Biomaterial Database

Site-specific 1,N6-ethenoadenylated single-stranded oligonucleotides as structural probes for the T4 gene 32 protein-ssDNA complex. 1991

D P Giedroc, and R Khan, and K Barnhart

Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128.

Bacteriophage T4 gene 32 protein (g32P) is a DNA replication accessory protein that binds single-stranded (ss) nucleic acids nonspecifically, independent of nucleotide sequence. G32P contains 1 mol of Zn(II)/mol of protein monomer, which can be substituted with Co(II), with maintenance of the structure and activity of the molecule. The Co(II) is coordinated via approximately tetrahedral ligand symmetry by three Cys sulfur atoms and therefore exhibits intense S(-)----Co(II) ligand to metal charge-transfer (LMCT) transitions in the near ultraviolet [Giedroc, D. P., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8452-8456]. A series of fluorescent 1,N6-ethenoadenosine (epsilon A)-containing oligonucleotides conforming to the structure (5'----3') d[(Tp)m epsilon A(pT)l-m-1] where 0 less than or equal to m less than or equal to l - 1 and length (l) six or eight nucleotides have been evaluated as dynamics probes and potential fluorescence energy transfer donors to Co(II) in mapping the spatial proximity of the (fixed) intrinsic metal ion and a variably positioned epsilon A-base in a series of protein-nucleic acid complexes. We provide spectroscopic evidence that the epsilon A-oligonucleotides bind to g32P-(A + B) with a fixed polarity of the phosphodiester chain. A Trp side chain(s) makes close approach to a epsilon A base positioned toward the 3' end of a bound l = 8 oligonucleotide. Six oligonucleotides of l = 8 and m = 0, 1, 3, 5, 6, or 7 were investigated as energy transfer donors to Co(II) at 0.1 M NaCl, pH 8.1, 25 degrees C upon binding to Co(II)-substituted or Zn(II) g32P-(A + B), i.e., in the presence and absence of an energy acceptor, respectively. Detectable quenching of the epsilon A-fluorescence by the Co(II)-LMCT acceptors was found to occur in all epsilon A-oligonucleotide-protein complexes, yielding energy transfer efficiencies (E) of 0.43, 0.31, 0.26, 0.26, 0.28, and 0.41 for l = 8 and m = 0, 1, 3, 5, 6, and 7 epsilon A-oligonucleotides, respectively. The two-dimensional distances R (in A) were found to vary as follows: d[epsilon A(pT)7] (m = 0), 16.0 (15.5-16.9); d[Tp epsilon A(pT)6] (m = 1), 17.7 (16.9-19.1); d[(Tp)3 epsilon A(pT)4] (m = 3), 20.7 (19.5-22.1); d[(Tp)5 epsilon A(pT)2] (m = 5), 20.5 (19.5-21.9); d[(Tp)6 epsilon ApT] (m = 6), 19.0 (18.0-20.4); and d[(Tp)7 epsilon A] (m = 7), 18.6 (17.8-19.8).(ABSTRACT TRUNCATED AT 400 WORDS)

UI	MeSH Term	Description	Entries
D009690	Nucleic Acid Conformation	The spatial arrangement of the atoms of a nucleic acid or polynucleotide that results in its characteristic 3-dimensional shape.	DNA Conformation,RNA Conformation,Conformation, DNA,Conformation, Nucleic Acid,Conformation, RNA,Conformations, DNA,Conformations, Nucleic Acid,Conformations, RNA,DNA Conformations,Nucleic Acid Conformations,RNA Conformations
D004268	DNA-Binding Proteins	Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases.	DNA Helix Destabilizing Proteins,DNA-Binding Protein,Single-Stranded DNA Binding Proteins,DNA Binding Protein,DNA Single-Stranded Binding Protein,SS DNA BP,Single-Stranded DNA-Binding Protein,Binding Protein, DNA,DNA Binding Proteins,DNA Single Stranded Binding Protein,DNA-Binding Protein, Single-Stranded,Protein, DNA-Binding,Single Stranded DNA Binding Protein,Single Stranded DNA Binding Proteins
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
D004279	DNA, Viral	Deoxyribonucleic acid that makes up the genetic material of viruses.	Viral 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
D005454	Fluorescence Polarization	Measurement of the polarization of fluorescent light from solutions or microscopic specimens. It is used to provide information concerning molecular size, shape, and conformation, molecular anisotropy, electronic energy transfer, molecular interaction, including dye and coenzyme binding, and the antigen-antibody reaction.	Anisotropy, Fluorescence,Fluorescence Anisotropy,Polarization, Fluorescence,Anisotropies, Fluorescence,Fluorescence Anisotropies,Fluorescence Polarizations,Polarizations, Fluorescence
D005814	Genes, Viral	The functional hereditary units of VIRUSES.	Viral Genes,Gene, Viral,Viral Gene
D000241	Adenosine	A nucleoside that is composed of ADENINE and D-RIBOSE. Adenosine or adenosine derivatives play many important biological roles in addition to being components of DNA and RNA. Adenosine itself is a neurotransmitter.	Adenocard,Adenoscan
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
D013604	T-Phages	A series of 7 virulent phages which infect E. coli. The T-even phages T2, T4; (BACTERIOPHAGE T4), and T6, and the phage T5 are called "autonomously virulent" because they cause cessation of all bacterial metabolism on infection. Phages T1, T3; (BACTERIOPHAGE T3), and T7; (BACTERIOPHAGE T7) are called "dependent virulent" because they depend on continued bacterial metabolism during the lytic cycle. The T-even phages contain 5-hydroxymethylcytosine in place of ordinary cytosine in their DNA.	Bacteriophages T,Coliphages T,Phages T,T Phages,T-Phage