Heat Shock Gene Expression Research Articles

Thermozymes

Synthetic biology approaches often combine natural building blocks to generate new cellular activities. Here, we make use of two RNA elements to design a regulatory device with novel functionality. The system is based on a hammerhead ribozyme (HHR) that cleaves itself to generate a liberated ribosome-binding site and, thus, permits expression of a downstream gene. We connected a temperature-responsive RNA hairpin to the HHR and, thus, generated a temperature-controlled ribozyme that we call thermozyme. Specifically, a Salmonella RNA thermometer (RNAT) known to modulate small heat shock gene expression by temperature-controlled base-pairing and melting was fused to the ribozyme. Following an in vivo screening approach, we isolated two functional thermozymes. In vivo expression studies and in vitro structure probing experiments support a mechanism in which rising temperatures melt the thermometer structure impairing the self-cleavage reaction of the ribozyme. Since RNA cleavage is necessary to liberate the RBS, these engineered thermozymes shut off gene expression in response to a temperature increase and, thus, act in a reverse manner as the natural RNAT. Our results clearly emphasize the highly modular nature and biotechnological potential of ribozyme-based RNA thermometers.

Regulatory Coordination between Two Major Intracellular Homeostatic Systems: HEAT SHOCK RESPONSE AND AUTOPHAGY

The eukaryotic cell depends on multitiered homeostatic systems ensuring maintenance of proteostasis, organellar integrity, function and turnover, and overall cellular viability. At the two opposite ends of the homeostatic system spectrum are heat shock response and autophagy. Here, we tested whether there are interactions between these homeostatic systems, one universally operational in all prokaryotic and eukaryotic cells, and the other one (autophagy) is limited to eukaryotes. We found that heat shock response regulates autophagy. The interaction between the two systems was demonstrated by testing the role of HSF-1, the central regulator of heat shock gene expression. Knockdown of HSF-1 increased the LC3 lipidation associated with formation of autophagosomal organelles, whereas depletion of HSF-1 potentiated both starvation- and rapamycin-induced autophagy. HSP70 expression but not expression of its ATPase mutant inhibited starvation or rapamycin-induced autophagy. We also show that exercise induces autophagy in humans. As predicted by our in vitro studies, glutamine supplementation as a conditioning stimulus prior to exercise significantly increased HSP70 protein expression and prevented the expected exercise induction of autophagy. Our data demonstrate for the first time that heat shock response, from the top of its regulatory cascade (HSF-1) down to the execution stages delivered by HSP70, controls autophagy thus connecting and coordinating the two extreme ends of the homeostatic systems in the eukaryotic cell.

Post-Transcriptional Regulation of the Trypanosome Heat Shock Response by a Zinc Finger Protein

In most organisms, the heat-shock response involves increased heat-shock gene transcription. In Kinetoplastid protists, however, virtually all control of gene expression is post-transcriptional. Correspondingly, Trypanosoma brucei heat-shock protein 70 (HSP70) synthesis after heat shock depends on regulation of HSP70 mRNA turnover. We here show that the T. brucei CCCH zinc finger protein ZC3H11 is a post-transcriptional regulator of trypanosome chaperone mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of insect-form trypanosomes from heat shock. ZC3H11 binds to mRNAs encoding heat-shock protein homologues, with clear specificity for the subset of trypanosome chaperones that is required for protein refolding. In procyclic forms, ZC3H11 was required for stabilisation of target chaperone-encoding mRNAs after heat shock, and the HSP70 mRNA was also decreased upon ZC3H11 depletion in bloodstream forms. Many mRNAs bound to ZC3H11 have a consensus AUU repeat motif in the 3′-untranslated region. ZC3H11 bound preferentially to AUU repeats in vitro, and ZC3H11 regulation of HSP70 mRNA in bloodstream forms depended on its AUU repeat region. Tethering of ZC3H11 to a reporter mRNA increased reporter expression, showing that it is capable of actively stabilizing an mRNA. These results show that expression of trypanosome heat-shock genes is controlled by a specific RNA-protein interaction. They also show that heat-shock-induced chaperone expression in procyclic trypanosome enhances parasite survival at elevated temperatures.

