ZAKα-driven ribotoxic stress response activates the human NLRP1 inflammasome.

Human NLRP1 (NACHT, LRR, and PYD domain-containing protein 1) is an innate immune sensor predominantly expressed in the skin and airway epithelium. Here, we report that human NLRP1 senses the ultraviolet B (UVB)- and toxin-induced ribotoxic stress response (RSR). Biochemically, RSR leads to the direct hyperphosphorylation of a human-specific disordered linker region of NLRP1 (NLRP1DR) by MAP3K20/ZAKα kinase and its downstream effector, p38. Mutating a single ZAKα phosphorylation site in NLRP1DR abrogates UVB- and ribotoxin-driven pyroptosis in human keratinocytes. Moreover, fusing NLRP1DR to CARD8, which is insensitive to RSR by itself, creates a minimal inflammasome sensor for UVB and ribotoxins. These results provide insight into UVB sensing by human skin keratinocytes, identify several ribotoxins as NLRP1 agonists, and establish inflammasome-driven pyroptosis as an integral component of the RSR.

Hedgehog signaling enables repair of ribosomal DNA double-strand breaks.

Ribosomal DNA (rDNA) consists of highly repeated sequences that are prone to incurring damage. Delays or failure of rDNA double-strand break (DSB) repair are deleterious, and can lead to rDNA transcriptional arrest, chromosomal translocations, genomic losses, and cell death. Here, we show that the zinc-finger transcription factor GLI1, a terminal effector of the Hedgehog (Hh) pathway, is required for the repair of rDNA DSBs. We found that GLI1 is activated in triple-negative breast cancer cells in response to ionizing radiation (IR) and localizes to rDNA sequences in response to both global DSBs generated by IR and site-specific DSBs in rDNA. Inhibiting GLI1 interferes with rDNA DSB repair and impacts RNA polymerase I activity and cell viability. Our findings tie Hh signaling to rDNA repair and this heretofore unknown function may be critically important in proliferating cancer cells.

Light Activates the Translational Regulatory Kinase GCN2 via Reactive Oxygen Species Emanating from the Chloroplast.

Cytosolic mRNA translation is subject to global and mRNA-specific controls. Phosphorylation of the translation initiation factor eIF2α anchors a reversible regulatory switch that represses cytosolic translation globally. The stress-responsive GCN2 kinase is the only known kinase for eIF2α serine 56 in Arabidopsis (Arabidopsis thaliana). Here, we show that conditions that generate reactive oxygen species (ROS) in the chloroplast, including dark-light transitions, high light, and the herbicide methyl viologen, rapidly activated GCN2 kinase, whereas mitochondrial and endoplasmic reticulum stress did not. GCN2 activation was light dependent and mitigated by photosynthesis inhibitors and ROS quenchers. Accordingly, the seedling growth of multiple Arabidopsis gcn2 mutants was retarded under excess light conditions, implicating the GCN2-eIF2α pathway in responses to light and associated ROS. Once activated, GCN2 kinase preferentially suppressed the ribosome loading of mRNAs for functions such as mitochondrial ATP synthesis, the chloroplast thylakoids, vesicle trafficking, and translation. The gcn2 mutant overaccumulated transcripts functionally related to abiotic stress, including oxidative stress, as well as innate immune responses. Accordingly, gcn2 displayed defects in immune priming by the fungal elicitor, chitin. Therefore, we provide evidence that reactive oxygen species produced by the photosynthetic apparatus help activate the highly conserved GCN2 kinase, leading to eIF2α phosphorylation and thus affecting the status of the cytosolic protein synthesis apparatus.

Limited Responsiveness of Chloroplast Gene Expression during Acclimation to High Light in Tobacco.

