Properties of purified ribonuclease P from Escherichia coli

The purified protein moiety of ribonuclease P (EC 3.1.26.5) from Escherichia coli, a single polypeptide of molecular weight approximately 17 500, has not catalytic activity by itself on several RNA substrates. However, when it is marked in vitro with an RNA species called M1 RNA, RNase P activity is reconstituted. The rate at which the purified RNase P cleaves any particular tRNA precursor molecule depends on the identity of that tRNA precursor.     

Evidence for rapid association-dissociation of ribonuclease T1 from a recombinant strain of Escherichia coli

On the basis of photon correlation experiments and computer simulations, we provide evidence for a rapid dimerization of the enzyme ribonuclease T1 isolated from an Escherichia coli overproducing strain. An attractive potential in addition to the usual repulsive hardcore and electrostatic potentials was found to be necessary for interpreting the concentration dependence of the diffusion coefficient of the enzyme. Computer searches of surface complementarity suggest that dimer formation of ribonuclease T1 takes place due to an extensive surface contact of approximately 700 A2. Energy minimization of the ribonuclease T1 dimer shows that large conformational changes are not induced upon self-association of the enzyme. The two molecules in the dimer are orientated back-to-back, and this is expected to lead to an active enzyme form.     

Fold recognition by combining sequence profiles derived from evolution and from depth-dependent structural alignment of fragments

Recognizing structural similarity without significant sequence identity has proved to be a challenging task. Sequence-based and structure-based methods as well as their combinations have been developed. Here, we propose a fold-recognition method that incorporates structural information without the need of sequence-to-structure threading. This is accomplished by generating sequence profiles from protein structural fragments. The structure-derived sequence profiles allow a simple integration with evolution-derived sequence profiles and secondary-structural information for an optimized alignment by efficient dynamic programming. The resulting method (called SP(3)) is found to make a statistically significant improvement in both sensitivity of fold recognition and accuracy of alignment over the method based on evolution-derived sequence profiles alone (SP) and the method based on evolution-derived sequence profile and secondary structure profile (SP(2)). SP(3) was tested in SALIGN benchmark for alignment accuracy and Lindahl, PROSPECTOR 3.0, and LiveBench 8.0 benchmarks for remote-homology detection and model accuracy. SP(3) is found to be the most sensitive and accurate single-method server in all benchmarks tested where other methods are available for comparison (although its results are statistically indistinguishable from the next best in some cases and the comparison is subjected to the limitation of time-dependent sequence and/or structural library used by different methods.). In LiveBench 8.0, its accuracy rivals some of the consensus methods such as ShotGun-INBGU, Pmodeller3, Pcons4, and ROBETTA. SP(3) fold-recognition server is available on http://theory.med.buffalo.edu.     

Purification of recombinant ribonuclease T1 expressed in Escherichia coli

A protocol for the rapid purification of ribonuclease T1 expressed from a chemically synthesized gene cloned into Escherichia coli is described. QAE ion-exchange and Sephadex G-50 chromatography are used to give over 300 mg (88% yield) of pure ribonuclease T1 from 61 of liquid culture in 3 days. We also report a new absorption coefficient for RNase T1: E1%278 nm = 15.4.     

Characterization of ribonuclease HII from Escherichia coli overproduced in a soluble form

Escherichia coli RNase HII is composed of 198 amino acid residues. The enzyme has been overproduced in an insoluble form, purified in a urea-denatured form, and refolded with poor yield [M. Itaya (1990) Proc. Natl. Acad. Sci. USA 87, 8587-8591]. To facilitate the preparation of the enzyme in an amount sufficient for physicochemical studies, we constructed an overproducing strain in which E. coli RNase HII is produced in a soluble form. The enzyme was purified from this strain and its biochemical and physicochemical properties were characterized. The good agreement in the molecular weights estimated from SDS-PAGE (23,000) and gel filtration (22,000) suggests that the enzyme acts as a monomer. From the far-UV circular dichroism spectrum, its helical content was calculated to be 23%. The enzyme showed Mn(2+)-dependent RNase H activity. Its specific activity determined using (3)H-labeled M13 RNA/DNA hybrid as a substrate was comparable to but slightly higher than that of the refolded enzyme, indicating that the enzyme overproduced and purified in a soluble form is more suitable for structural and functional analyses than the refolded enzyme.     

Molecular requirements for degradation of a modified sense RNA strand by Escherichia coli ribonuclease H1

