Mark L. Pearson

THE ULTRAVIOLET PHOTOCHEMISTRY OF THYMIDYLYL-(3'-5')-THYMIDINE.

Irradiation of 32 P-labelled TpT‡ by monochromatic light in the wavelength range 225 to 289 m μ . results in the production of four chromatographically separable photoproducts. Two isomeric dimers, T p T 1 ¯ and T p T 2 ¯ , approach an equilibrium with TpT with 2·5% in the dimer form at 225 m μ and 95% at 289 m μ . The relative yields of the dimers are 5 : 1 and independent of wavelength. One of the unknown photoproducts, TpT 4 , which has an absorption maximum at 325 m μ , is produced irreversibly from TpT at a yield comparable to the least plentiful dimer, T p T 2 ¯ . This photoproduct can in turn be converted to a second unknown, TpT 3 , by irradiation at 313 m μ . TpT 3 can be reconverted to TpT 4 by re-irradiation at 240 m μ . Since TpT 3 and TpT 4 have an extinction at 267 m μ which is about one-third of one thymine, it is likely that in both these compounds both thymine moieties are altered. None of the photoproducts is hydrolysed by venom phosphodiesterase; this also suggests that the thymine moieties in all four photoproducts are joined together. None of the photoproducts is completely stable at 85°C at pH 1 or 13. No conclusive evidence for the production of a heat-labile hydrate photoproduct of TpT has been found. The cross-sections and quantum yields for the production of photoproducts from TpT, the reversal of dimers to TpT, and the interconversion of TpT 4 and TpT 3 have been determined as a function of wavelength, using chromatographically purified photoproducts. Mathematical analysis of the dose-effect curves permits the calculation of the cross-sections.

Protein degradation in E. coli: The ion mutation and bacteriophage lambda N and cll protein stability

The Ion gene of E. coli controls the stability of two bacteriophage lambda proteins. The functional half-life of the phage N gene product, measured by complementation, is increased about 5-fold in Ion mutant strains, from 2 min to 10 min. The chemical half-life of N protein, determined by its disappearance on polyacrylamide gels following pulse-chase labeling, increases about three-fold in Ion cells. In contrast to its effect on the N protein, the Ion mutation produces a 50% decrease in the chemical half-life of cII protein. The decay rate of many other phage proteins, including the unstable gene O product, remains unaffected by a host Ion defect. A Ion mutation alters lambda physiology in two ways. First, upon infection, the phage enters the lytic pathway predominantly. This may result from the deficiency of cII protein caused by its decreased stability, since cII product is required for establishment of lysogeny. Second, brief thermal induction of a Ion (lambda c1857) lysogen leads irreversibly to lysis; repression cannot be restablished and the treated cells are committed to forming infective centers. Although N product is normally required for rapid commitment, Ion lysogens become committed more rapidly than Ion+ lysogens, even in the absence of N function. These results identify for the first time native proteins whose stability is affected by the Lon proteolytic pathway. They also indicate that the Lon system may be important in regulating gene expression in E. coli.

Suppression of hydrate and dimer formation in ultraviolet-irradiated poly (A + U) relative to poly U

Abstract [ 32 P]Poly U and poly (A + U), containing a 1:1 mixture of poly A and [ 32 P]poly U, were irradiated at 280 mμ to study the influence of secondary structure on photoproduct formation in the uracil residues. Samples were removed as a function of exposure, hydrolyzed to completion with RNase, and chromatographed to give U, H ‡ , DU and DH as the major products. The rates of formation of DU and H photoproducts in poly U and poly (A + U) were compared. The rate of hydration in poly (A + U) is suppressed by a factor of ten relative to poly U. The rate of DU formation in poly (A + U) is one-fifth that of poly U. These results provide a direct proof of the importance of secondary structure in determining the photochemical behavior of U in polynucleotides. In addition, the kinetics of product formation in poly (A + U) leads us to postulate a localized melting of hydrogen bonds in the region of dimers.