Acclimation to changing light intensities poses major challenges to plant metabolism and has been shown to involve regulatory adjustments in chloroplast gene expression. However, this regulation has not been examined at a plastid genome-wide level and for many genes, it is unknown whether their expression responds to altered light intensities. Here, we applied comparative ribosome profiling and transcriptomic experiments to analyze changes in chloroplast transcript accumulation and translation in leaves of tobacco (Nicotiana tabacum) seedlings after transfer from moderate light to physiological high light. Our time-course data revealed almost unaltered chloroplast transcript levels and only mild changes in ribosome occupancy during 2 d of high light exposure. Ribosome occupancy on the psbA mRNA (encoding the D1 reaction center protein of PSII) increased and that on the petG transcript decreased slightly after high light treatment. Transfer from moderate light to high light did not induce substantial alterations in ribosome pausing. Transfer experiments from low light to high light conditions resulted in strong PSII photoinhibition and revealed the distinct light-induced activation of psbA translation, which was further confirmed by reciprocal shift experiments. In low-light-to-high-light shift experiments, as well as reciprocal treatments, the expression of all other chloroplast genes remained virtually unaltered. Altogether, our data suggest that low light-acclimated plants upregulate the translation of a single chloroplast gene, psbA, during acclimation to high light. Our results indicate that psbA translation activation occurs already at moderate light intensities. Possible reasons for the otherwise mild effects of light intensity changes on gene expression in differentiated chloroplasts are discussed.

Multilevel effects of light on ribosome dynamics in chloroplasts program genome-wide and psbA-specific changes in translation.

Plants and algae adapt to fluctuating light conditions to optimize photosynthesis, minimize photodamage, and prioritize energy investments. Changes in the translation of chloroplast mRNAs are known to contribute to these adaptations, but the scope and magnitude of these responses are unclear. To clarify the phenomenology, we used ribosome profiling to analyze chloroplast translation in maize seedlings following dark-to-light and light-to-dark shifts. The results resolved several layers of regulation. (i) The psbA mRNA exhibits a dramatic gain of ribosomes within minutes after shifting plants to the light and reverts to low ribosome occupancy within one hour in the dark, correlating with the need to replace damaged PsbA in Photosystem II. (ii) Ribosome occupancy on all other chloroplast mRNAs remains similar to that at midday even after 12 hours in the dark. (iii) Analysis of ribosome dynamics in the presence of lincomycin revealed a global decrease in the translation elongation rate shortly after shifting plants to the dark. The pausing of chloroplast ribosomes at specific sites changed very little during these light-shift regimes. A similar but less comprehensive analysis in Arabidopsis gave similar results excepting a trend toward reduced ribosome occupancy at the end of the night. Our results show that all chloroplast mRNAs except psbA maintain similar ribosome occupancy following short-term light shifts, but are nonetheless translated at higher rates in the light due to a plastome-wide increase in elongation rate. A light-induced recruitment of ribosomes to psbA mRNA is superimposed on this global response, producing a rapid and massive increase in PsbA synthesis. These findings highlight the unique translational response of psbA in mature chloroplasts, clarify which steps in psbA translation are light-regulated in the context of Photosystem II repair, and provide a foundation on which to explore mechanisms underlying the psbA-specific and global effects of light on chloroplast translation.

Transmission electron microscopy method providing high resolution in the cell section without radiation damage.

Several new features of mitochondrial nucleoid and its surroundings in mammalian cells were described previously (Prachar, 2016). Very small details were observed using the improved transmission electron microscopy method, as described in the article. In the meantime, the method has again been improved to 2 Å resolutions in the cell section. The method described in detail in the present work is documented on the same records that were published in lower resolution in the work Prachar (2016), enabling comparison of the achieved resolution with the previous one. New records are also presented, showing extremely high resolution and thus implying the importance of the method. Potential use of this method in different fields is suggested.

Antibacterial Mechanism of 405-Nanometer Light-Emitting Diode against Salmonella at Refrigeration Temperature.

