Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1

Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA ligase 1 has a temperature optimum of 60–64°C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.     

Isolation of highly purified RNA ligase from bacteriophage T4

A technique for isolation of RNA-ligase of bacteriophage T4 was proposed. It is mainly based on the using of Soviet materials and sorbents and includes seven purification stages. The technique enables to isolate about 80 000 units of active enzyme from 100 g of E. coli B cells infected with the phage. T4am N82; that makes up 20% of the activity of the cell extract. The obtained preparations of RNA-ligase are homogeneous by the data of electrophoresis and practically, free of endo- and exonuclease admixtures.     

Purification and properties of the bacteriophage T4 gene 61 RNA priming protein

The bacteriophage T4 gene 61 protein is required, together with the gene 41 protein and single-stranded DNA, for the synthesis of the pentaribonucleotides that are used as primers for the start of each new Okazaki DNA fragment during T4 DNA replication. Using this priming activity as an assay, we have purified the 61 protein to essential homogeneity in milligram amounts. The priming activity was identified with the product of T4 gene 61 by using two-dimensional polyacrylamide gel electrophoresis to compare all of the T4-induced proteins in wild-type and mutant infections; the purified protein co-migrates with the only detectable protein missing in a 61- mutant infection. The purified 61 protein is shown to bind to the T4 helix-destabilizing protein (gene 32 protein) and to both single-stranded and double-stranded DNA. We have failed to detect any ribonucleotide polymerizing activity in either the 61 protein or the 41 protein alone; both the 61 and 41 proteins must be present to observe any synthesis of oligoribonucleotides.     

RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2

Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3'-OH/5'-PO(4) nick in a double-stranded RNA or an RNA.DNA hybrid. The critical role of the template strand in approximating the reactive 3'-OH and 5'-PO(4) termini is underscored by the drastic reductions in the RNA-sealing activity of Rnl2 when the duplex substrates contain gaps or flaps instead of nicks. RNA nick joining requires ATP and a divalent cation cofactor (either Mg or Mn). Neither dATP, GTP, CTP, nor UTP can substitute for ATP. We identify by alanine scanning seven functionally important amino acids (Tyr-5, Arg-33, Lys-54, Gln-106, Asp-135, Arg-155, and Ser-170) within the N-terminal nucleotidyl-transferase domain of Rnl2 and impute specific roles for these residues based on the crystal structure of the AMP-bound enzyme. Mutational analysis of 14 conserved residues in the C-terminal domain of Rnl2 identifies 3 amino acids (Arg-266, Asp-292, and Glu-296) as essential for ligase activity. Our findings consolidate the evolutionary connections between bacteriophage Rnl2 and the RNA-editing ligases of kinetoplastid protozoa.     

Purification of histidine-tagged T4 RNA ligase from E. coli

Here we report the construction of a histidine-tagged T4 RNA ligase expression plasmid (pRHT4). The construct, when overexpressed in BL21 (DE3) cells, allows the preparation of large quantities of T4 RNA ligase in high purity using only a single purification column. The histidine affinity tag does not inhibit enzyme function, and we were able to purify 1-3 mg pure protein/g cell pellet. A simple purification procedure ensures that the enzyme is de-adenylated to levels comparable to those found for many commercial preparations. The purified protein has very low levels of RNase contamination and functioned normally in a variety of activity assays.     

Purification and various properties of exonuclease from bacteriophage T4

Exonuclease A was isolated from bacteriophage T4-infected cells of E. coli. The molecular mass of the enzyme is approximately 42,000 Da, pH optimum is 7-8.5, pI is 4.05. The enzyme activity depends on Mg2+, the optimal concentration of Mg2+ being 1-5 mM. The enzyme splits one- and two-helical DNA in the direction of 3'----5' and is a deoxyribonuclease splitting 5'-deoxynucleotides. The enzyme shows a practically equal affinity for one and two-helical DNA. The Km value for one- and two-helical DNA is 10 +/- 1 and 11 +/- 1 pmole of chain DNA, respectively. The Vmax value for one- and two-helical DNA is 61 +/- 5 and 45 +/- 5 pmole of nucleotides per min. Exonuclease A may be used for preparing substrates for DNA-polymerase T4 and Klenow fragment, i.e., during labeling of DNA at 3'-ends.     

Kinetic characteristics of the ATP-pyrophosphate isotope exchange catalyzed by RNA ligase from T4 bacteriophage

The dependence of initial rate v0 of ATP--PPi exchange reaction catalyzed by RNA-ligase of bacteriophage T4 on the concentration of ATP(s), pyrophosphate (z) and Mgcl2 has been determined. The dependence of v0 on s and z described by the equation v0 = k-1k2E0/(k-1 + K2) (1 + K1/s + k2/z) has been obtained for the reaction of E + S in equilibrium ES in equilibrium E1 + Z, where E--enzyme, E1--adenylylenzyme, S--ATP, Z--pyrophosphate, K1 and K2--constants of equilibrium, k-1, k2--velocity constants of transition of ES to E + S and E1 + Z, E0--complete concentration of enzyme. The low inhibition of the ATP--PPi exchange by the acceptor A(pA)2 and donors pAp, p(Ap)3, pCp has been shown. The dependence of v0 on the concentration of MgCl2 is consent with the incorporation of only dimagnesium salts of substrates in the isotope-exchange reaction.     