Thermal adaptation and acclimation of ectotherms from differing aquatic climates

To elucidate the mechanisms of thermal adaptation and acclimation in ectothermic aquatic organisms from differing climates, we used a common-garden experiment for thermal stress to investigate the heat shock response of redband trout (Oncorhynchus mykiss gairdneri) from desert and montane populations. Evidence for adaptation was observed as expression of heat shock genes in fish from the desert population was more similar to control (unstressed) fish and significantly different (P ≤ 0.05) from those from the montane population, while F1 crosses were intermediate. High induction of heat shock proteins (Hsps) in the montane strain appeared to improve short-term survival during first exposure to high water temperatures, but high physiological costs of Hsp production may have led to lower long-term survival. In contrast, the desert strain had significantly lower heat shock response than the montane fish and F1 crosses, suggesting that these desert fish have evolved alternative mechanisms to deal with thermal stress that provide better balance of physiological costs. Genomewide tests of greater than 10 000 SNPs found multiple SNPs that were significantly associated with survival under thermal stress, including Hsp47 which consistently appeared as a strong candidate gene for adaption to desert climates. Candidate SNPs identified in this study are prime targets to screen more broadly across this species' range to predict the potential for adaptation under scenarios of climate change. These results demonstrate that aquatic species can evolve adaptive responses to thermal stress and provide insight for understanding how climate change may impact ectotherms.

Induction of heat shock gene expression in RAT1 primary fibroblast cells by ELF electric fields

Recent studies have demonstrated that the Ku70 gene fragment can be placed in the anti-sense orientation under the control of a heat-inducible heat shock protein 70 (HSP70) promoter and activated through heat shock exposure. This results in attenuation of the Ku70 protein expression, inhibiting cellular repair processes, and sensitizing the transfected cells to exposures such as the ionizing radiation exposures used clinically. However, achieving the tissue temperatures necessary to thermally induce the HSP70 response presents significant limitations to the clinical application of this strategy. Previous findings suggest an alternative approach to inducing a heat shock response, specifically through the use of extremely low frequency (ELF) electrical field stimulation. To further pursue this approach, we investigated HSP70 responses in transfected rat primary fibroblast (RAT1) cells exposed to 10 Hz electric fields at intensities of 20-500 V/m. We confirmed that low frequency electric fields can induce HSP70 heat shock expression, with peak responses obtained at 8 h following a 2 h field exposure. However, the approximate threefold increase in expression is substantially lower than that obtained using thermal stimulation, raising questions of the clinical utility of the response.

In vivo imaging of induction of heat-shock protein-70 gene expression with fluorescence reflectance imaging and intravital confocal microscopy following brain ischaemia in reporter mice

Stroke induces strong expression of the 72-kDa heat-shock protein (HSP-70) in the ischaemic brain, and neuronal expression of HSP-70 is associated with the ischaemic penumbra. The aim of this study was to image induction of Hsp-70 gene expression in vivo after brain ischaemia using reporter mice. A genomic DNA sequence of the Hspa1b promoter was used to generate an Hsp70-mPlum far-red fluorescence reporter vector. The construct was tested in cellular systems (NIH3T3 mouse fibroblast cell line) by transient transfection and examining mPlum and Hsp-70 induction under a challenge. After construct validation, mPlum transgenic mice were generated. Focal brain ischaemia was induced by transient intraluminal occlusion of the middle cerebral artery and the mice were imaged in vivo with fluorescence reflectance imaging (FRI) with an intact skull, and with confocal microscopy after opening a cranial window. Cells transfected with the Hsp70-mPlum construct showed mPlum fluorescence after stimulation. One day after induction of ischaemia, reporter mice showed a FRI signal located in the HSP-70-positive zone within the ipsilateral hemisphere, as validated by immunohistochemistry. Live confocal microscopy allowed brain tissue to be visualized at the cellular level. mPlum fluorescence was observed in vivo in the ipsilateral cortex 1day after induction of ischaemia in neurons, where it is compatible with penumbra and neuronal viability, and in blood vessels in the core of the infarction. This study showed in vivo induction of Hsp-70 gene expression in ischaemic brain using reporter mice. The fluorescence signal showed in vivo the induction of Hsp-70 in penumbra neurons and in the vasculature within the ischaemic core.

