Architecture of a complex between the sigma70 subunit of Escherichia coli RNA polymerase and the nontemplate strand oligonucleotide. Luminescence resonance energy transfer study. 1999

E Heyduk, and T Heyduk
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical School, St. Louis, Missouri 63104, USA. heydukt@wpogate.slu.edu

We used luminescence energy transfer measurements to determine the localization of 5'- and 3'-ends of a 12-nucleotide nontemplate strand oligonucleotide bound to sigma70 holoenzyme. Five single reactive cysteine mutants of sigma70 (cysteine residues at positions 1, 59, 366, 442, and 596) were labeled with a europium chelate fluorochrome (donor). The oligonucleotide was modified at the 5'- or at the 3'-end with Cy5 fluorochrome (acceptor). The energy transfer was observed upon complex formation between the donor-labeled sigma70 holoenzyme and the acceptor-labeled nontemplate strand oligonucleotide, whereas no interaction was observed with the template strand oligonucleotide. The oligonucleotide was bound in one preferred orientation. This observation together with the sequence specificity of single-stranded oligonucleotide interaction suggests that two mechanisms of discrimination between the template and nontemplate strand are used by sigma70: sequence specificity and strand polarity specificity. The bound oligonucleotide was found to be close to residue 442, confirming that the single-stranded DNA binding site of sigma70 is located in an alpha-helix containing residue 442. The 5'-end of the oligonucleotide was oriented toward the COOH terminus of the helix.

UI MeSH Term Description Entries
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D004277 DNA, Single-Stranded A single chain of deoxyribonucleotides that occurs in some bacteria and viruses. It usually exists as a covalently closed circle. Single-Stranded DNA,DNA, Single Stranded,Single Stranded DNA
D004735 Energy Transfer The transfer of energy of a given form among different scales of motion. (From McGraw-Hill Dictionary of Scientific and Technical Terms, 6th ed). It includes the transfer of kinetic energy and the transfer of chemical energy. The transfer of chemical energy from one molecule to another depends on proximity of molecules so it is often used as in techniques to measure distance such as the use of FORSTER RESONANCE ENERGY TRANSFER. Transfer, Energy
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D012321 DNA-Directed RNA Polymerases Enzymes that catalyze DNA template-directed extension of the 3'-end of an RNA strand one nucleotide at a time. They can initiate a chain de novo. In eukaryotes, three forms of the enzyme have been distinguished on the basis of sensitivity to alpha-amanitin, and the type of RNA synthesized. (From Enzyme Nomenclature, 1992). DNA-Dependent RNA Polymerases,RNA Polymerases,Transcriptases,DNA-Directed RNA Polymerase,RNA Polymerase,Transcriptase,DNA Dependent RNA Polymerases,DNA Directed RNA Polymerase,DNA Directed RNA Polymerases,Polymerase, DNA-Directed RNA,Polymerase, RNA,Polymerases, DNA-Dependent RNA,Polymerases, DNA-Directed RNA,Polymerases, RNA,RNA Polymerase, DNA-Directed,RNA Polymerases, DNA-Dependent,RNA Polymerases, DNA-Directed
D012808 Sigma Factor A protein which is a subunit of RNA polymerase. It effects initiation of specific RNA chains from DNA. Sigma Element,Sigma Initiation Factor,Sigma Subunit,Minor Sigma Factor,RNA Polymerase Sigma Factor H,Factor, Sigma,Factor, Sigma Initiation,Initiation Factor, Sigma,Sigma Factor, Minor,Subunit, Sigma
D013698 Templates, Genetic Macromolecular molds for the synthesis of complementary macromolecules, as in DNA REPLICATION; GENETIC TRANSCRIPTION of DNA to RNA, and GENETIC TRANSLATION of RNA into POLYPEPTIDES. Genetic Template,Genetic Templates,Template, Genetic

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