Arthur Landy

Duplicate genes for tyrosine transfer RNA in Escherichia coli

Abstract Genetic and biochemical studies of Escherichia coli and the new transducing phage φ80psuIII+ have been used to characterize the tRNA genes of E. coli. The transducing phage stimulates the production of both suIII+ and suIII− tyrosine tRNA upon infection, and in hybridization experiments its DNA is saturated with 1.4 tyrosine tRNA molecules per genome. One of its derivatives, selected for its genetic properties, stimulates only suIII+ tyrosine tRNA, and its DNA is saturated by 0.6 tyrosine tRNA molecule per genome. We conclude that the original phage carries two tyrosine tRNA genes, one suIII+ and one suIII−, while the derivative carries a single suIII+ gene. The single-gene derivative apparently arises by unequal recombination involving the two genes of the original phage; the reciprocal recombination product, carrying three tyrosine tRNA genes, is also detected. Entirely analogous single-gene and three-gene derivatives of E. coli are found, and we conclude that E. coli normally carries a pair of closely-linked genes specifying its minor, or suIII tyrosine tRNA.

Cellular factors couple recombination with growth phase: Characterization of a new component in the λ site-specific recombination pathway

Here we characterize FIS (factor for inversion stimulation), a new cellular component of the lambda site-specific recombination pathway. This host protein binds to a specific region in the lambda attP overlapping the Xis binding sites and can bind cooperatively with Xis to these sites. FIS stimulates lambda excision up to 20-fold in vitro in the presence of suboptimal Xis concentrations, but has no effect in the presence of saturating Xis; FIS has no effect on integrative recombination. FIS can replace one Xis molecule in a series of cooperative and competitive interactions but cannot carry out excision in the absence of Xis. FISs role in the regulation of recombination has been inferred from in vivo modification of DNA. In exponentially growing cells the lambda FIS site is fully occupied, whereas in stationary-phase cells this binding site is vacant.

A structural basis for allosteric control of DNA recombination by lambda integrase.

Site-specific DNA recombination is important for basic cellular functions including viral integration, control of gene expression, production of genetic diversity and segregation of newly replicated chromosomes, and is used by bacteriophage λ to integrate or excise its genome into and out of the host chromosome. λ recombination is carried out by the bacteriophage-encoded integrase protein (λ-int) together with accessory DNA sites and associated bending proteins that allow regulation in response to cell physiology. Here we report the crystal structures of λ-int in higher-order complexes with substrates and regulatory DNAs representing different intermediates along the reaction pathway. The structures show how the simultaneous binding of two separate domains of λ-int to DNA facilitates synapsis and can specify the order of DNA strand cleavage and exchange. An intertwined layer of amino-terminal domains bound to accessory (arm) DNAs shapes the recombination complex in a way that suggests how arm binding shifts the reaction equilibrium in favour of recombinant products.

Mechanistic and structural complexity in the site-specific recombination pathways of Int and FLP

This review focuses on two of the approximately 30 members of the diverse Int family of site-specific recombinases. The lambda recombination system represents those reactions involving accessory proteins and a complex higher-order structure. The FLP system represents the most streamlined reactions and has been the subject of detailed and informative studies on the mechanisms of DNA cleavage and ligation.

DNA Looping Generated by DNA Bending Protein IHF and the Two Domains of Lambda Integrase

The multiprotein-DNA complexes that participate in bacteriophage lambda site-specific recombination were used to study the combined effect of protein-induced bending and protein-mediated looping of DNA. The protein integrase (Int) is a monomer with two autonomous DNA binding domains of different sequence specificity. Stimulation of Int binding and cleavage at the low affinity core-type DNA sites required interactions with the high affinity arm-type sites and depended on simultaneous binding of the sequence-specific DNA bending protein IHF (integration host factor). The bivalent DNA binding protein is positioned at high affinity sites and directed, by a DNA bending protein, to interactions with distant lower affinity sites. Assembly of this complex is independent of protein-protein interactions.

Site-specific Recombination in Phage Lambda

INTRODUCTION Cells lysogenic for λ carry a quiescent form of the viral chromosome called prophage. The prophage differs from the DNA of viral particles in two important ways: (1) The ends of the prophage and of the viral particle DNA are at different points in the nucleotide sequence, and (2) the prophage ends are covalently joined to host DNA. Campbell (1962) proposed that viral particle DNA is converted to prophage by the joining of the ends followed by insertion of the resulting circular molecule into the host DNA. Insertion occurs by reciprocal recombination at specific sites (attachment or att sites) in each chromosome (Fig. 1). This proposal has received extensive experimental support and, indeed, was generally accepted when the first edition of this book was written (see Gottesman and Weisberg 1971). A stable lysogen is formed when an infecting viral particle succeeds both in repressing lytic functions and in inserting its DNA. The inserted prophage is then replicated as part of the bacterial chromosome. In the rare event that repression is not followed by insertion (abortive lysogeny), the viral chromosome cannot replicate and so is lost by dilution as the host cell divides. Excision of the prophage from the chromosome is rare during normal bacterial growth but occurs readily following repressor inactivation. If the repressor is inactivated only briefly (abortive or transient derepression), prophage excision can occur without lytic growth. The excised DNA is then frequently lost as the cell divides (prophage curing). Intricate controls ensure that insertion occurs only...