The aim of this study was to elucidate the antibacterial mechanism of 405 ± 5-nm light-emitting diode (LED) illumination against Salmonella at 4°C in phosphate-buffered saline (PBS) by determining endogenous coproporphyrin content, DNA oxidation, damage to membrane function, and morphological change. Gene expression levels, including of oxyR, recA, rpoS, sodA, and soxR, were also examined to understand the response of Salmonella to LED illumination. The results showed that Salmonella strains responded differently to LED illumination, revealing that S. enterica serovar Enteritidis (ATCC 13076) and S. enterica subsp. enterica serovar Saintpaul (ATCC 9712) were more susceptible and resistant, respectively, than the 16 other strains tested. There was no difference in the amounts of endogenous coproporphyrin in the two strains. Compared with that in nonilluminated cells, the DNA oxidation levels in illuminated cells increased. In illuminated cells, we observed a loss of efflux pump activity, damage to the glucose uptake system, and changes in membrane potential and integrity. Transmission electron microscopy revealed a disorganization of chromosomes and ribosomes due to LED illumination. The levels of the five genes measured in the nonilluminated and illuminated S Saintpaul cells were upregulated in PBS at a set temperature of 4°C, indicating that increased gene expression levels might be due to a temperature shift and nutrient deficiency rather than to LED illumination. In contrast, only oxyR in S Enteritidis cells was upregulated. Thus, different sensitivities of the two strains to LED illumination were attributed to differences in gene regulation.IMPORTANCE Bacterial inactivation using visible light has recently received attention as a safe and environmentally friendly technology, in contrast with UV light, which has detrimental effects on human health and the environment. This study was designed to understand how 405 ± 5-nm light-emitting diode (LED) illumination kills Salmonella strains at refrigeration temperature. The data clearly demonstrated that the effectiveness of LED illumination on Salmonella strains depended highly on the serotype and strain. Our findings also revealed that its antibacterial mechanism was mainly attributed to DNA oxidation and a loss of membrane functions rather than membrane lipid peroxidation, which has been proposed by other researchers who studied the antibacterial effect of LED illumination by adding exogenous photosensitizers, such as chlorophyllin and hypericin. Therefore, this study suggests that the detailed antibacterial mechanisms of 405-nm LED illumination without additional photosensitizers may differ from that by exogenous photosensitizers. Furthermore, a change in stress-related gene regulation may alter the susceptibility of Salmonella cells to LED illumination at refrigeration temperature. Thus, our study provides new insights into the antibacterial mechanism of 405 ± 5-nm LED illumination on Salmonella cells.

The Cyanobacterial Ribosomal-Associated Protein LrtA Is Involved in Post-Stress Survival in Synechocystis sp. PCC 6803.

A light-repressed transcript encodes the LrtA protein in cyanobacteria. We show that half-life of lrtA transcript from Synechocystis sp. PCC 6803 is higher in dark-treated cells as compared to light-grown cells, suggesting post-transcriptional control of lrtA expression. The lrtA 5´ untranslated leader region is involved in that darkness-dependent regulation. We also found that Synechocystis sp. PCC 6803 LrtA is a ribosome-associated protein present in both 30S and 70S ribosomal particles. In order to investigate the function of this protein we have constructed a deletion mutant of the lrtA gene. Cells lacking LrtA (∆lrtA) had significantly lower amount of 70S particles and a greater amount of 30S and 50S particles, suggesting a role of LrtA in stabilizing 70S particles. Synechocystis strains with different amounts of LrtA protein: wild-type, ∆lrtA, and LrtAS (overexpressing lrtA) showed no differences in their growth rate under standard laboratory conditions. However, a clear LrtA dose-dependent effect was observed in the presence of the antibiotic tylosin, being the LrtAS strains the most sensitive. Similar results were obtained under hyperosmotic stress caused by sorbitol. Conversely, after prolonged periods of starvation, ∆lrtA strains were delayed in their growth with respect to the wild-type and the LrtAS strains. A positive role of LrtA protein in post-stress survival is proposed.

Mechanistic aspects of the photodynamic inactivation of vancomycin-resistant Enterococci mediated by 5-aminolevulinic acid and 5-aminolevulinic acid methyl ester.

Vancomycin-resistant Enterococci (VRE) is a serious concern for public health. Serious infections with VRE have very limited effective antimicrobial therapy, and alternative treatment approaches are highly desirable. One promising approach might be the photodynamic antimicrobial chemotherapy. In the present study, we investigated the photodynamic inactivation (PDI) of two VRE strains mediated by 5-aminolevulinic acid (5-ALA) and its derivative 5-ALA methyl ester (MAL). The photodynamic damages to bacteria on the level of genomic DNA, the leakage of cell components, and the changes of membrane structure were investigated. After treated with 10 mM 5-ALA and irradiated by the 633 ± 10 nm LED for 60 min, 5.37 and 5.22 log10 reductions in bacterial survival were achieved for the clinical isolate of VRE and E. faecalis (ATCC 51299), respectively. After treated with 10 mM MAL and irradiated by the LED for 60 min, 5.02 and 4.91 log10 reductions in bacterial survival were observed for the two VRE strains, respectively. In addition, the photocleavage on genomic DNA and the rapid release of intracellular biopolymers were detected in PDI-treated bacteria. The intensely denatured cytoplasm and the aggregated ribosomes were also found in PDI-treated bacteria by transmission electron microscopy. Although 5-ALA and MAL-mediated PDI could induce the photocleavage on genomic DNA, the PDI of the two VRE strains might be predominantly attributed to the envelope injury, the intracellular biopolymers leakage, and the cytoplasm denature.

