Structure of influenza a virus promoter and its implications for viral RNA synthesis.
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Genome, Viral↗
Biomedical subjects
B S Choi
Publications and source records attributed to B S Choi.
Structural features of an influenza virus promoter and their implications for viral RNA synthesis.
The influenza A virus, a severe pandemic pathogen, has a segmented RNA genome consisting of eight single-stranded RNA molecules. The 5' and 3' ends of each RNA segment recognized by the influenza A virus RNA-dependent RNA polymerase direct both transcription and replication of the virus's RNA genome. Promoter binding by the viral RNA polymerase and formation of an active open complex are prerequisites for viral replication and proliferation. Here we describe the solution structure of this promoter as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. Our studies show that the viral promoter has a significant dynamic nature and reveal an unusual displacement of an adenosine that forms a novel (A-A) x U motif and a C-A mismatch stacked in a helix. The characterized structural features of the promoter imply that the specificity of polymerase binding results from an internal RNA loop. In addition, an unexpected bending (46 +/- 10 degrees ) near the initiation site suggests the existence of a promoter recognition mechanism similar to that of DNA-dependent RNA polymerase and a possible regulatory function for the terminal structure during open complex formation.
Influenza A virus↗
SeqA protein aggregation is necessary for SeqA function.
The binding of SeqA protein to hemimethylated GATC sequences is important in the negative modulation of chromosomal initiation at oriC, and in the formation of SeqA foci necessary for Escherichia coli chromosome segregation. Using gel-filtration chromotography and glycerol gradient sedimentation, we demonstrate that SeqA exists as a homotetramer. SeqA tetramers are able to aggregate or multimerize in a reversible, concentration-dependent manner. Using a bacterial two-hybrid system, we demonstrate that the N-terminal region of SeqA, especifically the 9th amino acid residue, glutamic acid, is required for functional SeqA-SeqA interaction. Although the SeqA(E9K) mutant protein, containing lysine rather than glutamic acid at the 9th amino acid residue, exists as a tetramer, the mutant protein binds to hemimethylated DNA with altered binding patterns as compared with wild-type SeqA. Aggregates of SeqA(E9K) are defective in hemimethylated DNA binding. Here we demonstrate that proper interaction between SeqA tetramers is required for both hemimethylated DNA binding and formation of active aggregates. SeqA tetramers and aggregates might be involved in the formation of SeqA foci required for the segregation of chromosomal DNA as well as the regulation of chromosomal initiation.
Bacterial Outer Membrane Proteins↗
The Dewar photoproduct of thymidylyl(3'-->5')- thymidine (Dewar product) exhibits mutagenic behavior in accordance with the "A rule".
In contrast to the highly mutagenic pyrimidine(6-4)pyrimidone photoproduct, its Dewar valence isomer (Dewar product) has low mutagenic potential and produces a broad range of mutations [LeClerc, J. E., Borden, A. & Lawrence, C. W. (1991) Proc. Natl. Acad. Sci. USA 88, 9685-9689]. To determine the origin of the mutagenic property of the Dewar product, we used experimental NMR restraints and molecular dynamics to determine the solution structure of a Dewar-lesion DNA decamer duplex. This DNA decamer duplex (DW/GA duplex) contains a mismatched base pair between the 3' T residue of the Dewar lesion (T6) and an opposed G residue (G15). The 3' T (T6) of the Dewar lesion formed stable hydrogen bonds with the opposing G15 residue. However, the helical bending and unwinding angles of the DW/GA duplex were much larger than those of a second duplex that contains the Dewar lesion and opposing A15 and A16 residues (DW/AA duplex). The DW/GA duplex showed poorer stacking interactions at the two bases of the Dewar product and at the adjacent A7 small middle dotT14 base pair than did the DW/AA duplex. These structural features imply that no thermal stability or conformational benefit is obtained by incorporating a G instead of an A opposite the 3' T of the Dewar lesion. These properties may thus facilitate the preferential incorporation of an A in accordance with the A rule during translesion replication and lead to the low frequency of 3' T-->C mutations observed at this site.