Expression of hsp70, hsp100 and ubiquitin in Aloe barbadensis Miller under direct heat stress and under temperature acclimation conditions

KEY MESSAGE : The study determined the tolerance of Aloe vera to high temperature, focusing on the expression of hsp70 , hsp100 and ubiquitin genes. These were highly expressed in plants acclimated at 35°C prior to a heat shock of 45°C. Aloe barbadensis Miller (Aloe vera), a CAM plant, was introduced into Chile in the semiarid IV and III Regions, which has summer diurnal temperature fluctuations of 25 to 40°C and annual precipitation of 40mm (dry years) to 170mm (rainy years). The aim of this study was to investigate how Aloe vera responds to water and heat stress, focusing on the expression of heat shock genes (hsp70, hsp100) and ubiquitin, which not studied before in Aloe vera. The LT(50) of Aloe vera was determined as 53.2°C. To study gene expression by semi-quantitative RT-PCR, primers were designed against conserved regions of these genes. Sequencing the cDNA fragments for hsp70 and ubiquitin showed a high identity, over 95%, with the genes from cereals. The protein sequence of hsp70 deduced from the sequence of the cDNA encloses partial domains for binding ATP and the substrate. The protein sequence of ubiquitin deduced from the cDNA encloses a domain for interaction with the enzymes E2, UCH and CUE. The expression increased with temperature and water deficit. Hsp70 expression at 40-45°C increased 50% over the controls, while the expression increased by 150% over the controls under a water deficit of 50% FC. The expression of all three genes was also studied under 2h of acclimation at 35 or 40°C prior to a heat shock at 45°C. Under these conditions, the plants showed greater expression of all genes than when they were subjected to direct heat stress.

Hyperosmotic Stress Response of Campylobacter jejuni

The diarrheal pathogen Campylobacter jejuni and other gastrointestinal bacteria encounter changes in osmolarity in the environment, through exposure to food processing, and upon entering host organisms, where osmotic adaptation can be associated with virulence. In this study, growth profiles, transcriptomics, and phenotypic, mutant, and single-cell analyses were used to explore the effects of hyperosmotic stress exposure on C. jejuni. Increased growth inhibition correlated with increased osmotic concentration, with both ionic and nonionic stressors inhibiting growth at 0.620 total osmol liter(-1). C. jejuni adaptation to a range of osmotic stressors and concentrations was accompanied by severe filamentation in subpopulations, with microscopy indicating septum formation and phenotypic diversity between individual cells in a filament. Population heterogeneity was also exemplified by the bifurcation of colony morphology into small and large variants on salt stress plates. Flow cytometry of C. jejuni harboring green fluorescent protein (GFP) fused to the ATP synthase promoter likewise revealed bimodal subpopulations under hyperosmotic stress. We also identified frequent hyperosmotic stress-sensitive variants within the clonal wild-type population propagated on standard laboratory medium. Microarray analysis following hyperosmotic upshift revealed enhanced expression of heat shock genes and genes encoding enzymes for synthesis of potential osmoprotectants and cross-protective induction of oxidative stress genes. The capsule export gene kpsM was also upregulated, and an acapsular mutant was defective for growth under hyperosmotic stress. For C. jejuni, an organism lacking most conventional osmotic response factors, these data suggest an unusual hyperosmotic stress response, including likely "bet-hedging" survival strategies relying on the presence of stress-fit individuals in a heterogeneous population.

