The influence of cold shock proteins on transcription and translation studied in cell-free model systems Roland Hofweber1, Gudrun Horn1, Thomas Langmann2, Jochen Balbach3, Werner Kremer1, Gerd Schmitz2 and Hans R Kalbitzer1 Institut fur Biophysik und Physikalische Biochemie, Universitat Regensburg, Germany ă ă Institut fur Klinische Chemie und Laboratoriumsmedizin, Klinikum der Universitat Regensburg, Germany ă ă Laboratorium fur Biochemie, Universitat Bayreuth, Germany ă ă Keywords cold shock protein; in vitro translation; RNA chaperone Correspondence H R Kalbitzer, Universitat Regensburg, ă Institut fur Biophysik und Physikalische ă Biochemie, Universitatsstraòe 31, ă 93053 Regensburg, Germany Fax: +49 941 9432479 Tel: +49 941 9432594 E-mail: hans-robert.kalbitzer@biologie uni-regensburg.de (Received 21 March 2005, revised June 2005, accepted 27 July 2005) doi:10.1111/j.1742-4658.2005.04885.x Cold shock proteins (CSPs) form a family of highly conserved bacterial proteins capable of single-stranded nucleic acid binding They are suggested to act as RNA chaperones during cold shock inhibiting the formation of RNA secondary structures, which are unfavourable for transcription and translation To test this commonly accepted theory, isolated CSPs from a mesophilic, thermophilic and a hyperthermophilic bacterium (Bacillus subtilis, Bacillus caldolyticus and Thermotoga maritima) were studied in an Escherichia coli based cell free expression system on their capability of enhancing protein expression by reduction of mRNA secondary structures The E coli based expression of chloramphenicol acetyltransferase and of H-Ras served as model systems We observed a concentration-dependent suppression of transcription and translation by the different CSPs which makes the considered addition of CSPs for enhancing the protein expression in in vitro translation systems obsolete Protein expression was completely inhibited at CSP concentrations present under cold shock conditions The CSP concentrations necessary for 50% inhibition were lowest (140 lm) for the protein of the hyperthermophilic and increased when the thermophilic (215 lm) or even the mesophilic protein (451 lm) was used Isolated in vitro transcription under the influence of CSPs showed that the transcriptory effect is independent from the rest of the cell It could be shown in a control experiment that the inhibition of protein expression can be removed by addition of hepta-2’-desoxy-thymidylate (dT7); a heptanucleotide that competitively binds to CSP The data are in line with a hypothesis that CSPs act on bulk protein expression not as RNA chaperones but inhibit their transcription and translation by rather unspecific nucleic acid binding Organisms have achieved many mechanisms to survive drastic changes in environmental conditions Bacteria are known to respond to alterations like extreme, unphysiological temperature, pH value, ionic strength and pressure in a specific regulation of protein synthesis and degradation In the case of cold adaptation many bacteria respond to a decrease in temperature with the reorganization of their protein expression pattern In general the protein synthesis of bulk proteins becomes inhibited whereas expression of cold shock proteins (CSPs) increases rapidly [1] CSPs have been proposed to act as regulators of gene expression for specific proteins during the acclimation phase after a downshift in temperature [2] Abbreviations Bc, Bacillus caldolyticus; Bs, Bacillus subtilis; CAT, chloramphenicol acetyltransferase; CSP, cold shock protein; dT7, hepta-2’-desoxythymidylate; IF, initiation factor; Tm, Thermotoga maritima FEBS Journal 272 (2005) 4691–4702 ª 2005 FEBS 4691 Influence of CSPs on transcription and translation Many CSPs have been shown to have a high affinity to single-stranded nucleic acids and therefore are thought to act on transcription and ⁄ or translation For the mesophilic bacterium Escherichia coli it is known that up to 25 different proteins are newly induced after shifting the temperature from 37 to 10 °C [3] CspA is one of the most prominent CSPs It has high amino acid sequence identity to the ‘cold-shock domain’ of eukaryotic Y-box proteins that are known to bind DNA and RNA [4] It is a member of the protein family of bacterial CSPs who form a group of highly homologous 7.4 kDa proteins known to bind to RNA and ssDNA with distinct sequence specificity [5] Cold shock induced CSP synthesis itself is controlled at different levels including transcription, RNA stability, translational and post-translational events The cspA-mRNA contains an 159 base 5¢-untranslated region bearing binding sites for regulatory proteins such as helicases, RNAses, CSPs, initiation factors (IFs) and others [7] This region plays also an essential role in mRNA stability and translation efficiency [8,9] CSP itself binds to the 5¢-untranslated region of their own mRNA to destabilize secondary structures of mRNA therein leading to higher expression yields Although the role of CspA in cold adaptation is not fully understood, it is known that they are up-regulated during the reorganization