Purification and characterization of bacteriophage N4-induced DNA polymerase

Coliphage N4 replication is independent of most host DNA replication functions except for the 5'----3' exonuclease activity of polA, DNA ligase, DNA gyrase, and ribonucleotide reductase (Guinta, D., Stambouly, J., Falco, S. C., Rist, J. K., and Rothman-Denes, L. B. (1986) Virology 150, 33-44). It is therefore expected that N4 codes for most of the functions required for replication of its genome. In this paper we report the purification of the N4-coded DNA polymerase from N4-infected cell extracts by following its activity on a gapped template and in an in vitro complementation system for N4 DNA replication (Rist, J. K., Pearle, M., Sugino, A., and Rothman-Denes, L. B. (1986) J. Biol. Chem. 261, 10506-10510). The enzyme is composed of one polypeptide, Mr 87,000. It is most active on templates containing short gaps synthesizing DNA with high fidelity in a quasi-processive manner. A strong 3'----5' exonuclease activity is associated with the DNA polymerase polypeptide. No 5'----3' exonuclease or strand-displacing activities were detected.     

Competition between bacteriophage f2 RNA and bacteriophage T4 messenger RNA

In an $\text{Escherichia coli}$ cell-free protein synthesis assay, mRNA isolated from cells late after infection by phage T4 out-competes bacteriophage f2 RNA. Addition of a saturating or subsaturating amount of T4 mRNA inhibits translation of f2 RNA, while even an excess of f2 RNA has no effect on translation of T4 mRNA. Peptide mapping of reaction products labeled with formyl-[³⁵S]-methionyl-tRNA was used to quantitate f2 and T4 protein products synthesized in the same reaction. We suggest that messenger RNA competition might be one mechanism by which T4 superinfection of cells infected with phage f2 blocks translation of f2 RNA and possibly host mRNA.     

Sequence and cloning of bacteriophage T4 gene 63 encoding RNA ligase and tail fibre attachment activities

The sequence of gene 63 of bacteriophage T4 was determined by a shotgun approach. Small DNA fragments, derived by sonication of a restriction fragment that encompasses the region of gene 63, were cloned in M13 vectors and sequenced by the 'dideoxy' method. The position of the gene was established by comparison with the sequence of a gene 63 amber mutant. Knowledge of the DNA sequence of gene 63 and surrounding regions has allowed the construction of a clone of gene 63 in which RNA ligase production is under the control of the lac promoter of bacteriophage M13mp8. Infected E. coli cells can be induced to produce a protein indistinguishable from commercially available RNA ligase.     

RNA ligase from bacteriophage T4. VIII. Solid phase enzymatic synthesis of oligoribonucleotides]

A method of the solid-phase enzymic synthesis of oligoribonucleotides has been suggested. The donor is fixed through its 3'-end on a water-insoluble matrix followed by the stepwise RNA ligase- and T4 polynucleotide kinase-assisted coupling of trinucleoside diphosphates in the 5'-direction. As an example, (pA)6pAox was immobilised on Biogel P-300 hydrazine and the RNA ligase-catalyzed addition of acceptor ApApA to the donor gave (Ap)9 with the 50% yield.     

Purification and properties of a DNA-dependent ATPase induced by bacteriophage T4.

A DNA-dependent ATPase formed after T4 phage infection is purified to apparent homogeneity. The molecular weight of the purified enzyme is 50 000 when determined by glycerol gradient centrifugation and by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The enzyme at an earlier stage in purification (prior to DEAE-cellulose chromatography) exists as a complex with a molecular weight of 100000. However, molecular weight determinations by Sephadex gel chromatography give considerably decreased molecular weights for the complex and for the enzyme after DEAE-cellulose chromatography. The enzyme is stimulated to varying degrees by a variety of single-stranded polydeoxyribonucleotides or by single-stranded DNA, but no chemical change in the polynucleotide has been detected as a result of the enzyme action.     

Structure-function analysis of T4 RNA ligase 2

Bacteriophage T4 RNA ligase 2 (Rnl2) exemplifies a polynucleotide ligase family that includes the trypanosome RNA-editing ligases and putative RNA ligases encoded by eukaryotic viruses and archaea. Here we analyzed 12 individual amino acids of Rnl2 that were identified by alanine scanning as essential for strand joining. We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of ligase adenylylation and phosphodiester bond formation. The essential residues of Rnl2 are located within conserved motifs that define a superfamily of nucleotidyl transferases that act via enzyme-(lysyl-N)-NMP intermediates. Our mutagenesis results underscore a shared active site architecture in Rnl2-like ligases, DNA ligases, and mRNA capping enzymes. They also highlight two essential signature residues, Glu(34) and Asn(40), that flank the active site lysine nucleophile (Lys(35)) and are unique to the Rnl2-like ligase family.     

Biotin and fluorescent labeling of RNA using T4 RNA ligase.

Biotin, fluorescein, and tetramethylrhodamine derivatives of P1-(6-aminohex-1-yl)-P2-(5'-adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase. In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3'-hydroxyl of an RNA acceptor to form a phosphodiester bond and the AMP portion is released. E. coli and D. melanogaster 5S RNA, yeast tRNAPhe, (Ap)3C, and (Ap)3A serve as acceptors with yields of products varying from 50 to 100%. Biotin-labeled oligonucleotides are bound selectively and quantitatively to avidin-agarose and may be eluted with 6 M guanidine hydrochloride, pH 2.5. Fluorescein and tetramethylrhodamine-labeled oligonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor. The diverse structures of the appended groups and of the chain lengths and compositions of the acceptor RNAs show that T4 RNA ligase will be a useful modification reagent for the addition of various functional groups to the 3'-terminus of RNA molecules.