Base Sequence↗
Solution structure of the DNA decamer duplex containing a 3'-T x T basepair of the cis-syn cyclobutane pyrimidine dimer: implication for the mutagenic property of the cis-syn dimer.
The cis - syn dimer is the major DNA photoproduct produced by UV irradiation. In order to determine the origin of the mutagenic property of the cis - syn dimer, we used NMR restraints and molecular dynamics to determine the solution structure of a DNA decamer duplex containing a wobble pair between the 3'-T of the cis - syn dimer and the opposite T residue (CS/TA duplex). The solution structure of the CS/TA duplex revealed that the 3'-T x T base pair of the cis - syn dimer had base pair geometry that was significantly different from the canonical Watson-Crick base pair and caused destabilization and conformational distortion of its 3'-region. However, a 3'-T x A base pair at the cis - syn dimer within this related DNA decamer maintains the normal Watson-Crick base pair geometry and causes little distortion in the conformation of its 3'-side. Our results show that in spite of its stable hydrogen bonding, the insertion of a T residue opposite the 3'-T of the cis - syn dimer is inhibited by structural distortion caused by the 3'-T x T base pair. This may explain why the frequency of the 3'-T-->A transversion, which is the major mutation produced by the cis - syn dimer, is only 4%.
Base Pairing↗
Synthesis and antimicrobial activity of squalamine analogue.
Synthesis and antimicrobial activity of squalamine analogue 2 are reported. The synthesis of 2 was accomplished from bisnoralcohol 3. The spermidine moiety was introduced via reductive amination of an appropriately functionalized 3beta-aminosterol with spermidinyl aldehyde 17 utilizing sodium triacetoxyborohydride as the reducing agent. Compound 2 shows weaker antimicrobial activity than squalamine.
Animals↗
Structures of ovine corticotropin-releasing factor and its Ala32 mutant as studied by CD and NMR techniques.
The corticotropin-releasing factor (CRF) is a 41-amino acid peptide-amide hormone, which mediates a general stress-response. It has been reported that the substitution of His-32 in the ovine CRF (oCRF) with Ala brings about a 4.5-fold increase in activity [Kornreich et al. (1992) J. Med. Chem. 35, 1870-76]. Here, we have determined the secondary structure of this Ala-substituted ovine CRF ([Ala32]oCRF) and compare it with that of oCRF using circular dichroism (CD) and NMR techniques in trifluoroethanol (TFE) solution, which is known to stabilize the alpha-helix formation. In contrast to an earlier report, it was observed the alpha-helical structure extends to the C-terminus of oCRF. By analyzing the CalphaH and NH chemical shifts, the properties of local structures of oCRF were elucidated. The oCRF and [Ala32]oCRF have stable alpha-helical structures in the middle region, regardless of pH and temperature, and the alpha-helix initiation regions of these peptides are stabilized as the pH is decreased. However, the [Ala32]oCRF has a more stable alpha-helical structure than oCRF in the vicinity of the substitution region, and it is thought that this is the cause of the increased activity of [Ala32]oCRF.
Alanine↗
Validity and cost-effectiveness of diagnostic procedures in CS2 poisoning.
To determine relatively useful diagnostic procedures of carbon disulfide (CS2) poisoning in terms of validity and cost-effectiveness, several diagnostic tests are evaluated on 1,552 people by prevalence ratio (exposed/nonexposed), dose-response relationship, sensitivity and specificity, and the cost of the tests. Several symptoms with high kappa indices are found to be useful in various combinations, showing a consistent dose-response relationship and high exposed-nonexposed ratio. In clinicopathologic tests on functions of the kidney, liver and hematopoietic systems, eight items out of 22 have been shown to have significant dose-response relationship, mostly in liver function tests. A thorough dental examination failed to identify any useful indicator peculiar to the CS2 poisoning. Unlike Western people, the prevalence of coronary heart disease among Koreans was too low to be useful in the diagnosis of CS2 poisoning. Among four elective tests, i.e., Minnesota Multiphasic Personality Inventory (MMPI), Brain magnetic resonance imaging (MRI), nerve conduction velocity (NCV), and fluorescent angiography (FAG), the NCV appeared to be a more sensitive and specific test than the others are. Combinations of the tests improved the probability of diagnosing CS2 poisoning cases when any one test out of four was positive. Addition of other valid tests increased the probability of excluding non-cases. It was concluded that diagnosis of CS2 poisoning could be made validly and inexpensively if the diagnostic tests were carefully chosen step by step.
