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.     

Self cleavage of a precursor RNA from bacteriophage T4

We found that a precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Spl; precursor of species 1) has the capacity to cleave itself in a specific position. This cleavage is similar to a cleavage carried out by the aid of a protein, RNase F, that has been previously identified. This cleavage could lead to the maturation of an RNA (species 1) found in T4-infected E. coli cells. The reaction is time and temperature-dependent and is relatively slow as compared to the protein-dependent reaction. It requires at least a monovalent cation and is aided by non-ionic detergents. In the absence of detergent the cleavage can occur but at a reduced rate. The substrate does not contain hidden nicks and a variety of experiments suggest that it does not contain a protein. Moreover, we found no indication that the cleavage is due to contaminating nucleases in the substrate or in the reagents. The intact secondary and tertiary structures of the molecule are necessary for the cleavage to occur. The finding of a self cleaving RNA molecule has interesting evolutionary implications.     

Partial exclusion of genes of bacteriophage T2 with T4-glucosylated DNA in crosses with bacteriophage T4

A recombinant strain (D41) between phage T2 and T4 was isolated which possessed the T2 region of the genome between genes 32 and 39 and both the T4 genesgt ⁺ andgt ⁺ for glucosyltransferase. D41 was crossed with T4amber mutants in the genes for early functions and in some genes for late funcitions. The progeny of the crosses was examined for the frequency of theam ⁺ markers from D41. Genes 32, 60 and 39 in the T2 region of the recombinant strain were as sensitive to exclusion as those in standard-type T2. The T4 glucosylation of the DNA of these T2 genes did not protect them against partial exclusion by T4. However, genes in the region from gene 56 to 55 in the recombinant were resistent to exclusion. In standard-type T2 this region of the genome is sensitive to partial exclusion by T4. There are at least four exclusion sensitive sites in T2: one near gene 32, one near gene 60, one linked to gene 56 and one between genes 42 and 55.This investigation was carried out partially within the frame of the Association between Euratom and the University of Leiden, contract nr. 052-64-1-BIAN.     

RNA priming of DNA replication by bacteriophage T4 proteins

Bacteriophage T4 DNA replication proteins have been shown previously to require ribonucleoside triphosphates to initiator new DNA chains on unprimed single-stranded DNA templates in vitro. This DNA synthesis requires a protein controlled by T4 gene 61, as well as the T4 gene 41, 43 (DNA polymerase), 44, 45, and 62 proteins, and is stimulated by the gene 32 (helix-destabilizing) protein. In this paper, the nature of the RNA primers involved in DNA synthesis by the T4 proteins has been determined, using phi X174 and f1 DNA as model templates. The T4 41 and "61" proteins synthesize pentanucleotides with the sequence pppA-C(N)3 where N in positions 3 and 4 can be G, U, C, or A. The same group of sequences is found in the RNA at the 5' terminus of the phi X174 DNA product made by the seven T4 proteins. The DNA product chains begin at multiple discrete positions on the phi X174 DNA template. The characteristics of the T4 41 and "61" protein priming reaction are thus appropriate for a reaction required to initiate the synthesis of discontinuous "Okazaki" pieces on the lagging strand during the replication of duplex DNA.     

Purification and properties of bacteriophage T4-induced RNA ligase

RNA ligase has been extensively purified by a new procedure in high yield from T4-infected Escherichia coli. The enzyme consists of a single polypeptide chain of molecular weight 47,000. It catalyzes the formation of a phosphodiester bond between a 5′-PO₄-terminated oligonucleotide and a 3′-OH terminated oligonucleotide. The purified enzyme catalyzes both the intramolecular formation of single-stranded circles with longer oligonucleotides of the type pAp(Ap)_nA_?OH, where n is about 15 or greater and the intermolecular joining of pAp(Ap)₃A_OH (where the 5′-PO₄-terminated oligonucleotide is short enough to prevent apposition of its 3′ and 5′ ends) to UpUpU_OH when high concentrations of the 3′-OH-terminated acceptor oligonucleotide are present. Preparations of RNA ligase at all stages of purification show an unusual dependence of specific activity of the enzyme on the concentration of enzyme present in the assay. However, when care is taken to determine meaningful specific activities at each step, the ligase is found to be very stable during chromatography on various ion-exchange columns and may be purified by conventional techniques.     

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.     

Three suppressor forms of bacteriophage T4 leucine transfer RNA

Three suppressor forms of bacteriophage T4 leucine transfer RNA were isolated and characterized. One suppresses U-A-G mutations, another suppresses U-A-G and U-A-A mutations, while the third suppresses U-G-A mutations. Each suppressor specifies a new anticodon sequence in leucine transfer RNA. Whereas wild-type leucine transfer RNA has the anticodon sequence N-A-A (N is a modified U), the suppressor forms have C-U-A, N-U-A or N-C-A, respectively.     

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.     

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.