period (adaptation phase) following cold shock Here a transient inhibition of bulk protein synthesis occurs and the cell growth stops for several hours A putative connection between these stages has not been shown to date [1,6] The mechanism of this inhibition is not yet known it could occur at transcriptional and ⁄ or translational level and is usually assumed to be regulated by specific interactions with specific factors involved in the regulation of these processes However, it has also been suggested that inhibition of protein synthesis is the effect of unspecific binding of CSPs to ssRNA or ssDNA [5,6] Using complex cellular systems it is difficult to distinguish on the molecular level between these different mechanisms of inhibition in an unequivocal manner Cold shock proteins have the putative function as ‘RNA chaperones’ [2,10,11] and are reported to generally inhibit RNA secondary structure formation that stabilize RNA by intramolecular base pairings These secondary structures, especially hairpin loops, are supposed to be the main reason why many genes can only be expressed heterologically in suboptimal amounts [12] For testing the chaperone hypothesis, for learning more about the biochemical function of CSPs, and for potentially improving the performance of the combined in vitro transcription ⁄ translation system, we 4692 R Hofweber et al investigated the influence of different CSPs on the cellfree protein synthesis with E coli cell free extracts For obtaining a more general view of the properties of CSPs we used CSPs from various organisms, which differ in their physiological growth temperature: Thermotoga maritima exhibits optimal growth at 80 °C, Bacillus caldolyticus at 60 °C and Bacillus subtilis at 35 °C These representative CSPs were chosen because they share a high homology and sequence identity among this protein family Using a cell-free expression system it is possible to control the concentration of CSPs accurately and thus their concentration dependent effects In addition, complex regulatory events, as they occur in complex cellular systems and can lead to a misinterpretation of the data, are less likely Therefore the system can complement data from whole cells by a more quantitative analysis of the direct action of CSPs on protein synthesis It is especially well-suited for testing the RNA chaperone function of the CSPs because RNA secondary structures are particularly critical; the widely used T7 RNA polymerase processes faster on DNA than E coli ribosomes interact with mRNA Therefore free mRNA accumulates in these cell-free systems [13] and excessive secondary structure formation takes place Results Effect of cold shock proteins on the combined cell-free transcription/translation of proteins RNA secondary structures, especially hairpin loops, are supposed to be the main reason why many genes can only be expressed in suboptimal amounts by combined in vitro transcription ⁄ translation systems CSPs have the putative function as ‘RNA chaperones’ [2] and therefore could possibly enhance the protein expression To test this hypothesis, a gene sequence which is only weakly expressed in the cell-free system and is supposed to form very stable secondary structures is required In this case a significant increase of the expression level by the RNA-chaperone activity can be expected The human H-Ras protein served here as a model, as the human ‘wild type’ coding sequence encoded on pET14bRasc¢ (C-terminal truncated H-Ras) or on pET14bRasfl (full-length Ras) is supposed to form extensive secondary structures, especially at the 3¢-region of the mRNA It can be compared with the chemically synthesized sequence encoded on pK7Ras which was optimized in terms of expression rate by silent mutations [14,15] These mutations were directed to maximize the cell-free H-Ras expression by reducing FEBS Journal 272 (2005) 4691–4702 ª 2005 FEBS R Hofweber et al the number of base-pairs in the RNA structure whilst influencing other critical factors for protein expression The free energy difference between the two most stable mRNA structures calculated by mfold [16] is 245 kJỈmol)1 Both plasmids, encoding either the wild-type or the synthetic sequence, were used in a cell-free expression system, as indicated in the Experimental procedures; 10 lL of the reaction volume were subjected to SDS ⁄ PAGE The difference in expression level of the two versions of H-Ras is shown in Fig The synthetic sequence of pK7Ras results in a prominent protein band on the SDS PAGE whereas the two wildtype sequences pET14bRasc¢ and pET14bRasfl not result in a visible band on the Coomassie stained gel and can only be detected by western blotting (Fig 2) We therefore deployed CSPs to our E coli cell-free system to investigate the effects of the putative RNA chaperones on the protein expression CSPs in different concentrations were used to monitor the concentration dependent effects on gene expression We analyzed the influence on expression rate of H-Ras by western blotting experiments and of chloramphenicol-acetyltransferase (CAT) by a colorimetric assay The expression of H-Ras