The structural requirements for DNA/RNA hybrids to be suitable substrates for RNase H1 are well described; however the tolerance level of this enzyme towards modifications that do not alter the duplex conformation is not clearly understood, especially with respect to the sense RNA strand. In order to investigate the molecular requirements of Escherichia coli RNase H1 (termed RNase H1 here) with respect to the sense RNA strand, we synthesized a series of oligonucleotides containing 2'-deoxy-2'-fluoro-beta-D-ribose (2'F-RNA) as a substitute for the natural beta-D-ribose sugars found in RNA. Our results from a series of RNase H1 binding and cleavage studies indicated that 2'F-RNA/DNA hybrids are not substrates of RNase H1 and ultimately led to the conclusion that the 2'-hydroxyl moiety of the RNA strand in a DNA/RNA hybrid is required for both binding and hydrolysis by RNase H1. Through the synthesis of a series of chimeric sense oligonucleotides of mixed RNA and 2'F-RNA composition, the gap requirements of RNase H1 within the sense strand were examined. Results from these studies showed that RNase H1 requires at least five or six natural RNA residues within the sense RNA strand of a hybrid substrate for both binding and hydrolysis. The RNase H1-mediated degradation patterns of these hybrids agree with previous suggestions on the processivity of RNase H1, mainly that the binding site is located 5' to the catalytic site with respect to the sense strand. They also suggest, however, that the binding and catalytic domains of RNase H1 might be closer than has been previously suggested. In addition to the above, physicochemical studies have revealed the thermal stabilities and relative conformations of these modified heteroduplexes under physiological conditions. These findings offer further insights into the physical binding and catalytic properties of the RNase H1-substrate interaction, and have been incorporated into a general model summarizing the mechanism of action of this unique enzyme.     

Modulation of Escherichia coli DNA methyltransferase activity by biologically derived GATC-flanking sequences

Escherichia coli DNA adenine methyltransferase (EcoDam) methylates the N-6 position of the adenine in the sequence 5'-GATC-3' and plays vital roles in gene regulation, mismatch repair, and DNA replication. It remains unclear how the small number of critical GATC sites involved in the regulation of replication and gene expression are differentially methylated, whereas the approximately 20,000 GATCs important for mismatch repair and dispersed throughout the genome are extensively methylated. Our prior work, limited to the pap regulon, showed that methylation efficiency is controlled by sequences immediately flanking the GATC sites. We extend these studies to include GATC sites involved in diverse gene regulatory and DNA replication pathways as well as sites previously shown to undergo differential in vivo methylation but whose function remains to be assigned. EcoDam shows no change in affinity with variations in flanking sequences derived from these sources, but methylation kinetics varied 12-fold. A-tracts immediately adjacent to the GATC site contribute significantly to these differences in methylation kinetics. Interestingly, only when the poly(A) is located 5' of the GATC are the changes in methylation kinetics revealed. Preferential methylation is obscured when two GATC sites are positioned on the same DNA molecule, unless both sites are surrounded by large amounts of nonspecific DNA. Thus, facilitated diffusion and sequences immediately flanking target sites contribute to higher order specificity for EcoDam; we suggest that the diverse biological roles of the enzyme are in part regulated by these two factors, which may be important for other enzymes that sequence-specifically modify DNA.     

Overproduction and preliminary crystallographic study of ribonuclease H from Escherichia coli

To facilitate the preparation of ribonuclease H from Escherichia coli in an amount sufficient for crystallographic studies, we have constructed an overproduction system for the enzyme. The structural gene for the enzyme was subcloned from pSK750 (Kanaya, S., and Crouch, R. J. (1983) J. Biol. Chem. 258, 1276-1281) to make a plasmid vector pPL801, in which the gene was under the control of bacteriophage lambda PL promoter. Thermal induction of the gene accumulated the enzyme in E. coli N4830-1 to approximately 8% of the total cytosolic protein. The level of production of the enzyme in N4830-1 harboring pPL801 was 14 mg/liter culture, which was 3000 times as high as that in the host cell. The enzyme was purified with a yield of more than 80% and crystallized by utilizing the property that the solubility of the enzyme decreased at pH values close to its isoelectric point (pI = 9). Crystals were grown by successive seeding (hanging drop method) for x-ray crystallographic analysis. The crystals belong to space group P212121 with unit cell dimensions of alpha = 44.1 A, b = 87.0 A, c = 35.5 A and contain one molecule in an asymmetric unit. They diffracted x-rays beyond 2.5 A resolution.     

Removal of deoxyinosine from the Escherichia coli chromosome as studied by oligonucleotide transformation

Deoxyinosine (dI) is produced in DNA by the hydrolytic or nitrosative deamination of deoxyadenosine. It is excised in a repair pathway that is initiated by endonuclease V, the product of the nfi gene. The repair was studied in vivo using high-efficiency oligonucleotide transformation mediated by the Beta protein of bacteriophage lambda in a mismatch repair-deficient host. Escherichia coli was transformed with oligonucleotides containing a selectable A-G base substitution mutation. When the mutagenic dG was replaced by a dI in the oligonucleotide, it lost 93-99% of its transforming ability in an nfi(+) cell, but it remained fully functional in an nfi mutant. Therefore, endonuclease V is responsible for most of the removal of deoxyinosine from DNA. New nfi mutants were isolated based on the strong selection provided by their tolerance for transformation by dI-containing DNA. The repair patch for dI was then measured by determining how close to the transforming dG residue a dI could be placed in the oligonucleotide before it interferes with transformation. At the endonuclease V cleavage site, three nucleotides were preferentially removed from the 3' end and two nucleotides were removed from the 5' end. dI:dT and dI:dC base pairs gave the same results. Caveats include possible interference by Beta protein and by mispaired bases. Thus, oligonucleotide transformation can be used to determine the relative importance of redundant repair pathways, to isolate new DNA repair mutants, and to determine with high precision the sizes of repair tracts in intact cells.