Temperature-dependent radiation sensitivity and order of 70S ribosome crystals.

All evidence to date indicates that at T = 100 K all protein crystals exhibit comparable sensitivity to X-ray damage when quantified using global metrics such as change in scaling B factor or integrated intensity versus dose. This is consistent with observations in cryo-electron microscopy, and results because nearly all diffusive motions of protein and solvent, including motions induced by radiation damage, are frozen out. But how do the sensitivities of different proteins compare at room temperature, where radiation-induced radicals are free to diffuse and protein and lattice structures are free to relax in response to local damage? It might be expected that a large complex with extensive conformational degrees of freedom would be more radiation sensitive than a small, compact globular protein. As a test case, the radiation sensitivity of 70S ribosome crystals has been examined. At T = 100 and 300 K, the half doses are 64 MGy (at 3 Šresolution) and 150 kGy (at 5 Šresolution), respectively. The maximum tolerable dose in a crystallography experiment depends upon the initial or desired resolution. When differences in initial data-set resolution are accounted for, the former half dose is roughly consistent with that for model proteins, and the 100/300 K half-dose ratio is roughly a factor of ten larger. 70S ribosome crystals exhibit substantially increased resolution at 100 K relative to 300 K owing to cooling-induced ordering and not to reduced radiation sensitivity and slower radiation damage.

Adaptation of the black yeast Wangiella dermatitidis to ionizing radiation: molecular and cellular mechanisms.

Observations of enhanced growth of melanized fungi under low-dose ionizing radiation in the laboratory and in the damaged Chernobyl nuclear reactor suggest they have adapted the ability to survive or even benefit from exposure to ionizing radiation. However, the cellular and molecular mechanism of fungal responses to such radiation remains poorly understood. Using the black yeast Wangiella dermatitidis as a model, we confirmed that ionizing radiation enhanced cell growth by increasing cell division and cell size. Using RNA-seq technology, we compared the transcriptomic profiles of the wild type and the melanin-deficient wdpks1 mutant under irradiation and non-irradiation conditions. It was found that more than 3000 genes were differentially expressed when these two strains were constantly exposed to a low dose of ionizing radiation and that half were regulated at least two fold in either direction. Functional analysis indicated that many genes for amino acid and carbohydrate metabolism and cell cycle progression were down-regulated and that a number of antioxidant genes and genes affecting membrane fluidity were up-regulated in both irradiated strains. However, the expression of ribosomal biogenesis genes was significantly up-regulated in the irradiated wild-type strain but not in the irradiated wdpks1 mutant, implying that melanin might help to contribute radiation energy for protein translation. Furthermore, we demonstrated that long-term exposure to low doses of radiation significantly increased survivability of both the wild-type and the wdpks1 mutant, which was correlated with reduced levels of reactive oxygen species (ROS), increased production of carotenoid and induced expression of genes encoding translesion DNA synthesis. Our results represent the first functional genomic study of how melanized fungal cells respond to low dose ionizing radiation and provide clues for the identification of biological processes, molecular pathways and individual genes regulated by radiation.

Genome-wide microarray analysis of human fibroblasts in response to γ radiation and the radiation-induced bystander effect.

Radiation-induced bystander effects have been studied extensively due to their potential implications for cancer therapy and radiation protection; however, a complete understanding of the molecular mechanisms remains to be elucidated. In this study, we monitored transcriptional responses to γ radiation in irradiated and bystander fibroblasts simultaneously employing a genome-wide microarray approach to determine factors that may be modulated in the generation or propagation of the bystander effect. For the microarray data we employed analysis at both the single-gene and gene-set level to place the findings in a biological context. Unirradiated bystander fibroblasts that were recipients of growth medium harvested from irradiated cultures 2 h after exposure to 2 Gy displayed transient enrichment in gene sets belonging to ribosome, oxidative phosphorylation and neurodegenerative disease pathways associated with mitochondrial dysfunctions. The response to direct irradiation was characterized by induction of signaling and apoptosis genes and the gradual formation of a cellular immune response. A set of 14 genes, many of which were regulated by p53, were found to be induced early after irradiation (prior to medium transfer) and may be important in the generation or propagation of the bystander effect.

Morphological changes in the pineal gland of rats under conditions of long-term exposure to bright light.