Patterns of λ int recognition in the regions of strand exchange

Int protein has two classes of binding sites within the phage att site: the arm-type recognition sequences are found in three specific sites that are distant from the region of strand exchange; the junction-type recognition sequences occur as inverted pairs around the crossover region in both attP and attB. During recombination between attP and attB each of the four DNA strands is cut at a homologous position within each of the junction-type Int binding sites. In all four junction-type sites Int protein interacts primarily with the same face of the DNA helix, as determined by those purine nitrogens that are protected against methylation by dimethylsulfate. Efficient secondary attachment sites for λ contain sequences with partial homology to the junction-type binding sites. In addition, the sequence between, but not part of, the two junction-type sites (the overlap region) is strongly conserved in secondary att sites. Thus, in the vicinity of strand exchange, attP and a recombining partner, such as attB, are very similar; each comprises two junction-type Int recognition sites and an overlap (crossover) region.

Mutant tyrosine transfer ribonucleic acids.

Three independent mutants of the suIII tyrosine suppressor transfer RNA gene have been isolated. These mutants are shown to produce mutant tRNAs differing from the wild-type suIII+ tRNA molecule in each case by a single base change. A mutant tRNA containing an A residue in place of a G in the “dihydrouracil loop” appears to be defective in a step in protein synthesis occurring after the acylated tRNA is bound to the ribosome. A mutant tRNA having an A residue in place of a G in the “anticodon stem” appears to be defective exclusively in its apparent affinity for the tyrosyl tRNA synthetase. The isolation of mutant tRNAs as well as their biological properties are discussed.

Role for DNA homology in site-specific recombination: The isolation and characterization of a site affinity mutant of coliphage λ*

Site-affinity (or saf) mutations change the specificity of prophage insertion. We have isolated a saf mutation of the bacteriophage lambda attachment site by inserting the phage chromosome into and then excising it from a secondary host attachment site. This causes reciprocal exchange of two seven base-pair segments (the overlap regions) that lie within the cores of the two sites. Since the two overlap regions differ from each other in nucleotide sequence, the recombinant sites are mutants. We have determined the effect of overlap region homology on recombination. We found that homology promotes integrative and excisive recombination. This suggests that the two overlap regions interact directly during recombination. The pattern of segregation of the saf mutation during site-specific recombination shows that it lies to the right of the point of genetic exchange about 95% of the time. This is a surprising result because lambda integrative recombination normally occurs by two staggered, reciprocal single-strand exchanges, one at each edge of the overlap region (Mizuuchi et al., 1981). Since saf lies within the overlap region, we might have expected that the point of genetic exchange would occur to the left of saf as often as to the right. We offer two models to account for this. (1) The mutation alters the location of one of the single-strand exchange points. (2) Efficient and strand-specific processing of mismatched base-pairs changes the expected segregation pattern.

Specific hybridization of tyrosine transfer ribonucleic acids with DNA from a transducing bacteriophage φ80 carrying the amber suppressor gene suIII

Abstract Transducing phages of φ80 have been isolated which carry the su III gene. We have used the technique of DNA-RNA hybridization to test the hypothesis that a tRNA Tyr molecule is the direct product of this gene. The following results strongly support this hypothesis: 1. (1) Upon infection of Escherichia coli by φ80 d su III , there is a tenfold increase in the fraction of the tRNA which will hybridize with φ80 d su III DNA. This is to be compared with a similar increase in the fraction of tRNA which will accept tyrosine following infection. 2. (2) Saturation experiments show that there is a single nucleotide sequence (a gene) which will hybridize with tRNA. 3. (3) As tRNA is purified for tyrosine-accepting activity, there is an increase in the fraction of the tRNA which will hybridize with φ80 d su III DNA. This has been shown both in saturation and in competition experiments. Our view of suppression dictates that there be at least two genes specifying tyrosine tRNAs in E. coli . One of the tRNAs, the major species, is not involved in suppression; the other, a minor species, recognizes the normal tyrosine codons in the su − cell, and UAG, a chain-terminating codon in the su + III cell. The present evidence shows that these two tRNA molecules must be very similar, as they both hybridize efficiently with φ80 d su III DNA.