ABSTRACT
The C-terminal domain of the alpha-subunit of Escherichia coli RNA polymerase (alphaCTD) is responsible for transcriptional activation through interaction with both activator proteins and UP element DNA. Previously, we determined the solution structure of alphaCTD. Here, we investigated the interaction between alphaCTD and UP element DNA by NMR. DNA titration curves and intermolecular NOE measurements indicate that alphaCTD can bind to multiple sites on the UP element DNA. Unlike many transcription factors, alphaCTD does not have a strict base sequence requirement for binding. There is a good correlation between the strength of the interaction and the extent of intrinsic bending of the DNA oligomer estimated from the gel retardation assay. We propose that alphaCTD recognizes the backbone structure of DNA oligomers responsible for the intrinsic bending. Moreover, NMR studies and drug competition experiments indicated that alphaCTD interacts with the UP element on the minor groove side of the DNA. The C-terminal end of helix-1, the N-terminal end of helix-4, and the loop between helices 3 and 4 are used for the interaction. Based on these observations, we propose a model for the UP element-alphaCTD complex.
Subject(s)
DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/metabolism , DNA/chemistry , DNA/metabolism , Escherichia coli/enzymology , Nucleic Acid Conformation , Base Sequence , Binding, Competitive , DNA/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Protein Conformation , Protein Structure, Tertiary , Protein Subunits , Substrate Specificity , ThermodynamicsABSTRACT
The structure of d(GGAGGAGGAGGA) containing four tandem repeats of a GGA triplet sequence has been determined under physiological K+ conditions by NMR. d(GGAGGAGGAGGA) folds into an intramolecular quadruplex composed of a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad. Four G-G segments of d(GGAGGAGGAGGA) are aligned parallel to each other due to seven successive turns of the main chain at each of the GGA and GAGG segments. Two quadruplexes form a dimer stabilized through a stacking interaction between the heptads of the two quadruplexes. On the basis of these results, the biological implications of naturally occurring GGA triplet repeat DNA are discussed.
Subject(s)
Adenosine/chemistry , DNA/chemistry , Guanine/chemistry , DNA Replication , Dimerization , G-Quadruplexes , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Repetitive Sequences, Nucleic AcidABSTRACT
The structure of d(GGAGGAGGAGGA) containing four tandem repeats of a GGA triplet sequence has been determined under physiological K(+) conditions. d(GGAGGAGGAGGA) folds into an intramolecular quadruplex composed of a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad. Four G-G segments of d(GGAGGAGGAGGA) are aligned parallel with each other due to six successive turns of the main chain at each of the GGA and GAGG segments. Two quadruplexes form a dimer stabilized through a stacking interaction between the heptads of the two quadruplexes. Comparison of the structure of d(GGAGGAGGAGGA) with the reported structure of d(GGAGGAN) (N=G or T) containing two tandem repeats of the GGA triplet revealed that although the two structures resemble each other to some extent, the extension of the repeats of the GGA triplet leads to distinct structural differences: intramolecular quadruplex for 12-mer versus intermolecular quadruplex for 7-mer; heptad versus hexad in the quadruplex; and three sheared G:A base-pairs versus two sheared G:A base-pairs plus one A:A base-pair per quadruplex. It was also suggested that d(GGAGGAGGAGGA) forms a similar quadruplex under low salt concentration conditions. This is in contrast to the case of d(GGAGGAN) (N=G or T), which forms a duplex under low salt concentration conditions. On the basis of these results, the structure of naturally occurring GGA triplet repeat DNA is discussed.
Subject(s)
DNA/chemistry , DNA/genetics , Nucleic Acid Conformation , Tandem Repeat Sequences/genetics , Trinucleotide Repeats/genetics , Base Pairing/drug effects , Base Sequence , Circular Dichroism , DNA/drug effects , Dimerization , G-Quadruplexes , Models, Molecular , Mutation/genetics , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation/drug effects , Potassium/pharmacology , Protons , Salts/pharmacologyABSTRACT
The structure of an RNA oligomer, r (GGAGGUUUUGGAGG) (R14-2) whose G-G steps are separated by adenine and uracil residues has been investigated by NMR. In the presence of 20 mM K+, a novel dimeric multiplex architecture is adopted by two strands of R14-2. In each strand a UUUU loop and two A residues connect four parallel G-G steps that pair-align into two tetrads. One of the tetrads is further pair-aligned by two A residues through the sheared mismatch and a novel hexad is subsequently formed. Two hexads coming from two different strands stack to make a dimeric multiplex. All of the guanosine and adenosine residues take an anti conformation.
Subject(s)
RNA/chemistry , Base Sequence , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Oligoribonucleotides/chemistryABSTRACT
An RNA aptamer for an HIV Tat protein has been isolated by the in vitro SELEX method. The RNA aptamer binds to the Tat protein 50-100 times more strongly than native TAR RNA does. Here, we have investigated the structure of the RNA aptamer complexed with ligands, partial peptide fragments of the Tat protein or argininamide, by multidimensional 1H/13C/15N NMR. It is strongly suggested that two U:A:U base triples are formed in the RNA aptamer upon binding of ligands. Specific hydrogen bonds between arginine side chains of ligands and guanine bases located adjacent to the base triples are identified. On the basis of many intramolecular and intermolecular NOEs, a structural model of the complex has been constructed.
Subject(s)
Gene Products, tat/chemistry , Gene Products, tat/metabolism , Oligoribonucleotides/chemistry , RNA/chemistry , RNA/metabolism , Base Sequence , Carbon Isotopes , Models, Molecular , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , Oligoribonucleotides/chemical synthesisABSTRACT
In vitro and in vivo antibacterial activities of MT-141, a new cephamycin, against anaerobic bacteria were compared with those of cefmetazole (CMZ), cefoxitin, cefotaxime, ceftizoxime, latamoxef and cefazolin to obtain the following results. MT-141 showed high activity against a wide variety of anaerobic bacteria including B. fragilis and C. difficile except for Eubacterium lentum, E. aerofaciens and B. furcosus. Antibacterial activity of MT-141 against anaerobic bacteria was almost independent of inoculum size, medium, pH and serum addition. In vitro development of resistance of MT-141 against P. variabilis and B. fragilis was comparable to that of CMZ. Successive parenteral administration of MT-141 into mice did not cause abnormal proliferation of C. difficile. MT-141 showed excellent therapeutic effect against experimental subcutaneous abscess in mice caused by B. fragilis.