Heat Shock and Caloric Restriction Have a Synergistic Effect on the Heat Shock Response in a sir2.1-dependent Manner in Caenorhabditis elegans

The heat shock response (HSR) is responsible for maintaining cellular and organismal health through the regulation of proteostasis. Recent data demonstrating that the mammalian HSR is regulated by SIRT1 suggest that this response may be under metabolic control. To test this hypothesis, we have determined the effect of caloric restriction in Caenorhabditis elegans on activation of the HSR and have found a synergistic effect on the induction of hsp70 gene expression. The homolog of mammalian SIRT1 in C. elegans is Sir2.1. Using a mutated C. elegans strain with a sir2.1 deletion, we show that heat shock and caloric restriction cooperate to promote increased survivability and fitness in a sir2.1-dependent manner. Finally, we show that caloric restriction increases the ability of heat shock to preserve movement in a polyglutamine toxicity neurodegenerative disease model and that this effect is dependent on sir2.1.

Bisphenol-A affects the developmental progression and expression of heat-shock protein genes in the moth Sesamia nonagrioides

The effects of bisphenol A (BPA) on the endocrine system of vertebrates have been demonstrated in several studies. Here, we report the impact of BPA on the developmental progression and expression of heat shock protein genes on the terrestrial insect Sesamia nonagrioides (Lepidoptera: Noctuidae). S. nonagrioides 1st instar larvae were exposed until the end of 6th (last) instar to selected concentrations of BPA (1 μg/L, 10 μg/L, 100 μg/L, 1 mg/L and 10 mg/L) applied in their artificial diets. The lower doses of BPA (1-10 μg/L) were found to decrease larvae's weight while the 100 μg/L dose increased it. The higher doses of BPA were found to induce various abnormal phenotypes during 5th instar larval molting, larval-pupal transformation and metamorphosis. The developmental and metamorphosis endpoints presented here may indicate the possible impact of BPA on terrestrial insects. Additionally, 6th instar larvae were injected with several concentrations of BPA. Semi-quantitative and Real-Time PCR assays were used to identify the effects of BPA in the transcriptional regulation of five heat shock protein genes (SnoHsp19.5, SnoHsp20.8, SnoHsp70, SnoHsc70 and SnoHsp83). Application of BPA by feeding or by injection induced the synthesis of the SnoHsp19.5 and SnoHsp20.8 mRNAs. The expression levels of SnoHsp70 were not affected. In contrast, SnoHsc70 and SnoHsp83, which play a pivotal role in vertebrate sex steroid signal transduction, were elevated by BPA. Our results suggest that SnoHsp19.5, SnoHsp20.8, SnoHsp83 and SnoHsc70 genes can be modulated by BPA.

Abstract 2261: Heat-shock and autophagy in cancer: Novel role for DGK iota

Abstract Autophagy is a cellular process by which damaged proteins, protein aggregates, and organelles are sequestered by lysosome-like compartments and degraded. Autophagy contributes to quality control, cell death, and senescence. Defects in autophagy cause the accumulation of cellular damage, inhibit cell death processes, and promote an inflammatory state that facilitates tumor development. In contrast, the heat shock response, which is mediated primarily by the transcription factor, HSF1 is frequently enhanced in cancer. Despite this inverse relationship between autophagy and heat-shock in cancer, the direct influence of heat-shock and HSF1-mediated gene expression on autophagy has not been investigated. Our study suggests that heat-shock mediated gene expression suppresses autophagy in a human colorectal cancer cell line. Our data show that diacylglycerol kinase iota (DGK iota) expression is regulated by HSF1. DGK iota phosphorylates diacylglycerol, generating phosphatidic acid (PA), which is a potent suppressor of autophagy through its activation of the serine/threonine kinase mTOR (mammalian target of rapamycin). The aim of our study was to evaluate the influence of HSF-1-mediated gene expression on autophagy, and to examine the specific contribution of DGK iota in this effect. We utilized RKO colorectal cancer cells for this investigation and assayed autophagy using complimentary techniques, including: conversion of LC3-I to LC3-II, which was monitored by Western blot; and the formation of autophagic vesicles (autophagosomes), which were visualized by high-content screening confocal microscopy of cells harboring an LC3-GFP expression construct. The influence of basal and heat-shock (42°C)-simulated HSF1 activity on autophagy was analyzed using siRNA. Silencing of HSF1 led to a profound increase in autophagic markers, suggesting a role for HSF1-driven gene expression in the suppression of autophagy. In HSF1-deficient cells we also observed increased phosphorylation of p70S6K, which is a surrogate marker for mTOR activation. We confirmed that DGK iota mRNA and protein expression is mediated by HSF1. Using biochemical inhibitors of DGK activity, we observed a significant increase in autophagy, supporting our hypothesis that DGK iota participates in suppressing autophagy. Additional siRNA experiments of DGK iota are being performed to confirm this effect, along with measurements of cellular PA levels. Taken together, these results indicate an important role for HSF1 in the suppression of autophagy, and provides mechanistic insight into a potential, tumor-promoting role for DGK iota. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2261. doi:1538-7445.AM2012-2261

