Laser cross-linking of nucleic acids to proteins. Methodology and first applications to the phage T4 DNA replication system

Single-pulse (approximately 8 ns) ultraviolet laser excitation of protein-nucleic acid complexes can result in efficient and rapid covalent cross-linking of proteins to nucleic acids. The reaction produces no nucleic acid-nucleic acid or protein-protein cross-links, and no nucleic acid degradation. The efficiency of cross-linking is dependent on the wavelength of the exciting radiation, on the nucleotide composition of the nucleic acid, and on the total photon flux. The yield of cross-links/laser pulse is largest between 245 and 280 nm; cross-links are obtained with far UV photons (200-240 nm) as well, but in this range appreciable protein degradation is also observed. The method has been calibrated using the phage T4-coded gene 32 (single-stranded DNA-binding) protein interaction with oligonucleotides, for which binding constants have been measured previously by standard physical chemical methods (Kowalczykowski, S. C., Lonberg, N., Newport, J. W., and von Hippel, P. H. (1981) J. Mol. Biol. 145, 75-104). Photoactivation occurs primarily through the nucleotide residues of DNA and RNA at excitation wavelengths greater than 245 nm, with reaction through thymidine being greatly favored. The nucleotide residues may be ranked in order of decreasing photoreactivity as: dT much greater than dC greater than rU greater than rC, dA, dG. Cross-linking appears to be a single-photon process and occurs through single nucleotide (dT) residues; pyrimidine dimer formation is not involved. Preliminary studies of the individual proteins of the five-protein T4 DNA replication complex show that gene 43 protein (polymerase), gene 32 protein, and gene 44 and 45 (polymerase accessory) proteins all make contact with DNA, and can be cross-linked to it, whereas gene 62 (polymerase accessory) protein cannot. A survey of other nucleic acid-binding proteins has shown that E. coli RNA polymerase, DNA polymerase I, and rho protein can all be cross-linked to various nucleic acids by the laser technique. The potential uses of this procedure in probing protein-nucleic acid interactions are discussed.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10(13) photons.cm-2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10(13)--10(16) photons-cm-2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Evidence for the formation of a quinone methide during the oxidation of the insect cuticular sclerotizing precursor 1,2-dehydro-N-acetyldopamine.

1,2-Dehydro-N-acetyldopamine (dehydro-NADA) is an important catecholamine derivative involved in the cross-linking of insect cuticular components during sclerotization. Since sclerotization is a vital process for the survival of insects, and is closely related to melanogenesis, it is of interest to unravel the chemical mechanisms participating in this process. The present paper reports on the mechanism by which dehydro-NADA is oxidatively activated to form reactive intermediate(s) as revealed by pulse radiolysis, electron spin resonance spectroscopy, high performance liquid chromatography, and ultraviolet-visible spectroscopic analysis. Pulse radiolytic one-electron oxidation of dehydro-NADA by N3. (k = 5.3 x 10(9) M-1 s-1) or Br2.- (k = 7.5 x 10(8) M-1 s-1) at pH6 resulted in the rapid generation of the corresponding semiquinone radical, lambda max 400 nm, epsilon = 20,700 M-1 cm-1. This semiquinone decayed to form a second transient intermediate, lambda max 485 nm, epsilon = 8000 M-1 cm-1, via a second order disproportionation process, k = 6.2 x 10(8) M-1 s-1. At pH 6 in the presence of azide, the first order decay of this second intermediate occurred over milliseconds; the rate decreases at higher pH. At pH 6 in the presence of bromide, the intermediate decayed much more slowly over seconds, k = 0.15 s-1. Under such conditions, the dependence of the first order decay constant upon parent dehydro-NADA concentration led to a second order rate constant of 8.5 x 10(2) M-1 s-1 for reaction of the intermediate with the parent, probably to form benzodioxan "dimers." (The term dimer is used for convenience; the products are strictly bisdehydrodimers of dehydro-NADA (see "Discussion" and Fig. 11)) Rate constants of 5.9 x 10(5), 4.5 x 10(5), 2.8 x 10(4) and 3.5 x 10(4) M-1 s-1 were also obtained for decay of the second intermediate in the presence of cysteine, cysteamine, o-phenylenediamine, and p-aminophenol, respectively. By comparison with the UV-visible spectroscopic properties of the two-electron oxidized species derived from dehydro-NADA and from 1,2-dehydro-N-acetyldopa methyl ester, it is concluded that the transient intermediate exhibiting absorbance at 485 nm is the quinone methide tautomer of the o-quinone of dehydro-NADA. Sclerotization of insect cuticle is discussed in the light of these findings.     

