Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP.

NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP. The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement. The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop. The second domain, consisting of five alpha-helices, forms the NMP binding pocket. A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding. Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination.     

Bacteriophage T4 deoxynucleotide kinase: gene cloning and enzyme purification.

Gene 1 of bacteriophage T4 has been cloned into a lambda pL expression vector, resulting in the overproduction of deoxynucleotide kinase. A procedure that includes affinity chromatography on Cibacron Blue F3GA-agarose has been used to purify milligram quantities of enzymes from a 2-liter culture. The enzyme has been partially characterized in vitro and in vivo, and it appears to be identical to the deoxynucleotide kinase isolated from T4-infected Escherichia coli. These results prove the earlier contention that the phosphorylation of three dissimilar deoxynucleotides (5-hydroxymethyldeoxycytidylate, dTMP, and dGMP), to the exclusion of most others, is catalyzed by a single protein.     

The distribution of functional bacteriophage T4 mRNA on polysomes.

Functional bacteriophage T4 deoxynucleotide kinase and α-glucosyl transferase mRNAs can be isolated from polysomes extracted from cells 8 min after infection. At least 55% of the 8-min deoxynucleotide kinase mRNA is associated with polysomes and is released from the cell membrane by deoxyribonuclease (DNase) treatment (soluble mRNA). Approximately 20% of the kinase mRNA remains tightly bound to membrane after DNase treatment (membrane mRNA) and 25% of the kinase mRNA is routinely lost during fractionation. The membrane-bound kinase mRNA is about three times as stable in vitro as the soluble kinase mRNA. Soluble kinase mRNA (14.5S) is found associated with as few as one ribosome and as many as 22 ribosomes; however, 14.5S α-glucosyl transferase mRNA is found predominantly in six ribosome polysomes. The size of the α-glucosyl transferase mRNA is heterogenous, ranging between 14.5 and 20S. The larger α-glucosyl transferase mRNAs are never found on small polysomes but appear only in polysomes containing at least nine ribosomes (18S α-glucosyl transferase mRNA). Maximum-size α-glucosyl transferase mRNA (approximately 20S) appears on polysomes containing at least 14 ribosomes. The relationships between decay of T4 mRNA and polysome size and the location of ribosome loading sites on the 20S α-glucosyl transferase message are also discussed.     

Gamma-T4 hybrid bacteriophage carrying the thymidine kinase gene of bacteriophage T4.

Among 32 lambda-T4 recombinant phages selected for growth on a thymidylate synthetase-deficient (thyA) host, 2 were shown to carry the T4 thymidine kinase (tk) gene. The lambda-T4tk phages contain two T4 HindIII DNA fragments (2.0 and 1.5 kilobases) that hybridize to restriction fragments of T4 DNA, encompassing the tk locus at 60 kilobases on the T4 map. The T4tk insert compensates for the simultaneous host deficiencies of thymidine kinase and thymidylate synthetase in a thymidine kinase-deficient (tdk) host growing in the presence of fluorodeoxyuridine when provided with thymidine and uridine. The lambda-T4tk hybrid phages specified five polypeptides with Mrs of 22,000 (22K), 21K, 14K, 11K, and 9K.     

Polynucleotide kinase mutant of bacteriophage T4

Summary We report the isolation and biological properties of two T4 phage mutants which are deficient in polynucleotide kinase. These two mutants are wild type with respect to replication, genetic recombination and DNA repair (as measured by sensitivity to UV irradiation, -irradiation and ³²P decay). These negative results suggest that this phage-induced enzyme is not a critical factor in DNA metabolism.     

Processing of bacteriophage T4 tRNAs. The role of RNAase III

In order to assess the contribution of the processing enzyme RNAase III to the maturation of bacteriophage T4 transfer RNA, RNAase III⁺ and RNAase III^? strains were infected with T4 and the tRNAs produced were analyzed. Infection of the RNAase III⁺ strains of Escherichia coli with T4Δ27, a deletion strain missing seven of the ten genes in the T4 tRNA cluster, results in the appearance of a transient 10.1 S RNA molecule as well as the three stable RNAs encoded by T4Δ27, species 1, rRNA^Leu and tRNA^Gln. Infection of an RNAase III^? strain results in the appearance of a larger, transient RNA molecule, 10.5 S, and a severe reduction in the accumulation of tRNA^Gln. The 10.5 S RNA is similar to 10.1 S RNA but contains extra nucleotides (about 50) at the 5′ end. (10.1 S contains all the three final molecules plus about 70 extra nucleotides at the 3′ end.) Both 10.5 S and 10.1 S RNAs can be processed in vitro into the three final molecules. When 10.1 S is the substrate, the three final molecules are obtained whether extracts of RNAase III⁺ or RNAase III^? cells are used. However, when 10.5 S is the substrate RNAase III⁺ extracts bring out normal maturation, while using RNAase III^? extracts the level of tRNA^Gln is severely reduced. When 10.5 S is used with RNAase III⁺ extracts maturation proceeds via 10.1 S RNA, while when RNAase III^? extracts were used 10.1 S is not detected. The 10.5 S RNA can be converted to 10.1 S RNA by RNAase III in a reaction which produces only two fragments. The sequence at the 5′ end of the 10.5 S suggests a secondary structure in which the RNAase III cleavage site is in a stem. These experiments show that the endonucleolytic RNA processing enzyme RNAase III is required for processing at the 5′ end of the T4 tRNA cluster where it introduces a cleavage six nucleotides proximal to the first tRNA, tRNA^Gln, in the cluster.     