from the wild-type coding sequence is only visible on the western blot (Fig 2) and not on a Coomassie-stained SDS ⁄ polyacrylamide gel (Fig 1) With the addition of the CSP Fig In vitro transcription ⁄ translation of different Ras-constructs The reaction was performed for h at 37 °C, 10 lL of each reaction mixture were acetone precipitated and subjected to SDS PAGE The gel was stained with Coomassie Brilliant Blue (A) Molecular mass standard; (B) blank; (C) pK7Ras; (D) pET14bRasc¢; (E) pET14bRas The arrow displays the prominent band of H-Ras expressed from pK7ras FEBS Journal 272 (2005) 4691–4702 ª 2005 FEBS Influence of CSPs on transcription and translation Fig Inhibition of Ras expression by TmCSP investigated by western blot with a-Ras Cell-free expression of H-Ras encoded on pET14brasc¢ in h batch reaction under the influence of different concentrations of TmCSP as indicated Ten microlitres of each reaction were acetone precipitated and subjected to SDS ⁄ PAGE followed by western blotting with a-Ras from T maritima, the expression level of the protein decreases so that at TmCSP concentrations higher than 200 lm, only traces of H-Ras protein can be detected The expression rate of CAT at 37 °C was monitored under the influence of the CSPs from T maritima, B subtilis and of B caldolyticus Having no effect in low concentrations up to a certain level (see below), the expression rate declines rapidly by adding more CSP until no expression of CAT was detectable (Fig 3) Fig Concentration-dependent inhibition of CAT- expression by CSPs from different microorganisms CAT was expressed under the influence of CSPs with the indicated concentrations from the different organisms The reaction products were analyzed according to Shaw [40] The measured values are displayed as rectangles (TmCSP), circles (BcCSP) or triangles (BsCSP) The line fit to the experimental data using Eqn (2) are shown (see Experimental procedures) The obtained apparent dissociation constants Kapp of CSP (Kapp given in Table 1, the other fit parameters for TmCSP, BcCSP, and BsCSP, respectively, are cDNAtotal ¼ 335 ± 244 lM, N ¼ 4.01 ± 1.35 lM, Kribo ¼ 32 ± 1.3 nM, and cribototal 0.69 ± 0.05 lM 4693 Influence of CSPs on transcription and translation R Hofweber et al In detail, the CSP of the hyperthermophilic organism T maritima has no significant effect up to a concentration of £ 100 lm, then the expression rate decreases rapidly so that at concentrations ‡ 230 lm the expression of CAT is fully suppressed The addition of the CSP from the thermophilic bacterium B caldolyticus shows no significant effect at concentrations £ 200 lm The expression rate also decreases until it is not detectable at BcCSP concentrations ‡ 420 lm Furthermore, the CSP CspB from the mesophilic bacterium B subtilis shows no effect at concentrations £ 300 lm followed by an equivalent decrease of protein expression as described for the other CSPs After reaching BsCSP concentrations of ‡ 800 lm no CAT expression is observable any more The optimal growth temperatures of the respective organisms and the necessary concentrations for 50% inhibition (c50) of gene expression are displayed in Table A complete quantitative evaluation of the data is not possible; however, it is worthwhile fitting the data with plausible models The simplest model would assume the interaction with one component of the system (a protein, DNA or RNA) which then directly abolishes the expression Formally this situation would be described by Eqn (1) (see Experimental procedures) It turns out that the initial constant part of the curve cannot be described sufficiently well with Eqn (1) whereas the second part of the data can be described well by this equation In a more evolved model, CSP would decrease the transcription by binding to DNA and the available mRNA would be limiting for the expression rate With this model (Eqn in Experimental procedures) the data are well described and apparent dissociation constants can be determined (Table 1) Table Inhibition of CAT-expression by CSPs from different microorganisms Source Topt (K)a c50 (lM)b c99 (lM)c Kapp (lM)d KD (lM)(dT)7 KD (lM)(dA)7 T maritima B caldolyticus B subtilis 353 333 308 139.5 215.0 451.8 230 420 800 0.59 2.33 4.51 0.02e –g 0.37h 8.0f –g –g a In vitro transcription-translation assay was performed at 310 K Optimal growth temperature c CSP concentration for 50% inhibition d CSP concentration for virtually complete inhibition (> 99%) e Apparent KD from the fit of the in vitro data using Eqn (2) (Fig 3) The obtained apparent dissociation constants are largely independent of the other free fit parameters used on Eqn (2) f KD for binding of (dT)x or (dA)7 to TmCSP interpolated to 310 K from the data of M Zeeb (Universitat Bayreuth, Germany; personal communicaă tion) using the relation lnK ¼ –DG0 ⁄ RT g No data available h KD for binding of (dT)7 to BsCSP interpolated to 310 K from the data of [17] b 4694 Note that the values obtained not critically depend on the other free parameters of the model, a property of the used function On the other hand, this means that the other parameters following