Evidence for post-transcriptional control of the morphogenetic genes of bacteriophage lambda☆

Abstract The transcription of the morphogenetic genes A to ifJ of bacteriophage λ has been measured using a two-step RNA-DNA hybridization method employing λ h λ i 80 and a set of λ gal DNAs. The results indicate that the amount of messenger RNA per unit length of DNA is constant within experimental error. No difference was found between pulse-labelled and steady-state labelled messenger RNA indicating that there is no differential chemical stability of the RNA. Taken together with previous determinations of the molar amounts of protein per gene in this region, these data indicate that the amounts of the structural proteins of λ are controlled at the level of protein synthesis or stability.

Dependence on wavelength of photoproduct yields in ultraviolet-irradiated poly U.

Abstract The yield of dimer and hydrate photoproducts in ultraviolet-irradiated 32 P-labeled poly U has been determined as a function of exposure over the wavelength range 225 to 289 mμ using a method involving ribonuclease hydrolysis and paper chromatography. The cross-sections for dimer and hydrate formation are the same as in UpU over the complete wavelength range. However, the dimers formed in poly U are not randomly spaced throughout the chain, but tend to appear in sequences of more than one dimer. As a result of this clustering of dimers, the cross-section for total dimer formation is about three times that for the formation of the product DU ‡ . These results indicate that pre-existing photoproducts can affect the rate of photochemically converting neighboring sites in a polynucleotide chain. It is speculated that the rapid formation of adjacent dimers may be due to energy transfer within the poly U random coil structure.

EXCISION OF DIMERS AND HYDRATES FROM ULTRAVIOLET-IRRADIATED POLY U BY PANCREATIC RIBONUCLEASE.

Hydrolysis of ultraviolet irradiated poly U by pancreatic RNase results in the formation of three major photoproducts as Hp‡, DpUp and DpHp, as well as unaffected Up and other minor products. Irradiation drastically reduces the rate of hydrolysis of poly IT by RNase. The hydrolysis products have been isolated from 32 P-labelled poly U by two-dimensional paper chromatography, thus allowing the determination of the amount of dimer and hydrate formed in irradiated poly U as a function of dose. As an example, the yields of the four major products formed by 280 m μ irradiation are given. Dimers were reversed by re-irradiation at 240 m μ , while hydrates were reversed by heating at 85°C. Hydrolysis of the major dimer-containing oligonucleotides with bacterial alkaline phosphatase established their chain length as three, while hydrolysis with snake venom phosphodiesterase released D, pU and pH. DpU was split at about 14 times the rate of DpH by the snake venom enzyme, while both DpU and DpH were stable to spleen phosphodiesterase. Alkaline hydrolysis of these trinucleotides confirmed their structures as determined enzymically. There are two isomeric dimers found in irradiated poly U corresponding to U p U ⌢ 1 and U p U ⌢ 3 ( Brown, Freeman & Johns, 1966 ); U p U ⌢ 2 appeared to be absent. Attempts at heat reversal of hydrates in dimer-containing oligonucleotides led to extensive breakdown of these products, emphasizing the need for controls on chain integrity during thermal reactivation of irradiated polynucleotides.

Amanitin binding to RNA polymerase II in α-amanitin-resistant rat myoblast mutants*

A series of independent α-amanitin-resistant (Ama R ) mutants have been isolated from diploid and tetraploid strains of L6 rat myoblast cells. With one exception, these mutants contain both α-amanitin-sensitive wild-type and α-amanitin-resistant mutant RNA polymerase II activities. The relative resistance of the mutant enzymes in extracts of the different Ama R isolates, determined by measuring the inhibition constant, K 1 , for α-amanitin, parallels their relative resistance to growth inhibition by the toxin. The binding affinities of the different mutant forms of RNA polymerase II for γ-amanitin, determined by measuring the equilibrium dissociation constant, K d , for γ-[ 3 H]amanitin§ bound to the enzyme, are also reduced relative to the wild-type enzyme, and the K d values are proportional to the K 1 values. Thus the spectrum of Ama R phenotypes in these mutant strains likely reflects the presence of mutations at different sites in the structural gene coding for the α-amanitin binding subunit of RNA polymerase II. No difference has been found between mutant and wild-type forms of the enzyme with respect to their turnover numbers, K m values for UTP, or thermal stabilities. Diploid mutants contain equal amounts of wild-type and mutant forms of RNA polymerase II. This indicates that they possess one wild-type and one mutant allele for the α-amanitin binding subunit of the enzyme, and that both alleles are expressed in a codominant fashion. Similarly, the tetraploid mutants possess three copies of the wild-type and one of the mutant allele. One tetraploid mutant has been isolated which is only slightly resistant to growth inhibition by α-amanitin and does not contain a detectably altered form of RNA polymerase II. This strain may be defective in α-amanitin transport.

