The 40S Ribosomal Protein S6 Response to Blue Light by Interaction with SjAUREO in Saccharina japonica.

Blue light (BL) plays an important role in regulation of the growth and development of aquatic plants and land plants. Aureochrome (AUREO), the recent BL photoreceptor identified in photosynthetic stramenopile algae, is involved in the photomorphogenesis and early development of Saccharina japonica porophytes (kelp). However the factors that interact with the SjAUREO under BL conditions specifically are not clear. Here in our study, three high quality cDNA libraries with CFU over 5 × 106 and a recombination rate of 100% were constructed respectively through white light (WL), BL and darkness (DK) treatments to the juvenile sporophytes. Based on the constructed cDNA libraries, the interactors of SjAUREO were screened and analyzed. There are eighty-four genes encoding the sixteen predicted proteins from the BL cDNA library, sixty-eight genes encoding eighteen predicted proteins from the DK cDNA library, and seventy-four genes encoding nineteen proteins from the WL cDNA library. All the predicted proteins are presumed to interact with SjAUREO when co-expressed with SjAUREO seperately. The 40S ribosomal protein S6 (RPS6), which only exists in the BL treated cDNA library except for two other libraries, and which is essential for cell proliferation and is involved in cell cycle progression, was selected for detailed analysis. We showed that its transcription was up-regulated by BL, and was highly transcribed in the basal blade (meristem region) of juvenile sporophytes but less in the distal part. Taken together, our results indicated that RPS6 was highly involved in BL-mediated kelp cellular division and photomorphogenesis by interacting with SjAUREO.

Candidate tumour suppressor Fau regulates apoptosis in human cells: an essential role for Bcl-G.

FAU, which encodes a ubiquitin-like protein (termed FUBI) with ribosomal protein S30 as a carboxy-terminal extension, has recently been identified as a pro-apoptotic regulatory gene. This activity may be mediated by Bcl-G (a pro-apoptotic member of the Bcl-2 family) which can be covalently modified by FUBI. FAU gene expression has been shown to be down-regulated in human breast, prostate and ovarian tumours, and this down-regulation is strongly associated with poor prognosis in breast cancer. We demonstrate here that ectopic FAU expression increases basal apoptosis in human T-cell lines and 293T/17 cells, whereas it has only a transient stimulatory effect on ultraviolet-C (UVC)-induced apoptosis. Conversely, siRNA-mediated silencing of FAU gene expression has no effect on basal apoptosis, but attenuates UV-induced apoptosis. Importantly, prior knockdown of Bcl-G expression ablates the stimulation of basal apoptosis by FAU, consistent with an essential downstream role for Bcl-G, itself a candidate tumour suppressor, in mediating the apoptosis regulatory role of FAU. In 293T/17 cells, Bcl-G knockdown also attenuates UV-induced apoptosis, so that Bcl-G may constitute a common factor in the pathways by which both FAU and UV-irradiation induce apoptosis. UV irradiation increases Bcl-G mRNA levels, providing an explanation for the transient nature of the effect of ectopic FAU expression on UV-induced apoptosis. Since failure of apoptosis is fundamental to the development of many cancers, the pro-apoptotic activity of the Fau/Bcl-G pathway offers an attractive explanation for the putative tumour suppressor role of FAU.

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.

Mutational effects of different LET radiations in rpsL transgenic Arabidopsis.

PURPOSE: In an effort to assess the characteristics of mutation induced by different linear energy transfer (LET) radiation in higher plants, the mutational effects of carbon-ion beams and gamma-rays were investigated in Arabidopsis. MATERIALS AND METHODS: The rpsL (Escherichia coli ribosomal protein small subunit S12) transgenic Arabidopsis (Arabidopsis/rpsL) mutation detection system was adopted. Dry seeds of Arabidopsis/rpsL were irradiated with gamma-rays and 208-MeV carbon ions (208-MeV (12)C(5+)), and the mutation frequency and mutation spectrum were examined. RESULTS: The frequency of mutant clones increased following irradiation with 208-MeV (12)C(5+) and gamma-rays. Mutation spectrum analysis showed that G:C to A:T transitions and >2 bp deletions/insertions were significantly induced by both 208-MeV (12)C(5+) and gamma-rays. -1 and -2 frameshift mutations were characteristic in the gamma-ray irradiated group. CONCLUSIONS: 208-MeV (12)C(5+) and gamma-rays induced different intragenic mutations in respect to the size of deletions, reflecting differences in the nature of the DNA damage induced. Our results also suggested that base substitutions derived from the generation of 8-oxoguanine were low in dry seeds. The mutation spectrum obtained in this study might have reflected the characteristic conditions of plant dry seeds such as low water content and cell proliferation activity.