Adolescent↗
Antifungal activities of recombinant antifungal protein by conjugation with polyethylene glycol.
Tenecin 3, an antifungal protein isolated from coleopteran insect Tenebrio molitor larvae, inhibited growth of the fungus Candida albicans. We have previously reported that tenecin 3 has a propensity of random structure with very loose turn-like elements by circular dichroism (CD) analysis and 2D nuclear overhauser effect spectroscopy [Lee et al. (1999)]. However, the antifungal mechanism of tenecin-3 has not yet been studied due to its very low availability from natural sources. As an initial step to study the antifungal mechanism of tenecin 3, recombinant tenecin 3 (RT-3) obtained from an expression system in Escherichia coli showed antifungal activity against C. albicans as did natural tenecin 3. To elucidate the antifungal mechanism of RT-3 and to explore the possibility of preparing polyethylene glycol (PEG) conjugated derivative, we synthesized PEG conjugated RT-3 (RT-3-PEG) and examined its antifungal activity against C. albicans in vitro. RT-3-PEG showed greater antifungal activity against C. albicans than RT-3 alone at the same dose. When C. albicans was treated with RT-3-PEG in vitro, K+ in the C. albicans cell was leaked out rapidly compared to the C. albicans treated with RT-3 alone. When the morphological change of RT-3-PEG treated C. albicans was examined by scanning electron microscopy, string-like substances, which may have been derived from the fungus, were stacked around the cell whose wall was damaged. Also, no appreciable hemolysis of mouse erythrocytes was detected under conditions in which 1% melittin caused 100% hemolysis. These results suggested that the RT-3-PEG derivative probably does not interact with mammalian cell appreciably, although it has antifungal activity.
Animals↗
Solution structure of a DNA decamer duplex containing the stable 3' T.G base pair of the pyrimidine(6-4)pyrimidone photoproduct [(6-4) adduct]: implications for the highly specific 3' T --> C transition of the (6-4) adduct.
The pyrimidine(6-4)pyrimidone photoproduct [(6-4) adduct] is one of the major photoproducts induced by UV irradiation of DNA and occurs at TpT sites. The (6-4) adduct is highly mutagenic and leads most often to a 3' T --> C transition with 85% replicating error frequency [LeClerc, J. E., Borden, A. & Lawrence, C. W. (1991) Proc. Natl. Acad. Sci. USA 88, 9685-9689]. To determine the origin of the specific 3' T --> C transition of the (6-4) adduct, we have used experimental NMR restraints and molecular dynamics to determine the solution structure of a (6-4)-lesion DNA decamer duplex that contains a mismatched base pair between the 3' T residue and an opposed G residue. Normal Watson-Crick-type hydrogen bonding is retained at the 5' T of the lesion site. The O2 carbonyl of the 3' T residue forms hydrogen bonds with the imino and amino protons of the opposed G residue. This potential hydrogen bonding stabilizes the overall helix and restores the highly distorted conformation of the (6-4) adduct to the typical B-form-like DNA structure. This structural feature can explain the marked preference for the insertion of an A residue opposite the 5' T and a G residue opposite the 3' T of the (6-4) lesion during trans-lesion synthesis. Thus these insertions yield the predominant 3' T --> C transition.
Animals↗
Inhibition of poly(ADP-ribose)polymerase binding to DNA by thymidine dimer.
The ability of poly(ADP-ribose)polymerase to bind damaged DNA was assessed by electrophoretic mobility shift assay. DNA binding domain of poly(ADP-ribose)polymerase (PARPDBD) binds to synthetic deoxyribonucleotide duplex 10-mer. However, the synthetic deoxyribonucleotide duplex containing cys-syn thymidine dimer which produces the unwinding of DNA helix structure lost its affinity to PARPDBD. It was shown that the binding of PARPDBD to the synthetic deoxyribonucleotide duplex was not affected by O6-Me-dG which causes only minor distortion of DNA helix structure. This study suggests that the stabilized DNA helix structure is important for poly(ADP-ribose)polymerase binding to DNA breaks, which are known to stimulate catalytic activity of poly(ADP-ribose)polymerase.