Changes in the diurnal light cycle affect the morphofunctional state of the pineal gland. The volume of the nucleus, Golgi apparatus, and mitochondria in pinealocytes decreases after 45-day exposure to bright light. After 90 days, the degree of nuclear polymorphism increased, the specific volume of the Golgi apparatus returned to normal, the volume of the granular endoplasmic reticulum decreased, while the volume of lysosomes, free ribosomes, and polysomes increased. These changes reflect plasticity of pinealocytes and adaptation of the gland to long-term 24-h light exposure.

Structure and Dynamics of Ribosomal Protein L12: An Ensemble Model Based on SAXS and NMR Relaxation.

Ribosomal protein L12 is a two-domain protein that forms dimers mediated by its N-terminal domains. A 20-residue linker separates the N- and C-terminal domains. This linker results in a three-lobe topology with significant flexibility, known to be critical for efficient translation. Here we present an ensemble model of spatial distributions and correlation times for the domain reorientations of L12 that reconciles experimental data from small-angle x-ray scattering and nuclear magnetic resonance. We generated an ensemble of L12 conformations in which the structure of each domain is fixed but the domain orientations are variable. The ensemble reproduces the small-angle x-ray scattering data and the optimized correlation times of its reorientational eigenmodes fit the (15)N relaxation data. The ensemble model reveals intrinsic conformational properties of L12 that help explain its function on the ribosome. The two C-terminal domains sample a large volume and extend further away from the ribosome anchor than expected for a random-chain linker, indicating that the flexible linker has residual order. Furthermore, the distances between each C-terminal domain and the anchor are anticorrelated, indicating that one of them is more retracted on average. We speculate that these properties promote the function of L12 to recruit translation factors and control their activity on the ribosome.

Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes.

The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation.

Internucleotide movements during formation of 16 S rRNA-rRNA photocrosslinks and their connection to the 30 S subunit conformational dynamics.

UV light-induced RNA photocrosslinks are formed at a limited number of specific sites in the Escherichia coli and in other eubacterial 16 S rRNAs. To determine if unusually favorable internucleotide geometries could explain the restricted crosslinking patterns, parameters describing the internucleotide geometries were calculated from the Thermus thermophilus 30 S subunit X-ray structure and compared to crosslinking frequencies. Significant structural adjustments between the nucleotide pairs usually are needed for crosslinking. Correlations between the crosslinking frequencies and the geometrical parameters indicate that nucleotide pairs closer to the orientation needed for photoreaction have higher crosslinking frequencies. These data are consistent with transient conformational changes during crosslink formation in which the arrangements needed for photochemical reaction are attained during the electronic excitation times. The average structural rearrangement for UVA-4-thiouridine (s4U)-induced crosslinking is larger than that for UVB or UVC-induced crosslinking; this is associated with the longer excitation time for s4U and is also consistent with transient conformational changes. The geometrical parameters do not completely predict the crosslinking frequencies, implicating other aspects of the tertiary structure or conformational flexibility in determining the frequencies and the locations of the crosslinking sites. The majority of the UVB/C and UVA-s4U-induced crosslinks are located in four regions in the 30 S subunit, within or at the ends of RNA helix 34, in the tRNA P-site, in the distal end of helix 28 and in the helix 19/helix 27 region. These regions are implicated in different aspects of tRNA accommodation, translocation and in the termination reaction. These results show that photocrosslinking is an indicator for sites where there is internucleotide conformational flexibility and these sites are largely restricted to parts of the 30 S subunit associated with ribosome function.

A 16S rRNA-tRNA product containing a nucleotide phototrimer and specific for tRNA in the P/E hybrid state in the Escherichia coli ribosome.

Ribosome complexes containing deacyl-tRNA1(Val) or biotinylvalyl-tRNA1(Val) and an mRNA analog have been irradiated with wavelengths specific for activation of the cmo5U nucleoside at position 34 in the tRNA1(Val) anticodon loop. The major product for both types of tRNA is the cross-link between 16S rRNA (C1400) and the tRNA (cmo5U34) characterized already by Ofengand and his collaborators [Prince et al. (1982) Proc. Natl Acad. Sci. USA, 79, 5450-5454]. However, in complexes containing deacyl-tRNA1(Val), an additional product is separated by denaturing PAGE and this is shown to involve C1400 and m5C967 of 16S rRNA and cmo5U34 of the tRNA. Puromycin treatment of the biotinylvalyl-tRNA1(Val) -70S complex followed by irradiation, results in the appearance of the unusual photoproduct, which indicates an immediate change in the tRNA interaction with the ribosome after peptide transfer. These results indicate an altered interaction between the tRNA anticodon and the 30S subunit for the tRNA in the P/E hybrid state compared with its interaction in the classic P/P state.

