Acid-induced denaturation of Escherichia coli ribonuclease HI analyzed by CD and NMR spectroscopies

Acid-induced denaturation of the ribonuclease HI protein from Escherichia coli was analyzed by CD and NMR spectroscopies. The CD measurement revealed that the acid denaturation at 10 degrees C proceeds from the native state (N-state) to a molten globule-like state (A-state), through an apparently more unfolded state (U(A)-state). In (1)H-(15)N heteronuclear single-quantum coherence (HSQC) spectra, cross peaks from the N-state and those from the other two states are distinctively observed, while the U(A)-state and A-state are not distinguished from each other. Cross peaks from the U(A)/A-states showed a small pH dependence, which suggests a similarity in the backbone structure between the two states. The direct hydrogen-deuterium (H-D) exchange measurement at pH with the largest population of U(A)-state revealed that at least alpha-helix I is highly protected in the structure of the U(A)-state. A pH-jump H-D exchange analysis showed that the protection of alpha-helix I is highest also in the A-state. The profile of hydrogen-bond protection indicated that the structure of the A-state is closely related to that of the kinetic folding intermediate.     

E. coli RNase HI and the phosphonate-DNA/RNA hybrid: molecular dynamics simulations

A model for the complex between E. coli RNase HI and the DNA/RNA hybrid (previously refined by molecular dynamics simulations) was used to determine the impact of the internucleotide linkage modifications (either 3-O-CH2-P-O-5' or 3-O-P-CH2-O-5) on the ability of the modified-DNA/RNA hybrid to create a complex with the protein. Modified internucleotide linkages were incorporated systematically at different positions close to the 3-end of the DNA strand to interfere with the DNA binding site of RNase H. Altogether, six trajectories were produced (length 1.5ns). Mutual hydrogen bonds connecting both strands of the nucleic acids hybrid, DNA with RNase H, RNA with RNase H, and the scissile bond with the Mg++. 4H2O chelate complex (bound in the active site) were analyzed in detaiL Many residues were involved in binding of the DNA (Arg88, Asn84, Trp85, Trp104, Tyr73, Lys99, Asn100, Thr43, and Asn 16) and RNA (Gln76, Gln72, Tyr73, Lys122, Glu48, Asn44, and Cys13) strand to the substrate-binding site of the RNase H enzyme. The most remarkable disturbance of the hydrogen bonding net was observed for structures with modified internucleotide linkages positioned in a way to interact with the Trp104, Tyr73, Lys99, and Asn100 residues (situated in the middle of the DNA binding site, where a cluster of Trp residues forms a rigid core of the protein structure).     

Strong nucleic acid binding to the Escherichia coli RNase HI mutant with two arginine residues at the active site

The biochemical properties of the mutant protein D10R/E48R of Escherichia coli RNase HI, in which Asp(10) and Glu(48) are both replaced by Arg, were characterized. This mutant protein has been reported to have metal-independent RNase H activity at acidic pH [Casareno et al. (1995) J. Am. Chem. Soc. 117, 11011-11012]. The far- and near-UV CD spectra of this mutant protein were similar to those of the wild-type protein, suggesting that the protein conformation is not markedly changed by these mutations. Nevertheless, we found that this mutant protein did not show any RNase H activity in vitro. Instead, it showed high-nucleic-acid-binding affinity. Protein footprinting analyses suggest that DNA/RNA hybrid binds to or around the presumed substrate-binding site of the protein. In addition, this mutant protein did not complement the temperature-sensitive growth phenotype of the rnhA mutant strain, E. coli MIC3001, even at pH 6.0, suggesting that it does not show RNase H activity in vivo as well. These results are consistent with a current model for the catalytic mechanism of the enzyme, in which Glu(48) is not responsible for Mg(2+) binding but is involved in the catalytic function.     

Binding of oxytetracycline to E. coli ribosomes

Binding isotherms of oxytetracycline to E. coli MRE-600 ribosomes were measured by equilibrium dialysis. The results are consistent with the presence of two classes of binding sites for the antibiotic on ribosomes having different reactivities. There is one strong binding site for the antibiotic on the ribosome, while the number of weak binding sites is about 500. The association constant for strong complexes is about 10³ times greater than the corresponding value for weak complexes. The strong binding of the antibiotic prevents the template dependent association of aminoacyl-tRNA with ribosomes.All-Union Institute of Antibiotics Research, Moscow 113105.     

Binding of N-methyl isatin beta-thiosemicarbazone-copper complexes to proteins and nucleic acids.

N-Methyl isatin beta-thiosemicarbazone-copper complexes interact with nucleic acids and proteins as shown by ultraviolet (UV) and visible spectroscopy and Sephadex exclusion chromatography. The Cu++ ions are most effective; Co++ ions have less albeit significant activity. Chelating agents, such as Tris and histidine, high NaCl concentration, and dimethyl sulfoxide reduce the binding of the drug-metal complex. The binding constant of the drug-copper complex to calf-thymus DNA was calculated to range between 6.9 x 10(4) and 2.7 x 10(5) M-1.     

