Larry Snyder

Phage-exclusion enzymes: a bonanza of biochemical and cell biology reagents?

Many parasitic DNA elements including prophages and plasmids synthesize proteins that kill the cell after infection by other phages, thereby blocking the multiplication of the infecting phages and their spread to other nearby cells. The only known function of these proteins is to exclude the infecting phage, and therefore to protect their hosts, and thereby the DNA elements themselves, against phage contagion. Many of these exclusions have been studied extensively and some have long been used in molecular genetics, but their molecular basis was unknown. The most famous of the phage exclusions are those caused by the Rex proteins of λ prophage. The Rex exclusions are still not completely understood, but recent evidence has begun to lead to more specific models for their action. One of the Rex proteins, RexA, may be activated by a DNA‐protein complex, perhaps a recombination or replication intermediate, produced after phage infection. In the activated state, RexA may activate RexB, which has been proposed to be a membrane ion channel that allows the passage of monovalent cations, destroying the cellular membrane potential, and killing the cell. We now understand two other phage exclusions at the molecular level which use strategies that are remarkably similar to each other. The parasitic DNA elements responsible for the exclusions both constitutively synthesize enzymes that are inactive as synthesized by the DNA element but are activated after phage infection by a short peptide determinant encoded by the infecting phage. In the activated state, the enzymes cleave evolutionarily conserved components of the translation apparatus, in one case EF‐Tu, and in the other case tRNALys. Translation is blocked and development of the phage is arrested. A myriad of different phage‐exclusion systems are known to exist and many of these may also be specific for highly conserved cellular components, furnishing generally useful enzymes for biochemical and biomedical research.

The optional E. coli prr locus encodes a latent form of phage T4-induced anticodon nuclease.

The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4‐induced anticodon nuclease, an enzyme which triggers the cleavage‐ligation of the host tRNALys. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr‐associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4‐encoded factors of anticodon nuclease counteract the prr‐encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage‐ligation pathway by two separate genetic systems which cohabitate E. coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins.

DNA supercoiling in vivo

DNA topoisomerase mutants of Escherichia coli and Saccharomyces cerevisiae were used to study the topological state of intracellular DNA. In E. coli, it is shown that switching off the gene topA encoding DNA topoisomerase I leads to an increase in the degree of negative supercoiling of intracellular DNA and inhibition of the growth of the cells: a d(pCpG)16.d(pCpG)16 sequence on a plasmid is also shown to flip from a right-handed B-helical structure to a left-handed Z-helical structure in vivo when topA is switched off. In S. cerevisiae, the topological state of intracellular DNA is little affected by the cellular levels of the topoisomerases.

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

Abstract Two bacteriophage T4-induced, nucleic acid-modifying activities, 5′ polynucleotide kinase and 3′ phosphatase, are both coded by the pse T 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 pse T gene, making it non-essential for phage multiplication on most laboratory strains of Escherichia coli .

Genetic and physiological studies of the role of the RNA ligase of bacteriophage T4.

Abstract The product of bacteriophage T4 gene 63 has two activities, one which catalyzes the attachment of tail fibers to base plates during morphogenesis (TFA) and one which catalyzes the joining of single-stranded polynucleotides (RNA ligase). The only phenotype attributed to mutations in gene 63 is a defect in attachment of tail fibers leading to fiberless T4 particles. However, it is suspected that TFA and RNA ligase are unrelated activities of the same protein since they have very different requirements in vitro . We have isolated new mutants which have lost the RNA ligase but have retained the TFA activity of the product of gene 63. These mutants exhibit defects in T4 DNA replication and late gene expression in some strains of Escherichia coli . This work allows us to draw three conclusions: (1) the TFA and RNA ligase activities are unrelated functions of the gene 63 product making this the prototype for a protein which has more than one unrelated function; (2) the RNA ligase is probably involved in DNA metabolism rather than RNA processing as has been proposed: (3) the RNA ligase and polynucleotide 5′ kinase 3′ phosphatase of T4 perform intimately related functions.