Dynamics inherent in helix 27 from Escherichia coli 16S ribosomal RNA.

The original interpretation of a series of genetic studies suggested that the highly conserved Escherichia coli 16S ribosomal RNA helix 27 (H27) adopts two alternative secondary structure motifs, the 885 and 888 conformations, during each cycle of amino acid incorporation. Recent crystallographic and genetic evidence has called this hypothesis into question. To ask whether a slippery sequence such as that of H27 may harbor inherent conformational dynamics, we have designed a series of model RNAs based on E. coli H27 for in vitro physicochemical studies. One-dimensional (1)H NMR spectroscopy demonstrates that both the 885 and 888 conformations are occupied to approximately the same extent (f(888) = 0.427 +/- 0.04) in the native H27 sequence at low pH (6.4) and low ionic strength (50 mM NaCl). UV irradiation assays conducted under conditions analogous to those used for assays of ribosomal function (pH 7.5 and 20 mM MgCl(2)) suggest that nucleotides 892 and 905, which are too far apart in the known 885 crystal structures, can approach each other closely enough to form an efficient cross-link. The use of a fluorescence resonance energy transfer (FRET)-labeled RNA together with a partially complementary DNA oligonucleotide that induces a shift to the 888 conformation shows that H27 interchanges between the 885 and 888 conformations on the millisecond time scale, with an equilibrium constant of 0.33 +/-0.12. FRET assays also show that tetracycline interferes with the induced shift to the 888 conformation, a finding that is consistent with crystallographic localization of tetracycline bound to the 885 conformation of H27 in the 30S ribosomal subunit. Taken together, our data demonstrate the innate tendency of an isolated H27 to exist in a dynamic equilibrium between the 885 and 888 conformations. This begs the question of how these inherent structural dynamics are suppressed within the context of the ribosome.

[Escherichia coli ribosomes as the model to test new photoactivated tRNA analogues, containing 6-thioguanosine]. / Ribosomy Escherichia coli kak model'dlia aprobattsii novykh fotoaffinnykh analogov tRNK, soderzhashchikh ostatki 6-tioguanozina.

Photoreactive derivatives of tRNAs, containing 6-thioguanosine or diazirine derivative of 5-methyleneaminouridine were compared as probes to modify Escherichia coli ribosomes. The derivatives of tRNA were synthesized by T7 transcription Proportion of the modified nucleotide analogues was optimised to obtain good yield, analogue incorporation and binding to the ribosome. Complexes of the tRNA analogues with the ribosomal P-site were irradiated with mild UV light. Cross-links were analysed by oligonucleotide-directed hydrolysis of rRNA by RNase H and reverse transcription. 6-thioguanosine was proved to be a perspective reagent for cross-linking studies of complex ribonucleoproteides.

Efficiency and pattern of UV pulse laser-induced RNA-RNA cross-linking in the ribosome.

Escherichia coli ribosomes were irradiated with a KrF excimer laser (248 nm, 22 ns pulse) with incident pulse energies in the range of 10-40 mJ for a 1 cm2 area, corresponding to fluences of 4.5 to 18 x 10(9) W m(-2), to determine strand breakage yields and the frequency and pattern of RNA-RNA cross- linking in the 16S rRNA. Samples were irradiated in a cuvette with one laser pulse or in a flow cell with an average of 4.6 pulses per sample. The yield of strand breaks per photon was intensity dependent, with values of 0.7 to 1.3 x 10(-3) over the incident intensity range studied. The yield for RNA-RNA cross-linking was 3 x 10(-4) cross-links/photon at the intensity of 4.5 x 10(9) W m(-2), an approximately 4-fold higher yield per photon than obtained with a transilluminator. The cross-link yield/photon decreased at higher light intensities, probably due to intensity-dependent photoreversal. The pattern of cross-linking was similar to that observed with low intensity irradiation but with four additional long-range cross-links not previously seen in E.coli ribosomes. Cross- linking frequencies obtained with one laser pulse are more correlated to internucleotide distances than are frequencies obtained with transilluminator irradiation.