Spectral sensitivity,absolute threshold,and visual field of two tick species,Hyalomma dromedarii andAmblyomma variegatum

1. The spectral sensitivity in the wavelength range of 340-750 nm was determined by both a behavioral approach based on spontaneous positive phototaxis and the electroretinogram (ERG). 2. Concerning phototaxis the camel tick, Hyalomma dromedarii, showed two sensitivity maxima, one in the UV range (ca. 380 nm) and another in the blue-green range (ca. 500 nm). At higher intensities the relative sensitivity was more pronounced in the UV and at lower intensities more pronounced in the blue-green (reverse Purkinje shift). In the tropical bont tick, Amblyomma variegatum, there was a single sensitivity maximum in the blue range (ca. 480 nm). 3. In the ERG there was a maximum in the blue range (ca. 470 nm) in both species and a weak secondary maximum in the UV in Hyalomma. 4. The absolute sensitivity was very high. The threshold irradiance of phototaxis was as low as 5.2 x 10(6) photons.s-1.cm-2 in Hyalomma and 5.2 x 10(8) photons.s-1.cm-2 in Amblyomma. 5. When the eyes of Hyalomma were covered, the threshold irradiance was still very low, namely 5.2 x 10(8) photons.s-1.cm-2, indicating high absolute sensitivity of the extraretinal photoreceptors. 6. The visual field of the eyes was determined by ERG measurements. In both species the optical axis of each eye, i.e., the center of the visual field, was directed somewhat to the side and above the horizon. In Hyalomma this direction was 35 degrees to the long axis of the animal and 30 degrees above the horizon for natural body posture during walking. In Amblyomma the corresponding angles were 39 degrees and 33 degrees, respectively. The size of the field (at 50% sensitivity) in Hyalomma was relatively small, namely 14 degrees in the horizontal and 25 degrees in the vertical direction, compared with that of Amblyomma with 43 degrees and 49 degrees, respectively. 7. This is the first demonstration in ticks of spectral and absolute sensitivity by the behavioral approach and of the visual field by ERG. The results suggest that tick eyes possess features of both spider eyes and insect ocelli.     

An attempt to stimulate cell division in Saccharomyces cerevisiae with weak ultraviolet light

Liquid cultures of the yeast Saccharomyces cerevisiae were irradiated with weak light having irradiances ranging from ca. 1 X 10(2) to 5 X 10(9) photons cm-2 s-1 and at wavelengths ranging from 200 to 700 nm. When particular care was taken to control the temperature of the cultures and the flow rate of oxygen, no evidence was obtained for stimulation of either yeast growth or division by the incident light. These results do not support the claims of early workers that very low intensity uv light can stimulate cell division in living organisms.     