Thiostrepton is an inducer of oxidative and proteotoxic stress that impairs viability of human melanoma cells but not primary melanocytes

Pharmacological induction of oxidative and proteotoxic stress has recently emerged as a promising strategy for chemotherapeutic intervention targeting cancer cells. Guided by a differential phenotypic drug screen for novel lead compounds that selectively induce melanoma cell apoptosis without compromising viability of primary human melanocytes, we have focused on the cyclic pyridinyl-polythiazolyl peptide-antimicrobial thiostrepton. Using comparative gene expression-array analysis, the early cellular stress response induced by thiostrepton was examined in human A375 metastatic melanoma cells and primary melanocytes. Thiostrepton displayed selective antimelanoma activity causing early induction of proteotoxic stress with massive upregulation of heat shock (HSPA6, HSPA1A, DNAJB4, HSPB1, HSPH1, HSPA1L, CRYAB, HSPA5, DNAJA1), oxidative stress (HMOX1, GSR, SOD1), and ER stress response (DDIT3) gene expression, confirmed by immunodetection (Hsp70, Hsp70B′, HO-1, phospho-eIF2α). Moreover, upregulation of p53, proapoptotic modulation of Bcl-2 family members (Bax, Noxa, Mcl-1, Bcl-2), and induction of apoptotic cell death were observed. Thiostrepton rapidly induced cellular oxidative stress followed by inactivation of chymotrypsin-like proteasomal activity and melanoma cell-directed accumulation of ubiquitinated proteins, not observed in melanocytes that were resistant to thiostrepton-induced apoptosis. Proteotoxic and apoptogenic effects were fully antagonized by antioxidant intervention. In RPMI 8226 multiple myeloma cells, known to be exquisitely sensitive to proteasome inhibition, early proteotoxic and apoptogenic effects of thiostrepton were confirmed by array analysis indicating pronounced upregulation of heat shock response gene expression. Our findings demonstrate that thiostrepton displays dual activity as a selective prooxidant and proteotoxic chemotherapeutic, suggesting feasibility of experimental intervention targeting metastatic melanoma and other malignancies including multiple myeloma.

The histone phosphatase inhibitory property of plant nucleosome assembly protein-related proteins (NRPs)

SET/I(2)(PP2A), a member of the family of nucleosome assembly proteins (NAPs), has been previously described as a multifunctional protein inhibiting protein phosphatase 2A (PP2A)-mediated histone H3((pSer10)) dephosphorylation during the heat shock response in animal cells. In the present work we demonstrate that its plant orthologs, designated as NAP-related proteins (NRPs), have a similar invitro biochemical activity and interact with PP2A and histone H3((pSer10))invivo. Although heat shock gene promoters were found to be associated with histone H3((pSer10))-marked chromatin following a high temperature treatment, heat shock gene expression was not affected in NRP-deficient mutant Arabidopsis thaliana (L.) plantlets. These observations indicate that NRPs are potential regulators of histone dephosphorylation in plants, but they are dispensable for gene expression reorganization in response to heat shock.

