protein as a primer, attaching the first nucleotide of the phage genome to the hydroxyl of a specific serine of the priming protein. The crystal structure of phi29 DNA polymerase determined at 2.2 A resolution provides explanations for its extraordinary processivity and strand displacement activities. Homology modeling suggests that downstream template DNA passes through a tunnel prior to entering the polymerase active site. This tunnel is too small to accommodate double-stranded DNA and requires the separation of template and nontemplate strands. Members of the B family of DNA polymerases that use protein primers contain two sequence insertions: one forms a domain not previously observed in polymerases, while the second resembles the specificity loop of T7 RNA polymerase. The high processivity of phi29 DNA polymerase may be explained by its topological encirclement of both the downstream template and the upstream duplex DNA.

Amino Acid Sequence↗

PubMed2004

Visualizing a circadian clock protein: crystal structure of KaiC and functional insights.

Circadian (daily) biological clocks express characteristics that are difficult to explain by known biochemical mechanisms, and will ultimately require characterizing the structures, functions, and interactions of their molecular components. KaiC is an essential circadian protein in cyanobacteria that forms the core of the KaiABC clock protein complex. We report the crystal structure of the KaiC homohexameric complex at 2.8 A resolution. The structure resembles a double doughnut with a central pore that is partially sealed at one end. The crystal structure reveals ATP binding, inter-subunit organization, a scaffold for Kai-protein complex formation, the location of critical KaiC mutations, and evolutionary relationships to other proteins. A key auto-phosphorylation site on KaiC (T432) is identified from the crystal structure, and mutation of this residue abolishes circadian rhythmicity. The crystal structure of KaiC will be essential for understanding this circadian clockwork and for establishing its links to global gene expression.

Adenosine Triphosphate↗

PubMed2004

Crystal structure of a self-splicing group I intron with both exons.

The discovery of the RNA self-splicing group I intron provided the first demonstration that not all enzymes are proteins. Here we report the X-ray crystal structure (3.1-A resolution) of a complete group I bacterial intron in complex with both the 5'- and the 3'-exons. This complex corresponds to the splicing intermediate before the exon ligation step. It reveals how the intron uses structurally unprecedented RNA motifs to select the 5'- and 3'-splice sites. The 5'-exon's 3'-OH is positioned for inline nucleophilic attack on the conformationally constrained scissile phosphate at the intron-3'-exon junction. Six phosphates from three disparate RNA strands converge to coordinate two metal ions that are asymmetrically positioned on opposing sides of the reactive phosphate. This structure represents the first splicing complex to include a complete intron, both exons and an organized active site occupied with metal ions.

Azoarcus↗

PubMed2004

Nucleotide-dependent domain motions within rings of the RecA/AAA(+) superfamily.

The oligomeric rings formed by RecA-fold proteins are mechanochemical motors that perform many important biological functions. Their RecA-fold domains convert the chemical energy of ATP into mechanical work through large nucleotide-dependent conformational changes. This review summarizes recent structural and mechanistic works on the F1-ATPase and HslU regarding to the force generation by individual RecA folds in the context of ring structures. The F1-ATPase ring for example generates the force perpendicular to the ring axis, while the HslU ring generates forces presumably parallel to it. There exists a strong correlation between the directions of forces generated and the orientation of the RecA folds, not only in these two proteins but also in T7 DNA helicase, suggesting that it should be possible to predict the direction of forces generated by other members of this family on the basis of the orientation of their RecA folds.

Adenosine Triphosphate↗

PubMed2004

The structure of a ribosomal protein S8/spc operon mRNA complex.

In bacteria, translation of all the ribosomal protein cistrons in the spc operon mRNA is repressed by the binding of the product of one of them, S8, to an internal sequence at the 5' end of the L5 cistron. The way in which the first two genes of the spc operon are regulated, retroregulation, is mechanistically distinct from translational repression by S8 of the genes from L5 onward. A 2.8 A resolution crystal structure has been obtained of Escherichia coli S8 bound to this site. Despite sequence differences, the structure of this complex is almost identical to that of the S8/helix 21 complex seen in the small ribosomal subunit, consistent with the hypothesis that autogenous regulation of ribosomal protein synthesis results from conformational similarities between mRNAs and rRNAs. S8 binding must repress the translation of its own mRNA by inhibiting the formation of a ribosomal initiation complex at the start of the L5 cistron.

Base Sequence↗

PubMed2004

Crystal structure of a group I intron splicing intermediate.

A recently reported crystal structure of an intact bacterial group I self-splicing intron in complex with both its exons provided the first molecular view into the mechanism of RNA splicing. This intron structure, which was trapped in the state prior to the exon ligation reaction, also reveals the architecture of a complex RNA fold. The majority of the intron is contained within three internally stacked, but sequence discontinuous, helical domains. Here the tertiary hydrogen bonding and stacking interactions between the domains, and the single-stranded joiner segments that bridge between them, are fully described. Features of the structure include: (1) A pseudoknot belt that circumscribes the molecule at its longitudinal midpoint; (2) two tetraloop-tetraloop receptor motifs at the peripheral edges of the structure; (3) an extensive minor groove triplex between the paired and joiner segments, P6-J6/6a and P3-J3/4, which provides the major interaction interface between the intron's two primary domains (P4-P6 and P3-P9.0); (4) a six-nucleotide J8/7 single stranded element that adopts a mu-shaped structure and twists through the active site, making critical contacts to all three helical domains; and (5) an extensive base stacking architecture that realizes 90% of all possible stacking interactions. The intron structure was validated by hydroxyl radical footprinting, where strong correlation was observed between experimental and predicted solvent accessibility. Models of the pre-first and pre-second steps of intron splicing are proposed with full-sized tRNA exons. They suggest that the tRNA undergoes substantial angular motion relative to the intron between the two steps of splicing.

Azoarcus↗

PubMed2004

RNA kink turns to the left and to the right.

A helix-loop-helix within the group I intron has most of the canonical sequence elements of a kink turn (K-turn), yet it bends in the opposite direction. The reverse K-turn kinks toward the major rather than the minor grooves of the flanking helices. This suggests that there are two distinct subclasses of tertiary structures that a K-turn secondary structure can adopt. The final structure may be specified by external factors, such as protein binding or the tertiary structural context, rather than the intrinsic conformation of the RNA.

Azoarcus↗