柚树(Citrus grandis)叶片光合作用对补增UV-B辐射的响应

生长在人工光照 4 0 0μmol m- 2 s- 1 下的柚树幼树光合速率的最大值为 1 0 .2± 0 .5μmol m- 2 s- 1 ;而补增UV-B辐射 ( 3.8-4 .2μW cm- 2 ,2 4 5～ 2 97nm,4 5d)的叶片则为 6.4± 0 .8μmol m- 2 s- 1 ,较对照植株降低37.2 %。对照植物的表观量子产率 (固定 mol CO2 mol- 1量子 )为 0 .0 75± 0 .0 1 2 ,而经 UV-B辐射处理植株则为0 .0 4 1± 0 .0 0 8,明显较对照植株低。UV-B辐射处理使植株叶片的光呼吸和不包括光呼吸的 CO2 补偿点增高。对照植株叶片的最大值的 CO2 羧化速率 (μmol m- 2 s- 1 )为 57.1± 1 .5μmol m- 2 s- 1 ,较 UV-B辐射处理的高30 .9% ,而 UV-B辐射处理的植株的光合电子传递速率较对照低 30 %。同时 UV-B辐射植株叶片有较低的光能转化效率 ,其较对照低 39.1 % ,叶片亦含有较低的叶绿素含量。结果表明 ,UV-B辐射明显抑制叶片光合羧化速率和光合电子传递速率 ,UV-B辐射可能抑制包括 Rubisco羧化作用在内的多个光合生理过程 ,降低叶片光合速率。柚树叶片对 UV-B辐射敏感 ,选育抗 UV-B辐射的柚树品种势在必行。     

Uridine 5'-phosphate photohydrate formation in irradiated Escherichia coli 30S ribosomes: photochemistry and relation to ribonucleic acid-protein cross-linking

Irradiation of aqueous buffered solutions of Escherichia coli 30S ribosomes with doses of 254-nm radiation greater than 10(19) quanta causes formation of uridine 5'-phosphate (UMP) photohydrates in ribosomal 16S RNA (rRNA). The number of molecules of UMP photohydrate formed at doses less than 2 x 10(20) quanta is linearly dependent on dose of absorbed 254-nm radiation. Maximum UMP photohydrate formation is dependent on initial ribosome concentration. When solutions containing 1 A260 unit of 30S ribosomes/mL were irradiated with greater than 2 x 10(20) quanta of 254-nm radiation, maximum photohydrate formation was equal to 47 residues/ribosome. Irradiation of solutions containing 2 A260 units/mL with greater than 7 x 10(20) quanta caused formation of 102 UMP photohydrates/ribosome. These values correspond to conversion of either 15 or 33%, respectively, of the total UMP content of 30S ribosome 16S rRNA to photohydrates. Target theory analysis of UMP photohydration in 30S ribosomes showed that UMP photohydrates are formed by single-hit kinetics from two photochemically distinct precursors. Of the total 16S rRNA UMP residues, 10% was included in the most rapidly (low dose) reacting fraction. The respective photohydration cross sections are 0.014 (low dose) and 0.0095 cm2/muEinstein (high dose) for ribosome solutions containing 2 A260 units/mL. UMP photohydrate content of irradiated 30S ribosomes was compared with that of previous data for the extent of RNA-protein cross-linking at equivalent doses of absorbed 254-nm radiation. This comparison showed that at least two UMP photohydrates form per RNA-protein cross-linking event in 30S ribosomes irradiated with a dose of 254-nm radiation (1.5 x 10(19) quanta), which causes cross-linking of only three ribosomal proteins to 16S rRNA.     

Covalent cross-linking of poly(A) to Escherichia coli ribosomes, and localization of the cross-link site within the 16S RNA.

Poly(A) can be cross-linked to E. coli 70S ribosomes in the presence of tRNALys by mild ultraviolet irradiation. The cross-linking reaction is exclusively with the 30S subunit, and involves primarily the RNA moiety. Following a partial nuclease digestion, cross-linked complexes containing poly(A) and fragments of the 16S RNA were isolated by affinity chromatography on oligo(dT)-cellulose. The complexes were purified by gel electrophoresis and subjected to oligonucleotide analysis, which revealed a single cross-link site within positions 1394-1399 of the 16S RNA. The same pattern of cross-linking, at about one-fifth of the intensity, was observed in the absence of tRNALys. The cross-link site to poly(A), together with other sites in the 16S RNA that have been implicated in ribosomal function, is discussed in the framework of our recent model for the three-dimensional structure of 16S RNA; all of the functional sites are clustered together in two distinct groups in the model.     

Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light (800 μmol photons/m2 per s)-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center (PSII RC) under aerobic conditions (in the absence of electron donors or acceptors) was studied using high-pressure liquid chromatography (HPLC), absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSll RC as determined by HPLC after light treatment were as follows: with increasing illumination time chlorophyll (Chi) a and β-carotene (β-car)content decreased. However, decreases in pheophytin (Pheo) could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illumination, the initial bleaching occurred maximally at 680 nm but that with increasing illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 min light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination.After illumination, the fluorescence emission intensity changed and the fluorescence maximum blue shifted,showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm-1 (υ1), 1 159 cm-1 (υ2), 1 006 cm-1 (υ3), 966 cm-1 (υ4) for 488.0 nm excitation and 1 525 cm-1 (υ1), 1 159 cm-1 (υ2), 1 007 cm-1 (υ3), 968 cm-1 (υ4) for 514.5 nm excitation.It was confirmed that two spectroscopically different β-car molecules exist in the PSII RC. After light treatment for 20 min, band positions and bandwidths were unchanged. This indicates that carotenoid configuration is not the parameter that regulates photoprotection in the PSII RC.     

A specific, UV-induced RNA-protein cross-link using 5-bromouridine-substituted RNA.

The well-characterized RNA binding site of the bacteriophage R17 coat protein has been used to investigate the cross-linking of protein to 5-bromouridine (BrU)-substituted RNA using medium-wavelength UV light. We have demonstrated a specific RNA-protein cross-link and identified the site on the RNA of protein attachment. Formation of the covalent complex is dependent upon the presence of BrU at position -5 of the RNA and specific binding of the RNA by coat protein. The amount of cross-linking increases with time and depends on the light source and conditions used. Irradiations using a broad-spectrum UV transilluminator (peak at 312 nm) or monochromatic XeCl excimer laser (308 nm) gave levels of cross-linking exceeding 20 and 50%, respectively. The quantum yield of photo-cross-linking, determined with 308-nm excitation, was 0.003. While little strand breakage or debromination of the RNA occurred, significant protein photodamage was observed.     

The ends of La Crosse virus genome and antigenome RNAs within nucleocapsids are base paired.

The three La Crosse virus genomes are found as circular structures in the electron microscope, and the RNA ends of at least the small (S) and medium (M) segments are highly complementary. When examined for psoralen cross-linking, about half of the S, at most 1 to 2% of the M, and none of the large (L) nucleocapsid RNAs could be cross-linked in virions or at late times intracellularly, under conditions in which each free RNA reacted completely. For the S segment, genomes and antigenomes first detected intracellularly could not be cross-linked at all, and their cross-linkability increased gradually with time. Antigenomes behaved similarly to genomes in all respects. It appears that the majority of all three segments are base paired at their ends and that the limited cross-linkability reflects the accessability of the RNA within nucleocapsids to psoralen. The gradual increase in cross-linkability may be important in persistent mosquito cell infection, in which it correlates with decreased S mRNA synthesis rates, and may be part of the mechanism which this infection becomes self-limiting. The implications of double-stranded RNA panhandles within nucleocapsids are discussed.     

RNA-protein cross-linking in Eschericia coli 30S ribosomal subunits: a method for the direct analysis of the RNA regions involved in the cross-links.