DNA↗
Structure of influenza virus panhandle RNA studied by NMR spectroscopy and molecular modeling.
The structure of a 34 nucleotide RNA molecule in solution, which contains the conserved panhandle sequences, was determined by NMR spectroscopy and molecular modeling. The partially double-strandedpanhandle structure of the influenza virus RNA serves to regulate initiation and termination of viral transcription as well as polyadenylation. The panhandle RNA consists of internal loop flanked by short helices. The nucleotides at or near the internal loop are crucial for polymerase binding and transcriptional activity. They show more flexible conformational character than the Watson-Crick base-paired region, especially for the backbone torsion angles of alpha, gamma and delta. Although residues A10 and A12 are stacked in the helix, the phosphodiester backbones are distorted. Residues A12, A13 and G25 show dynamic sugar conformations and the backbone conformations of these nucleotides are flexible. This backbone conformation and its associated flexibility may be important for protein-RNA interactions as well as base-specific interactions.
Magnetic Resonance Spectroscopy↗
Chemical shift perturbation studies of the interactions of the second RNA-binding domain of the Drosophila sex-lethal protein with the transformer pre-mRNA polyuridine tract and 3' splice-site sequences.
The interactions of the second RNA-binding domain of the Drosophila melanogaster Sex-lethal protein (Sxl RBD2) with the oligoribonucleotides, GUUUUUUUU (GU8) and CUAGUG, representing the sequences surrounding an alternative 3'-splicing site of the transformer pre-mRNA (GU8CUAGUG), were studied using heteronuclear two-dimensional NMR techniques. The 1H and 15N chemical shifts of the backbone amide resonances upon titration of Sxl RBD2 with each of these RNAs were recorded. It was found that Sxl RBD2 can bind not only to the polyuridine tract, GU8, but also to the downstream 3' splice-site sequence, CUAGUG, with similar affinities. In contrast, a nonspecific sequence, C8, did not bind to Sxl RBD2. This result is consistent with previous in vitro RNA-selection and UV-cross-linking results which indicated that the Sex-lethal protein binds to the uridine stretch and the AG dinucleotide in the consensus sequence, AUnNnAGU. In both cases, the chemical-shift perturbations were significant for almost the same amino acid residues, including the two central beta-strands formed by the RNP2-motif and RNP1-motif with the two highly conserved aromatic residues (Y214 and F256) in the middle. As the first RNA-binding domain of Sex-lethal (Sxl RBD1) has a characteristic aliphatic residue at one of the two corresponding positions (I128 and F170), Y214 of Sxl RBD2 was replaced by Ile using site-directed mutagenesis. On the one hand, the 1H and 15N chemical-shift perturbations indicated that GU8 binds to the same interface of mutant Sxl RBD2 as of wild-type Sxl RBD2, although its binding affinity was decreased significantly. On the other hand, the specific binding of Sxl RBD2 to CUAGUG was abolished almost completely by the Y-->I mutation. Taken together, the present results indicate that the interface residues that bind with GU8 and CUAGUG are much the same, but the role of the Y214 residue is clearly different between these two target sequences.
Amides↗
Structural and functional implications of a proline residue in the antimicrobial peptide gaegurin.