Gene expression modulation by heat stress in Acidithiobacillus ferrooxidans LR

During bioleaching, Acidithiobacillus ferrooxidans is subjected to different types of stress, including heat stress, which affect bacterial growth. In this work, real time quantitative PCR was used to analyze the expression of heat shock genes, as well as genes that encode proteins related to several functional categories in A. ferrooxidans. Cells were submitted to long-term growth and heat shock, both at 40°C. The results showed that heat shock affected the expression levels of most genes investigated, whilst long-term growth at 40°C resulted in minor changes in gene expression, except for certain genes related to iron transport, which were strongly down-regulated, suggesting that the iron processing capability of A. ferrooxidans was affected by long-term growth at 40°C. A bioinformatic analysis of the genes' promoter regions indicated a putative transcriptional regulation by the σ(32) factor in 12 of the 31 genes investigated, suggesting the involvement of other regulatory mechanisms in the response of A. ferrooxidans to heat stress.

Cloning, expression, purification, crystallization and preliminary X-ray analysis of the 31 kDa Vibrio cholerae heat-shock protein VcHsp31.

The Gram-negative bacterium Vibrio cholerae, which is responsible for the diarrhoeal disease cholera in humans, induces the expression of numerous heat-shock genes. VcHsp31 is a 31 kDa putative heat-shock protein that belongs to the DJ-1/PfpI superfamily, functioning as both a chaperone and a protease. VcHsp31 has been cloned, overexpressed and purified by Ni(2+)-NTA affinity chromatography followed by gel filtration. Crystals of VcHsp31 were grown in the presence of PEG 6000 and MPD; they belonged to space group P2(1) and diffracted to 1.9 Å resolution. Assuming the presence of six molecules in the asymmetric unit, the Matthews coefficient was estimated to be 1.97 Å(3) Da(-1), corresponding to a solvent content of 37.4%.

Stress-Induced PARP Activation Mediates Recruitment of Drosophila Mi-2 to Promote Heat Shock Gene Expression

Eukaryotic cells respond to genomic and environmental stresses, such as DNA damage and heat shock (HS), with the synthesis of poly-[ADP-ribose] (PAR) at specific chromatin regions, such as DNA breaks or HS genes, by PAR polymerases (PARP). Little is known about the role of this modification during cellular stress responses. We show here that the nucleosome remodeler dMi-2 is recruited to active HS genes in a PARP–dependent manner. dMi-2 binds PAR suggesting that this physical interaction is important for recruitment. Indeed, a dMi-2 mutant unable to bind PAR does not localise to active HS loci in vivo. We have identified several dMi-2 regions which bind PAR independently in vitro, including the chromodomains and regions near the N-terminus containing motifs rich in K and R residues. Moreover, upon HS gene activation, dMi-2 associates with nascent HS gene transcripts, and its catalytic activity is required for efficient transcription and co-transcriptional RNA processing. RNA and PAR compete for dMi-2 binding in vitro, suggesting a two step process for dMi-2 association with active HS genes: initial recruitment to the locus via PAR interaction, followed by binding to nascent RNA transcripts. We suggest that stress-induced chromatin PARylation serves to rapidly attract factors that are required for an efficient and timely transcriptional response.

Glycinebetaine enhances the tolerance of tomato plants to high temperature during germination of seeds and growth of seedlings

Tomato (Lycopersicon esculentum cv. 'Moneymaker') was transformed with a codA gene, from Arthrobacter globiformis, for choline oxidase that had been modified to allow targeting to both chloroplasts and the cytosol. Glycinebetaine (GB) accumulated in seeds of transformed plants up to 1 µmol g(-1) dry weight (DW), while no detectable GB was found in wild-type (WT) seeds. The codA-transgenic seeds germinated faster and at higher frequency than WT seeds with high temperature treatment. After heat stress, levels of expression of a mitochondrial small heat-shock protein (MT-sHSP), heat-shock protein 70 (HSP70) and heat-shock cognate 70 (HSC70) were higher in transgenic seeds than in WT seeds during heat stress, and the accumulation of HSP70 was more prominent in codA-transgenic seeds than in WT seeds. Addition of GB to the germination medium or imbibition of seeds in a solution of GB enhanced the tolerance of WT seeds to high temperatures. WT seeds treated with exogenous GB also expressed heat-shock genes at elevated levels and accumulated more HSP70 than controls. Our results suggest that GB, either applied exogenously or accumulated in vivo in codA-transgenic seeds, enhanced the expression of heat-shock genes in and improved the tolerance to high temperature of tomato seeds during germination.