A prerequisite for topographical studies on ribosomal subunits involving RNA-protein cross-linking is that the cross-linking sites on the RNA should be determined. Methodology is presented which offers a solution to this problem, using as a test system 30S subunits in which protein S7 has been cross-linked to the 16S RNA by ultraviolet irradiation. The method is based on a gel separation system in the presence of a non-ionic detergent. When a ribonucleoprotein fragment containing RNA-protein cross-links is applied to this system, non-cross-linked protein is removed, and simultaneously the cross-linked RNA-protein complex is separated from non-cross-linked RNA. Oligonucleotide analysis of the S7-RNA complex isolated in this manner showed it to consist of a region of RNA from sections P-A of the 16S RNA. A single characteristic oligonucleotide was absent from this region, and it was tentatively concluded that this missing oligonucleotide contains the actual site of cross-linking.     

Rapid spectral scan and stopped-flow studies of carbon monoxide binding to bovine adrenocortical cytochrome P-450scc

Carbon monoxide binding with both cholesterol-free (low-spin) and cholesterol-bound (high-spin) reduced forms of purified cytochrome P-450scc has been investigated by rapid-scan and stopped-flow spectrometry. CO binding occurs within 150 ms at 25 degrees C for both forms of P-450scc, with a typical absorption maximum at 450 nm. Isosbestic points occur at the following wavelengths: between reduced-CO and reduced cholesterol-free P-450scc at 434 and 471 nm; between reduced-CO and reduced cholesterol-bound P-450scc at 433 and 469 nm. Both the 'on' (k1) and 'off' rate constants (k-1) are found to be independent of pH between pH 5 and 9. The mean values of k1 for cholesterol-free (1.8 +/- 0.2) X 10(5) M-1 X s-1) and cholesterol-bound [1.9 +/- 0.1) X 10(5) M-1 X s-1) P-450scc are almost identical, while the mean value of k-1 for the former [2.3 +/- 0.3) X 10 s-1) is about double that of the latter [1.2 +/- 0.1) X 10 s-1). This suggests the instability of the reduced-CO complex in the absence of cholesterol.     

Ssh10b, a conserved thermophilic archaeal protein, binds RNA in vivo

Proteins of the Sac10b family, which is highly conserved among hyperthermophilic archaea, have been regarded as DNA-binding proteins. Based on their in vitro DNA-binding properties, these proteins are thought to be involved in chromosomal organization or DNA repair/recombination. We show that Ssh10b, a member of the Sac10b family from Sulfolobus shibatae, bound with similar affinities to double-stranded DNA, single-stranded DNA and RNA in vitro. However, the protein was exclusively bound to RNA in S. shibatae cells, as revealed by in vivo UV cross-linking and co-immunoprecipitation. Ribosomal RNAs were among the RNA species co-immunoprecipitated with Ssh10b. Consistent with this observation, Ssh10b was co-purified with ribosomes under low salt conditions. Furthermore, we demonstrate by UV-cross-linking hybridization that, when the cells were irradiated with UV, Ssh10b became cross-linked to 16S, 23S rRNAs and mRNAs. Our data indicate that RNA is the physiological binding target of the Sac10b family.     

Exciton Annihilation in the Two Photosystems in Chloroplasts at 100°K

The fluorescence yield (F) of spinach chloroplasts at 100°K measured at 735 nm (photosystem I fluorescence—F 735) and at 685 nm (photosystem II fluorescence—F 685) has been determined with different modes of laser excitation. The modes of excitation included a single picosecond pulse, sequences of picosecond pulses (4, 22, and 300 pulses spaced 5 ns apart) and a single nonmode-locked 2-μs pulse (MP mode). The F 735/F 685 intensity ratios decrease from 1.62 to 0.61 when a single picosecond pulse (or low-power continuous helium-neon laser) is replaced by excitation with the 300-ps pulse train (PPT mode) or MP mode. In the PPT mode of excitation, the 735-nm fluorescence band is quenched by a factor of 45 as the intensity is increased from 10¹⁵ to 10¹⁸ photons/cm² per pulse train and the 685-nm fluorescence is quenched by a factor of 10. In the MP mode, the quenching factors are 25 and 7, respectively, in the same intensity range. Fluorescence quantum yield measurements with different picosecond pulse sequences indicate that relatively long-lived quenching species are operative, which survive from one picosecond pulse to another within the pulse train. The excitonic processes possible in the photosynthetic units are discussed in detail. The differences in the quenching factors between the MP and PPT modes of excitation are attributed to singlet-singlet annihilation, possible when picosecond pulses are utilized, but minimized in the MP mode of excitation. The long-lived quenchers are identified as triplets and/or bulk chlorophyll ions formed by singlet-singlet annihilation. The preferential quenching in photosystem I is attributed to triplet excitons. The influence of heating effects, photochemistry, bleaching, and two-photon processes is also considered and is shown to be negligible.     