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.

Crosslinking of ribosomal proteins to RNA in maize ribosomes by UV-B and its effects on translation.

Ultraviolet-B (UV-B) photons can cause substantial cellular damage in biomolecules, as is well established for DNA. Because RNA has the same absorption spectrum for UV as DNA, we have investigated damage to this cellular constituent. In maize (Zea mays) leaves, UV-B radiation damages ribosomes by crosslinking cytosolic ribosomal proteins S14, L23a, and L32, and chloroplast ribosomal protein L29 to RNA. Ribosomal damage accumulated during a day of UV-B exposure correlated with a progressive decrease in new protein production; however, de novo synthesis of some ribosomal proteins is increased after 6 h of UV-B exposure. After 16 h without UV-B, damaged ribosomes were eliminated and translation was restored to normal levels. Ribosomal protein S6 and an S6 kinase are phosphorylated during UV-B exposure; these modifications are associated with selective translation of some ribosomal proteins after ribosome damage in mammalian fibroblast cells and may be an adaptation in maize. Neither photosynthesis nor pigment levels were affected significantly by UV-B, demonstrating that the treatment applied is not lethal and that maize leaf physiology readily recovers.

Three-dimensional placement of the conserved 530 loop of 16 S rRNA and of its neighboring components in the 30 S subunit.

Nucleotides 518-533 form a loop in ribosomal 30 S subunits that is almost universally conserved. Both biochemical and genetic evidence clearly implicate the 530 loop in ribosomal function, with respect both to the accuracy control mechanism and to tRNA binding. Here, building on earlier work, we identify proteins and nucleotides (or limited sequences) site-specifically photolabeled by radioactive photolabile oligoDNA probes targeted toward the 530 loop of 30 S subunits. The probes we employ are complementary to 16 S rRNA nucleotides 517-527, and have aryl azides attached to nucleotides complementary to nucleotides 518, 522, and 525-527, positioning the photogenerated nitrene a maximum of 19-26 A from the complemented rRNA base. The crosslinks obtained are used as constraints to revise an earlier model of 30 S structure, using the YAMMP molecular modeling package, and to place the 530 loop region within that structure.

Photochemical cross-linking of the anticodon loop of yeast tRNA(Phe) to 30S-subunit protein S7 at the ribosomal A and P sites.

Yeast tRNA(Phe), containing the photoreactive nucleoside 2-azidoadenosine at position 37 within the anticodon loop, has been cross-linked to the aminoacyl-tRNA (A) and peptidyl-tRNA (P) binding sites of the Escherichia coli ribosome. The 30S subunit was exclusively labeled in each case, and cross-linking occurred to both protein and 16S rRNA. Electrophoretic and immunological analyses demonstrated that S7 was the only 30S-subunit protein covalently attached to the tRNA. However, digestion of the A and P site-labeled S7 with trypsin revealed a unique pattern of cross-linked peptide(s) at each site. Thus, while the anticodon loop of tRNA is in close proximity to protein S7 at both the A and P sites, it neighbors a different portion of the protein molecule in each. The placement of the aminoacyl- and peptidyl-tRNA binding sites is discussed in relationship to recent models of the 30S ribosomal subunit.

Study of several factors in RNA-protein cross-link formation induced by ionizing radiations within 70S ribosomes of E. coli MRE 600.

The induction of RNA-protein crosslinks in E. coli 70S ribosomes by gamma-irradiation was studied by measuring the dependence of cross-link formation on ribosome concentration. The inverse dependence of cross-link percentage upon concentration up to at least 20 A260 nm units ml-1 indicate that indirect effects seem to play a more major part than direct effects for these ribosome concentrations. The effect of various gases and free radical scavengers was used to determine the roles of the radicals H., CO2-., OH. and e-aq and to estimate their relative efficiencies for cross-links. They were found to be: 7.2(H.), 6(CO2-.), 2(OH.) and 1(e-aq). The extent of RNA-protein cross-link production in 70S ribosomes induced by gamma-rays and neutrons in the presence and absence of oxygen was also investigated. Cross-link formation was estimated by separation of linked and unlinked material on nitrocellulose filters or after separation by SDS-sucrose gradient centrifugation under dissociating conditions. Oxygen inhibited cross-link formation by both neutrons and gamma-rays. However, very few cross-links were formed in de-aerated solutions by exposure to neutrons, compared to those produced by gamma-rays under the same conditions. This suggests that molecular oxygen generated along the secondary particle track can reduce the formation of RNA-protein cross-links.