Binding of reticulocyte elongation factor 1 to ribosomes and nucleic acids.

The present study has examined the requirements for the binding of rabbit reticulocyte elongation factor 1 (EF-1) to ribosomes under different assay conditions. When a centrifugation procedure was used to separate the ribosome EF-1 complex, the binding of EF-1 to ribosomes required GTP and Phe-tRNA, but not poly(U). The results suggested that undr these conditions a ternary complex, EF-1 . GTP . aminoacyl-tRNA, is necessary for the formation of a ribosome . EF-1 complex. However, when gel filtration was used to isolate the ribosome . EF-1 complex, only template and tRNA were required. These studie emphasize the fact that the procedure used to isolate the ribosome . EF-1 complex determines the requirements for stable complex formation. EF-1 can also interact with nucleic acids such as 28 S and 18 S rRNA, messenger RNA and DNA. In contrast to the binding to ribosomes, EF-1 binding to nucleic acids requires only Mg2+.     

Secondary structure of nucleic acids in the folded chromosome from E. coli.

The circular dichroism of membrane-free folded chromosomes from E. coli was measured and analyzed. The spectrum can be explained as a simple linear combination of the individual spectra of E. coli RNA, and E. Coli DNA in the B form. No contribution from A form or C form DNA was detected. There was evidently some real variation in the ratio of the two nucleic acids from preparation to preparation, but the average value was 24% RNA and 76% DNA. No significant light scattering was observed and the analyses indicated no contribution to the circular dichroism from scattering artifacts. Apparently, combining DNA, RNA, and protein into membrane-free folded chromosomes does not change the secondary structure of the DNA or RNA from that found for the free nucleic acid in the same solvent system.     

Rapid preparation of total nucleic acids from E. coli for multi-purpose applications

Separate protocols are commonly used to prepare plasmid DNA, chromosomal DNA, or total RNA from E. coli cells. Various methods for the rapid preparation of plasmid DNA have been developed previously, but the preparation of the chromosomal DNA and total RNA are usually laborious. We report here a simple, fast, reliable, and cost-effective method to extract total nucleic acids from E. coli by direct lysis of the cells with phenol. Five distinct and sharp bands, which correspond to chromosomal DNA, plasmid DNA, 23S rRNA, 16S rRNA, and a mixture of small RNA, were observed when analyzing the prepared total nucleic acids on a regular 1-2% agarose gel. The simple and high-quality preparation of the total nucleic acids in a single tube allowed us to rapidly screen the recombinant plasmid, as well as to simultaneously monitor the change of the plasmid copy number and rRNA levels during the growth of E. coli in the liquid medium.     

Substrate-induced conformational changes in Escherichia coli arginyl-tRNA synthetase observed by 19F NMR spectroscopy

The 19F nuclear magnetic resonance (NMR) spectra of 4-fluorotryptophan (4-F-Trp)-labeled Escherichia coli arginyl-tRNA synthetase (ArgRS) show that there are distinct conformational changes in the catalytic core and tRNA anticodon stem and loop-binding domain of the enzyme, when arginine and tRNA(Arg) are added to the unliganded enzyme. We have assigned five fluorine resonances of 4-F-Trp residues (162, 172, 228, 349 and 446) in the spectrum of the fluorinated enzyme by site-directed mutagenesis. The local conformational changes of E. coli ArgRS induced by its substrates observed herein by 19F NMR are similar to those of crystalline yeast homologous enzyme.     

Structure and stability of complexes formed by nucleic acids. I. Binding of acridine to DNA

Nuclear magnetic resonance spectra of acridine have been measured in aqueous methanol solutions over a wide concentration range in the presence and absence of dissolved DNA. In solutions containing DNA the acridine spectra show a marked line broadening and intensity decrease at temperatures lower than 50°C. These line-shape changes can be associated with two types of binding interactions: (1) a tight, irrotational binding of the acridine at low acridine:phosphate ratios and (2) a weaker, rotationally less restrictive binding at high acridine concentrations. At temperatures above 50°C. a marked line narrowing is noted for the acridine spectrum and is attributed to an increase in mobility of the bound acridine as the DNA complex undergoes a helix–coil transition. A loose association of acridine molecules with the purine and pyrimidine bases in heat-denatured DNA is indicated by chemical shift changes in the acridine spectrum. The NMR measurements also show that the presence of acridine in denatured DNA solutions greatly reduces renaturation of the DNA.     

Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy

Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150+/-10 kDa, regardless of the conditions investigated-protein concentration, 0.18-1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4-20 degrees C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 microg/mL-1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s(0)20,w=7.7+/-0.3 and 8.3+/-0.4 S, respectively. The molecular mass of the sedimenting particle (146+/-30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.