Although it is commonly known as a helix breaker, proline residues have been found in the alpha-helical regions of many peptides and proteins. The antimicrobial peptide gaegurin displays alpha-helical structure and has a central proline residue (P14). The structure and activity of gaegurin and its alanine derivative (P14A) were determined by various spectroscopic methods, restrained molecular dynamics, and biological assays. Both P14 and P14A exhibited cooperative helix formation in solution, but the helical stability of P14 was reduced substantially when compared to that of P14A. Chemical-shift analysis indicated that both of the peptides formed curved helices and that P14 showed diminished stability in the region around the central proline. However, hydrogen-exchange data revealed remarkable differences in the location of stable amide protons. P14 showed a stable region in the concave side of the curved helix, while P14A exhibited a stable region in the central turn of the helix. The model structure of P14 exhibited a pronounced kink, in contrast to the uniform helix of P14A. Both peptides showed comparable binding affinities for negatively charged lipids, while P14 had a considerably reduced affinity for a neutral lipid. With its destabilized alpha-helix, P14 exhibited greater antibacterial activity than did P14A. Hence, electrostatic interaction between helical peptides and lipid membranes is believed to be the dominant factor for antibacterial activity. Moreover, helical stability can modulate peptide binding to membranes that is driven by electrostatic interactions. The observation that P14 is a more potent antibacterial agent than P14A implies that the helical kink of P14 plays an important role in the disruption of bacterial membranes.
Amides↗
Structural characteristics of tenecin 3, an insect antifungal protein.
Tenecin 3, an antifungal protein, previously isolated from the insect Tenebrio molitor, inhibits growth of the fungus Candida albicans. However, the antifungal mechanism and functions of tenecin 3 remain unknown. As an initial step to study the mechanism and functions, physical and structural properties of tenecin 3 were examined by circular dichroism (CD) analysis and 2D nuclear overhauser effect spectroscopy. These analyses suggest that tenecin 3 has a propensity of random structure with very loose turn-like elements. The CD results also indicate that this random structural propensity is not significantly affected by temperature, pH, and by the presence of organic solvents or sodium dodecyl sulfate (SDS) micelles. However, the hydrodynamic studies suggest that tenecin 3 is not in extended form in spite of its random structural feature.
Animals↗
Bacterial expression of tenecin 3, an insect antifungal protein isolated from Tenebrio molitor, and its efficient purification.
Tenecin 3, an antifungal protein isolated from the insect Tenebrio molitor larvae, inhibits growth of the fungus Candida albicans. However, the antifungal mechanism and functions of tenecin 3 have not yet been studied due to its very low availability from the natural source. Here we report an expression system of the recombinant tenecin 3 in E. coli, whose amino acid composition is the same with that of the natural tenecin 3. We also devised a simple and easy procedure to isolate the recombinant protein from the bacterial cell extracts. The recombinant tenecin 3 showed an antifungal activity against C. albicans as the natural tenecin 3 did. Therefore large quantities of tenecin 3 can be easily obtained by the expression and purification system of tenecin 3 described in this report.
Amino Acid Sequence↗
The solution structure of DNA decamer duplex containing the Dewar product of thymidylyl(3-->5')thymidine by NMR and full relaxation matrix refinement.
The (6-4) adducts and their Dewar isomers play an important role in cytotoxicity and mutation in skin cells exposed to sunlight. Structural study of the DNA duplex containing a site-specific photoproduct is an essential step toward understanding the molecular mechanism of the mutagenesis and the repair activity of UV-irradiated DNA. Here we use 1H NMR spectroscopy and full relaxation matrix refinement to investigate the solution structure of the duplex Dewar decamer. We find that the isomerization of the (6-4) adduct to its Dewar form induces a substantial change in overall structure of the oligonucleotide duplex. Contrasting base stacking of two lesion sites results in a large difference in the structural impacts induced by the two photoproducts, such as differential disruption of hydrogen bonding at the lesion sites and overall helical bending of 44 degrees by the (6-4) lesion and 21 degrees by the Dewar lesion.
Base Composition↗
Proton exchange kinetics in [d(ACGTATACGT)]2-echinomycin and [d(ACGTTAACGT)]2-echinomycin complexes.
Based on imino proton exchange catalysis, base-pair lifetimes and apparent dissociation constants are reported on the complexes formed by bisintercalation of echinomycin at the CpG steps of the d(ACGTATACGT)2 and d(ACGTTAACGT)2 duplexes. The lifetimes of the four central A x T base pairs between two echinomycin binding sites are much shorter than in the free duplexes. The destabilization of base pairs adjacent to the binding sites is propagated one additional base pair away from the binding site.
Base Composition↗