The role of structural changes in T4 bacteriophage tail proteins

Conformational changes in bacteriophage tail proteins after heating and ionic strength alteration leading to dissociation of tail sheath have been studied using protein fluorescence, differential scanning microcalorimetry and electron microscopy methods. Autonomous structural changes in tube-baseplate proteins have been revealed. They take place under the same conditions as those which release the bonds holding the sheath protein subunits to those of the tube in isolated sheathed tails. The conformational changes in the tube-baseplates are reversible similarly to the process of assembly and disassembly of the extended sheath. Morphological changes in the tube have been found at the temperature above the transition registered by protein fluorescence but not by calorimetry. This suggests that revealed spectral alterations reflect changes in quaternary structure of tail tube in particular.     

A role in true-late gene expression for the T4 bacteriophage 5' polynucleotide kinase 3' phosphatase.

Two bacteriophage T4-induced, nucleic acid-modifying activities, 5′ polynucleotide kinase and 3′ phosphatase, are both coded by the pseT gene. Therefore, the product of this gene is an enzyme which can remove phosphates from 3′ termini and add them to 5′-hydroxyl termini and thus could be said to “shuttle” phosphates on polynucleotides. This enzyme is sometimes required for T4 true-late gene expression, probably by helping establish the required intracellular DNA structure. Our data suggest that a host gene product normally can substitute for the product of the pseT gene, making it non-essential for phage multiplication on most laboratory strains of Escherichia coli.     

Recognition of DNA substrates by T4 bacteriophage polynucleotide kinase

T4 phage polynucleotide kinase (PNK) displays 5′-hydroxyl kinase, 3′-phosphatase and 2′,3′-cyclic phosphodiesterase activities. The enzyme phosphorylates the 5′ hydroxyl termini of a wide variety of nucleic acid substrates, a behavior studied here through the determination of a series of crystal structures with single-stranded (ss)DNA oligonucleotide substrates of various lengths and sequences. In these structures, the 5′ ribose hydroxyl is buried in the kinase active site in proper alignment for phosphoryl transfer. Depending on the ssDNA length, the first two or three nucleotide bases are well ordered. Numerous contacts are made both to the phosphoribosyl backbone and to the ordered bases. The position, side chain contacts and internucleotide stacking interactions of the ordered bases are strikingly different for a 5′-GT DNA end than for a 5′-TG end. The base preferences displayed at those positions by PNK are attributable to differences in the enzyme binding interactions and in the DNA conformation for each unique substrate molecule.     

The role of the bacteriophage T4 gene 32 protein in homologous pairing

The gene 32 protein of the bacteriophage T4 is required for efficient genetic recombination in infected Eschericia coli cells and strongly stimulates in vitro pairing catalyzed by the phage uvsX protein, a RecA-like strand transferase. This helix-destabilizing factor is known to bind tightly and cooperatively to single-stranded DNA and to interact specifically with the uvsX protein as well as other phage gene products. However, its detailed role in homologous pairing is not well understood. I show here that when the efficiency of uvsX protein-mediated pairing is examined at different gene 32 protein and duplex DNA concentrations, a correlation between the two is found, suggesting that the two interact in a functionally important manner during the reaction. These and other data are consistent with a model in which the gene 32 protein binds to the strand displaced from the recipient duplex during pairing, thereby stabilizing the heteroduplex product. An alternative model in which the gene 32 protein replaces UvsX on the invading strand, thereby freeing the strand transferase to bind to the displaced strand, is also considered.     

Regulation of early mRNA synthesis after bacteriophage T4 infection of Escherichia coli.

Regulation of T4-specific mRNA synthesis was studied during leucine starvation of a leucine-requiring stringent Escherichia coli B strain. This was done by imposing starvation prior to T4 infection and then letting RNA synthesis proceed for different time periods. Rifampin or streptolydigin was added to stop further RNA synthesis, and protein synthesis was restored by addition of leucine. Samples were withdrawn at different times, and the enzyme-forming capacities found that, during conditions which elicit the stringent response in uninfected bacteria, immediate early mRNA is not stringently regulated. This conclusion contradicts the earlier conclusion of others, obtained by measuring incorporation of radioactive uracil; this is explained by the observation of Edlin and Neuhard (1967), confirmed and extended by us to the T4-infected cell, that the incorporation of uracil into RNA of a stringent strain is virtually blocked by amino acid starvation, whereas that of adenine continues at 30 to 50% of the rate seen in the presence of the required amino acid.     

UV-induced mutation in bacteriophage T4.

Two late gene am mutants of bacteriophage T4 that can be induced to revert by UV were crossed to a temperature-sensitive ligase mutant. In the double mutants, UV-induced reversion was eliminated at a semirestrictive temperature. When the single am mutants were irradiated and then allowed a single passage in a permissive host, the UV-induced reversion frequency was increased by 15- to 25-fold. This increased mutagenesis was also abolished by the presence of the ligase allele. When the UV-irradiated single am mutants multiply infected a permissive host, allowing multiplicity reactivation to occur, the induced reversion frequency was reduced similarly to the reduction in lethality. The mutagenesis that remained was again abolished by the presence of the ligase allele. It is concluded that UV induces mutations in phage T4 through the action of a pathway that includes polynucleotide ligase. The increase in mutation frequency after growth in a permissive host implies that mutagenesis can occur at more than one stage of the infection rather than only in an early stage before expression of the mutant genome. The process of multiplicity reactivation appears to be error-free since it overcomes lethal lesions without inducing new mutations.