Ultraviolet photoproducts in ordered structures of poly U and their effects on secondary structure

Poly II was irradiated with ultraviolet light in the following ordered complexes: (I) poly (A + 2U), (II) poly U + spermine (with spermine at 14 the monomer concentration of poly U), and (III) poly U in 0.01 m-MgCl2. Yields of photoproducts were analysed by RNase hydrolysis of complexes containing 32P-labelled poly U followed by Chromatographic separation and liquid scintillation counting. Poly (A + 2U) was irradiated in two forms, as poly ((A + U) + U∗) or as poly ((A + U∗) + U) where U∗ represents the 32P-labelled poly U strand. No difference was detected in the photochemical behaviour of these two forms and the behaviour of poly (A + 2U) was almost the same as that of poly (A + U). Complexes II and III were irradiated at 235 mμ and 280 mμ above and below their melting temperatures. Absorbance at 260 mμ was found to increase with irradiation of III below its melting point. Absorbance-temperature profiles were plotted after various exposures, and, from these, the fraction of nucleotides “melted” was calculated. Hydrate production is suppressed in all the ordered structures studied. Dimer formation is suppressed in I but favoured in complexes II and III below their respective melting points. Both dimers and hydrates destroy secondary structure; the resultant fractional melting is nearly proportional to the fraction of Us converted to photoproducts.

Association of Bacteriophage Lambda N Gene Protein with E. coli RNA Polymerase

INTRODUCTION Gene expression in prokaryotes appears to be regulated primarily at the level of transcription (Chamberlin 1974). In some systems, alterations in transcriptional activity can be correlated with physical changes in the structure of RNA polymerase, such as modifications of the core subunits or association of new polypeptides with the enzyme (Stevens 1972; Greenleaf, Linn and Losick 1973; Horvitz 1973; Goff 1974; Ratner 1974). We are interested in the molecular details of the regulation of gene expression in bacteriophage λ, a system known to exhibit an intricate pattern of transcriptional control. One essential λ gene required for proper early transcription is the N gene, the product of which appears to be required for the elongation of “immediate-early” transcripts into the adjacent “delayed-early” genes (see Figure 1) (Herskowitz 1973). Although the mechanism of N action remains unclear, several models of N function have been proposed, including suggestions that N protein antagonizes the host transcription termination factor rho (Roberts 1971) and that N protein stabilizes distal messenger from rapid degradation (Brunel and Davison 1975). The isolation of several host mutations that specifically block N function (Georgopoulos 1971; Ghysen and Pironio 1972; Friedman, Jolly and Mural 1973) and the finding that some of these mutations map in the cistron coding for the β subunit of RNA polymerase have led to the suggestion that the N protein interacts with RNA polymerase. Such an interaction has also been postulated by Friedman, Wilgus and Mural (1973) to explain the apparent immunity specificities of N λ and N 21 ,...

Downstream regulation of int gene expression by the b2 region in phage lambda.

Expression of the int gene after phage lambda infection normally requires the products of genes cII and cIII. However, when the phage carries a deletion in the nonessential b2 region adjacent to int, efficient synthesis of active Int protein does not require cII and cIII function. This inhibition of Int synthesis by nucleotide sequences downstream from the int structural gene behaves in a cis-dominant fashion in mixed infections. It is specific for PL- and not pI-initiated transcripts. Based on these observations, and those of others, a model is proposed in which Int translation from the pL transcript is inhibited by the interaction of downstream b2 nucleotide sequences and nucleotide sequences in the int region. The data imply a novel temporal mechanism regulating prophage lambda induction: circularization of the prophage genome results in the transposition of inhibitory b2 region sequences next to int and blocks further Int protein synthesis beyond the low level required for excision. As a consequence of this process, the control of int expression is transferred from the pL promoter to pI and the cII/cIII system. Such a genetic regulatory mechanism involving the rearrangement of genetic elements downstream from a structural gene may be of general use during development in other systems.