A site in the T4 bacteriophage major head protein gene that can promote the inhibition of all translation in Escherichia coli

The cryptic DNA element, e14, synthesizes a protein, Lit, which can inhibit gene expression late in T4 bacteriophage development. This inhibition is due to the interaction between the Lit protein and a short region, the gol region, within gene 23, the major head protein gene of phage T4. We have constructed plasmids in which the gol region is transcribed from the lac promoter and fused translationally and transcriptionally to lacZ and cat (chloramphenicol acetyltransferase). These fusion plasmids were used to demonstrate that, in the presence of Lit protein, the gol region inhibits the expression of genes downstream in the same transcription unit. This local inhibition does not require the gene 23 polypeptide from the gol region. In addition, inducing the transcription and translation of the gol region in the presence of Lit protein causes an immediate global inhibition of all translation in Escherichia coli. This global inhibition does require the gene 23 polypeptide. No more than 75 base-pairs of DNA from the gol region are required for both the local and global inhibitions. The gol region sequence contains a short dyad symmetry. However, it is the sequence of bases in the region of dyad symmetry and not the ability to form a hairpin in the RNA that is required for gol region activity.

Phage and host genetic determinants of the specific anticodon loop cleavages in bacteriophage T4-infected Escherichia coli CTr5X☆

Anticodon loop cleavages of two host tRNA species occur in bacteriophage T4-infected Escherichia coli CTr5X, a host strain restricting phage mutants deficient in polynucleotide kinase (pnk) or RNA ligase (rli). The cleavage products accumulate with the mutants but are further processed in wt infection through polynucleotide kinase and RNA ligase reactions. Inactivating mutations in stp suppress pnk- or rli- mutations in E. coli CTr5X and, as shown here, also abolish the anticodon nuclease, implicating the stp product with this activity. We show also that there exist other suppressing mutations of a pnk- (pseT2) mutation that appear not to affect the anticodon nuclease and are not in stp. It has been shown that a single locus in E. coli CTr5X, termed prr, determines the restriction of pnk- or rli- mutants. A transductant carrying prr featured upon infection the anticodon nuclease reaction products, suggesting that prr determines the specific manifestation of this activity. However, prr does not encode the tRNA species that are vulnerable to the anticodon nuclease.

Regulation of anti-late RNA synthesis in bacteriophage T4: A delayed early control☆

Abstract Bacteriophage T4 make mutually complementary RNA transcripts. The antisense RNA is made in the late region but at early times after infection. It is complementary in base sequence to late messenger RNA and, hence is called anti-late RNA. We have studied some of the physical characteristics and possible regulatory mechanisms involved in the synthesis of these unique early RNA species. Anti-late RNA sediments on 5% to 20% sucrose gradients with an average sedimentation coefficient of 20 to 22 S, comparable to that of late mRNA. The fact that anti-late synthesis becomes refractory to rifampicin before one minute after infection, suggests that it occurs from a class of immediate early promoters in the late regions. Anti-late RNA production is examined under several different conditions of altered early gene expression. In all circumstances where the delayed early gene transcription is altered, anti-late synthesis is also altered. From the data presented here, we postulate that anti-late RNA synthesis is controlled by the same mechanism as that regulating delayed early genes.

The gol site: A Cis-acting bacteriophage T4 regulatory region that can affect expression of all the T4 late genes☆

Abstract We have shown that mutations in the Escherichia coli lit gene can prevent the expression of the late genes of bacteriophage T4 at temperatures below 34 °C. The defect in late gene expression occurs, at least partially, at the level of transcription, and neither DNA replication nor DNA encapsidation into phage heads is significantly affected. Rare T4 “ gol ” mutations overcome the defect in late transcription. Refined mapping experiments place gol mutations within gene 23, but an altered gene 23 protein is not responsible for the phenotype. Rather, gol mutations seem to alter a cis -acting site in T4 DNA, the wild-type form of which interferes with late transcription in lit − hosts.