Ribonucleic acid-protein cross-linking within the intact Escherichia coli ribosome, utilizing ethylene glycol bis[3-(2-ketobutyraldehyde) ether], a reversible, bifunctional reagent: synthesis and cross-linking within 30S and 50S subunits

We have used the reversible, bifunctional reagent ethylene glycol bis[3-(2-ketobutyraldehyde) ether] to cross-link RNA to protein within intact ribosomal subunits from Escherichia coli. Here we describe the synthesis of this compound (termed bikethoxal) and demonstrate its ability to form covalent attachments between RNA and protein in the 5S RNA-L18 complex and within 30S and 50S ribosomal subunits. The reagent is a symmetrical dicarbonyl compound and reacts with guanine in single-stranded RNA and with arginine in protein. RNA-protein cross-links generated with this reagent are stable, as demonstrated by the comigration of 35S-labeled ribosomal proteins with ribosomal RNA on neutrally buffered sodium dodecyl sulfate (SDS)-agarose gels. However, the cross-linked product is unstable in mildly basic conditions, allowing the identification of the linked macromolecules by conventional techniques. The reagent is potentially capable of cross-linking any combination of single-stranded RNA, single-stranded DNA, or protein; it should prove a useful probe of the RNA-protein proximities within the E. coli ribosome, since the SDS-agarose gel system we describe provides a rapid method of optimizing this RNA--protein cross-linking reaction.     

A backflow of electrons around photosystem II in Chlorella cells.

A study was made with a modulated oxygen electrode of the effect of variations of oxygen concentration on photosynthetic oxygen evolution from algal cells. When Chlorella vulgaris is examined with a modulated 650 nm light at 22 degrees C, both the oxygen yield and the phase lag between the modulated oxygen signal and the light modulations have virtually constant values between 800 and 120 ergs . cm-1 . s-1 if the bathing medium is in equilibrium with the air. Similar results are obtained at 32 degrees C between 1600 and 120 ergs . cm-2 . s-1. Under anaerobic conditions both the oxygen yield and the phase lag decrease if the light intensity is lowered below about 500 ergs . cm-2 . s-1 at 22 degrees C or about 1000 ergs . cm-2 . s-1 at 32 degrees C. A modulated 706 nm beam also gives rise to these phenomena but only at significantly lower rates of oxygen evolution. The cells of Anacystis nidulans and Porphyridium cruentum appear to react in the same way to anaerobic conditions as C. vulgaris. An examination of possible mechanisms to explain these results was performed using a computer simulation of photosynthetic electron transport. The simulation suggests that a backflow of electrons from a redox pool between the Photosystems to the rate-limiting reaction between Photosystem II and the water-splitting act can cause a decrease in oxygen yield and phase lag. If the pool between the Photosystems is in a very reduced state a significant cyclic flow is expected, whereas if the pool is largely oxidized little or no cyclic flow should occur. It is shown that the effects of 706 nm illumination and removal of oxygen can be interpreted in accordance with these proposals. Since a partial inhibition of oxygen evolution by 3-(3.4-dichlorophenyl)-1,1-dimethylurea (10(-8) M) magnifies the decreases in oxygen yield and phase lag, it is proposed that the pool which cycles back electrons is in front of the site of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition and is probably the initial electron acceptor pool after Photosystem II.