Induction of polynucleotide-protein cross-linkages by ultraviolet irradiation. Peculiarities of the high-intensity laser pulse irradiation.

The efficiency and specificity of RNA-protein cross-linking in the 30S subunit of Escherichia coli ribosomes, induced by low-intensity (10(15) photons cm-2 s-1, 254 nm) and high-intensity [(1.6-6.8) X 10(24) photons cm-2 s-1, 266 nm, pulse duration 10(-8) s] ultraviolet radiation, are studied. Under the former conditions proteins S4, S7 and S9/S11, and under the latter conditions these proteins together with S3, S18 and S20, are cross-linked to 16S RNA. Biphotonic processes operate in the latter case. In the presence of 2-mercaptoethanol cross-linking occurs either directly, via a higher excited state or via activated intermediates with life-times less than 25 ns. Cross-links thus formed are specific, i.e. they are formed between regions of macromolecules which are in contact in the native (non-disturbed) complex prior to excitation. The efficiency of cross-linking (per photon absorbed) is 20-100 times higher upon two-step excitation as compared with single-step excitation and an analysable number of cross-links are produced in a single pulse. Only base U-1239 of 16S RNA is cross-linked to protein S7 by low-intensity radiation, whereas the adjacent base, G-1240 is also involved in laser-induced cross-linking. A transition from the former to the latter conditions allows one to reduce the duration of irradiation from several minutes to several nanoseconds.

Effects of actinomycin D on mouse thymus in vivo chromatin degradation to nucleosomes and inhibition of RNA and protein synthesis.

The administration of actinomycin D in toxic doses (4 mg/kg i.p.) to mice induces a progressive degradation of thymus chromatin. The course of this damage is slower than the radiation-induced chromatin degradation; it starts at 6-8h and culminates at 24 h after actinomycin D injection, when already most of the treated mice are dying. The inhibition of the [2-14C] orotic acid incorporation into rapidly labelled RNA by actinomycin D is substantially less expressed in the thymus than in the liver. The course of chromatin degradation correlates with the onset of the inhibition of protein synthesis in the thymuses of actinomycin D-treated mice. This demonstrates that, starting from the 6th h after actinomycin D administration, irreversible changes appear in the thymocytes that lead to chromatin degradation and cellular death. Contrary to the action of cycloheximide, the treatment with actinomycin D aggravates the chromatin degradation induced by irradiation. A comparative electrophoretic analysis of the DNA fragments (in native state and denatured) isolated from thymus chromatin of mice treated with actinomycin D (and/or irradiated) demonstrated that the chromatin DNA is degraded only in the internucleosomal segments yielding DNA fragments of nucleosomal size. Similarly to the effect of other agents leading to chromatin damage in vivo, the actinomycin D-induced degradation does not affect the DNA in the nucleosomal segments of chromatin.

Identification of yeast 60 S ribosomal proteins crosslinked to rRNA by 2-iminothiolane.

Saccharomyces carlsbergensis 60 S ribosomal subunits were treated with the hetero-bifunctional crosslinking agent 2-iminothiolane and then subjected to mild UV irradiation to introduce protein-rRNA crosslinks. The major crosslinked products were identified as proteins L2, L3, L5, L19 and L23 of which L5 was found to be crosslinked at a 3-5-fold higher efficiency than the other four. Several additional proteins were cross-linked to a detectable but much lower extent.

Intersubunit RNA-protein contacts in pre- and post-translocated E. coli ribosome.

Ribosomal proteins participating in intersubunit RNA-protein contacts (directly interacting with RNA of the opposite subunit) were determined by means of ultraviolet-induced cross-links in pre- and post-translocated ribosomal complexes, as well as in the free 70 S ribosome (tight couple) of E. coli. In these 3 complexes at least L1 and L9 proteins interact with 16 S RNA, while S6, S9/11 and S15 react with 23 S RNA. All these proteins ('hinge-joint' proteins) are clustered on the small protuberance of the 50 S subunit and on the platform of the 30 S subunit. Reduction in the number of other (variable) intersubunit RNA-protein contacts in the course of transition from the tight couple to the pre- and, finally, to the post-translocated state, demonstrates gradual loosening of intersubunit interactions in 70 S ribosome. Such a loosening ('opening') of the 70 S ribosome is determined by conformational changes in ribosomal subunits and/or in their relative arrangement, conjugated with alteration of the functional state of the ribosomal complex.

Photo-induced protein cross-linking to 5S RNA and 28-5.8S RNA within rat-liver 60S ribosomal subunits.