Modulation of the stability of the Salmonella fourU-type RNA thermometer.

RNA thermometers are translational control elements that regulate the expression of bacterial heat shock and virulence genes. They fold into complex secondary structures that block translation at low temperatures. A temperature increase releases the ribosome binding site and thus permits translation initiation. In fourU-type RNA thermometers, the AGGA sequence of the SD region is paired with four consecutive uridines. We investigated the melting points of the wild-type and mutant sequences. It was decreased by 5°C when a stabilizing GC basepair was exchanged by an AU pair or increased by 11°C when an internal AG mismatch was converted to a GC pair, respectively. Stabilized or destabilized RNA structures are directly correlated with decreased or increased in vivo gene expression, respectively. Mg2+ also affected the melting point of the fourU thermometer. Variations of the Mg2+ concentration in the physiological range between 1 and 2 mM translated into a 2.8°C shift of the melting point. Thus, Mg2+ binding to the hairpin RNA is regulatory relevant. Applying three different NMR techniques, two Mg2+ binding sites were found in the hairpin structure. One of these binding sites could be identified as outer sphere binding site that is located within the fourU motif. Binding of the two Mg2+ ions exhibits a positive cooperativity with a Hill coefficient of 1.47. Free energy values ΔG for Mg2+ binding determined by NMR are in agreement with data determined from CD measurements.

Heat shock response in olive (Olea europaea L.) twigs: Identification and analysis of a cDNA coding a class I small heat shock protein

The objective of this study was to investigate at the molecular level the heat shock response in olive by identifying sequences coding for heat shock proteins (HSPs). Young twigs of Olea europaea trees (cv Cellina di Nardò) were subjected to different temperature treatments in order to induce the expression of heat shock genes. In order to identify genes induced by heat treatment, we used a PCR-based approach to amplify specific HS cDNAs. Search for low molecular weight HSP sequences was performed in public domain databases in order to design specific primers based upon multisequence alignments. By this approach, we isolated the first full length cDNA encoding a low-molecular weight HSP from O. europaea. This is a class I low molecular weight HSP of 18.3 kDa, which is highly expressed in young twigs subjected to heat stress.

Site-Pecific Fluorescnce Dynamics in an RNA ‘THERMOMETER’ Reveals the Mechanism of Temperature-Sensitive Translation

The ROSE (Repression Of heat Shock gene Expression) element of mRNA present in the 5’-UTR of small heat-shock genes in many Gram-negative bacteria is known to function as a ‘RNA thermometer’ by controlling protein translation in a temperature range of 30 - 42° C where the translation is blocked till 30°C and allowed at 42°C and beyond, perhaps due to an unfolding transition of the ROSE hair-pin motif.In this work, we have used site-specific fluorescence labeling and pico-second time-domain fluorescence spectroscopy to unravel the mechanism. The ‘ROSE RNA’ was site-specially labeled with 2-aminopurine (2-AP), a fluorescent analog of adenine. Observables such as fluorescence lifetime, fluorescence anisotropy decay kinetics and dynamic fluorescence quenching revealed properties such as the level of base stacking, rotational motion of the bases, segmental dynamics of the backbone and the level of exposure of base to solvent. As expected, all read-outs of 2-AP residue that were studied showed remarkable position-dependence/sensitivity in the RNA sequence at 25°C. The striking result was the persistence of the same position-dependence of the parameters even at 45°C albeit at a measurably reduced levels. However the same position-dependence was nearly ‘wiped out’ in the presence of urea where all intra-molecular interactions in RNA are undone. These observations have prompted us to revise the existing model of ROSE RNA action: we now suggest that unlike proposed earlier, the thermometer action of ROSE emanates not from its unfolding structural transition between 25 and 45°C, but rather from its propensity to enhance structural dynamics without “melting” the structure. We hypothesize that either the enhanced dynamics of the structure it self or its full melting due to an extrinsic factor (perhaps a protein interaction) might be the basis of its thermometer action.