Rat liver 60S ribosomal subunits were irradiated with 254-nm ultraviolet light (1.26 X 10(4) quanta/subunit), under conditions which preserved their functional activity. Cross-linked RNA-protein complexes were recovered after unreacted proteins had been removed by repeated acetic acid extractions. Proteins linked to the whole rRNA, to 5S RNA and to 28-5.8 S RNAs were identified by two-dimensional gel electrophoresis after RNA hydrolysis by ribonucleases T1 and A. Our results showed that numerous proteins interact with rRNAs (at least ten with 28-5.8 S RNA, eight with 5S RNA and among these three are common to both) and have been discussed in the light of all the available data.

[Changes in subunit conformation and their reciprocal configuration in the transition from the pretranslocated to the posttranslocated state]. / Izmenenie konformatsii subchastits i ikh vzaimnogo raspolozheniia pri perekhode ot pretranslotsirovannogo sostoianiia k posttranslotsirovannomu.

RNA-protein contacts in pretranslocated and posttranslocated states of E. coli ribosomes have been determined by means of UV-induced cross-linking. In the two functional states as well as in free 70C ribosome, the same proteins are involved in RNA-protein intersubunit contacts, located in the region of L1 protuberance (left side of 70S ribosome). The transition from pre- to posttranslocated state is accompanied by disappearance of RNA-protein contacts in the region of L7/L12 stalk. This favours the locking-unlocking model of the translating ribosome.

Ribosomal RNA-protein cross-links, induced by gamma-irradiation of 40S and 60S ribosomal subunits of L cells.

The 40S and 60S ribosomal subunits of L cells are gamma-irradiated in the absence of oxygen at low radiation doses to keep the integrity of the ribosomal structure. We show that under these experimental conditions, specific cross-links are induced in situ between rRNA and ribosomal proteins due to close contact between their reactive groups. We found that about 15 proteins are cross-linked to the 28S RNA. Most of them belong to the core proteins of the 60S ribosomal subunits. A few high-molecular mass proteins which might be factors are also bound to 28S RNA. Between 8 and 11 proteins are covalently linked to 18S RNA; 8 of these have been previously described as transferable proteins from one subunit to the other. Only 3 are core proteins of the small subunit. The contribution of these results to the study of the three-dimensional ribosomal structure is also discussed.

Identification of the proteins interacting with tRNA in rat liver small ribosomal subunits.

Irradiation at 254 nm of the rat liver 40 S ribosomal subunit-poly(U)-Phe[32P]tRNA complex induced a covalent linkage of tRNA with a limited number of ribosomal proteins. After RNA hydrolysis, S10 was found to be the protein most highly labeled by radioactive nucleotides. Some radioactivity was also associated with protein S6-6a, S3a, S2 and S13-15.

Proteins of the 30-S subunit of Escherichia coli ribosomes which interact directly with natural mRNA.

Ultraviolet irradiation (254 nm) of the complexes of MS2 phage RNA (mRNA) and the 30-S subunits of Escherichia coli ribosomes prepared at 0 degrees C and 37 degrees C in the presence and absence of initiation factor 3 (IF-3) causes cross-linking of mRNA with proteins S3, S4, S5, S7, S9, S18 and IF-3. Hence, these proteins interact directly with mRNA within the complex 30-S-subunit . mRNA. Addition of IF-3 results in an increase of the rate of complex formation and decrease of its dissociation rate. The addition of IF-3 changes the relative amounts of cross-linked proteins (mainly S3, S4 and S18). Decreasing the temperature from 37 degrees C to 0 degrees C not only decelerates the complex formation rate but also changes the relative amount of cross-linked proteins, indicating the influence of the conditions on the structure of the complex.

Photochemical cross-linking of translation initiation factor 3 to Escherichia coli 50S ribosomal subunits.

Translation initiation factor 3 (IF-3) was bound noncovalently to Escherichia coli 50S ribosomal subunits. Irradiation of such complexes with near-ultraviolet light (greater than 285 nm) resulted in covalent attachment of initiation factor 3 to the 50S subunit. Photo-cross-linking attained its maximum level of 40% of that which was noncovalently bound after 90 min of irradiation. Cross-linking was abolished in the presence of either 0.5 M NH4C1 or 0.25 mM aurintricarboxylic acid, indicating that specific binding of initiation factor 3 to the ribosome was a prerequisite for subsequent covalent attachment. Further analysis showed that all the IF-3 was covalently bound to a small number of 50S subunit proteins. The major cross-linked proteins were identified as L2, L7/L12, L11, and L27 by immunochemical techniques. These results are discussed in light of the proposed mechanism for IF-3 function.