from the fit of the data cannot be determined with high accuracy Inhibition of transcription by cold shock proteins To elucidate whether the effect shown above occurs on the transcriptional or on the translational level, the system of combined transcription ⁄ translation was decoupled The influence on the transcriptional level was investigated by in vitro transcription of the CAT gene using T7 RNA polymerase and nucleotides CAT was encoded on pK7cat or on pET14bcat when His6tagged CAT was used for western blotting analysis The transcript was isolated by digestion of RQ1 RNAse-free DNAse and was followed by separation of nucleotides with nucleospin columns The purified transcript was analyzed using an AGILENT 2100 Bioanalyzer In the absence of CSPs and in the presence of the equivalent amount of BSA in the according buffer a distinct band of transcription product was also present as a small amount of undigested plasmid The addition of CSP resulted in the loss of these distinct bands and a distribution of different bands appears which could not be assigned (data not shown) In a second test to determine whether transcription is still working in the presence of CSPs, in vitro transcription in the presence of [32P]CTP[aP] was performed and subjected to polyacrylamide electrophoresis The autoradiogram is shown in Fig As a result of this experiment, no transcription could be observed when CSPs of T maritima, B subtilis or B caldolyticus were added in concentrations which led to a complete suppression of protein expression in the combined transcription ⁄ translation assay (Table 1) Inhibition of translation by CSPs The effect of CSPs on the translatory process was investigated by in vitro translation of mRNA encoding chloramphenicol acetyltransferase As a starting point, the set up for the combined transcription ⁄ translation experiment was used omitting plasmid and T7 RNA polymerase as the components for transcription Instead of these, mRNA was used as template for the translation With this system, the effect of the CSPs on the translational level was studied In the absence of CSP the expression rate was approximately 11 lgỈmL)1 CAT in h using an mRNA concentration of 33 lgỈmL)1 When CSPs were present in concentrations where complete inhibition of the combined in vitro FEBS Journal 272 (2005) 4691–4702 ª 2005 FEBS R Hofweber et al Fig Influence of CSPs on the in vitro transcription of CAT In vitro transcription was performed in the presence of [32P]CTP[aP] Reaction products (equal amounts of radioactivity) were subjected to denaturing polyacrylamide gel electrophoresis with following autoradiography (time of exposure: 45 min); lane A: standard (F · 174 DNA ⁄ Hinf I from Promega); lane B: transcription experiment under the influence of buffer A (pH 6.5) with mgỈmL)1 BSA as control protein; lane C: transcription experiment under the influence of buffer B (pH 7.8) with mgỈmL)1 BSA as control protein; lane D: addition of 230 lM TmCSP in buffer A; lane E: addition of 420 lM BcCSP in buffer B; lane F: addition of 800 lM BsCSP in buffer B Fig Influence of CSPs on the in vitro translation of CAT Activity level of CAT after in vitro translation of mRNA transcribed from pET14bCAT in the presence of mgỈmL)1 BSA (control), 230 lM TmCSP, 420 lM BsCSP or 800 lM BcCSP The insert displays a western blot of these samples Samples (5 lL) were acetone precipitated and subjected to SDS ⁄ PAGE [41] following immunodetection of the His6-tagged CAT by His-Probe translation ⁄ transcription was observed (Table 1), translation was completely abolished (Fig 5) Reversal of the inhibitory effect of TmCSP by single-stranded DNA We suggest that the effects shown above are based on the binding of the CSPs to DNA and RNA However, other possible mechanisms are the inhibition of FEBS Journal 272 (2005) 4691–4702 ª 2005 FEBS Influence of CSPs on transcription and translation Fig Suppression of the CSP inhibition of CAT-expression by ssDNA Titration of poly dT7 to the combined transcription ⁄ translation of pK7CAT in the presence of TmCSP Poly(dT7) of different concentrations (final concentrations in expression as indicated) was incubated with TmCSP (final concentration was 139.5 lM) for 20 at 25 °C and then subjected to CAT expression transcription and ⁄ or translation by interaction with proteins involved in this process or an increased degradation of nucleic acids by an increased nuclease activity It was shown for TmCSP (M Zeeb, Universitat ă Bayreuth, Germany; personal communication) that it binds tightly to the oligodesoxynucleotide hepta-2’-desoxy-thymidylate (dT7) Therefore, we tested if binding of this nucleotide to TmCSP can interfere with the suggested DNA or RNA interaction in the combined transcription ⁄ translation assay At a TmCSP concentration of 139.5 lm (the TmCSP concentration for 50% inhibition of the protein expression), the protein expression was tested in dependence on different concentrations of dT7 The CSP was incubated with the nucleotide before cell-free expression was performed, so that part of the TmCSP molecules were inactivated with regard to their influence on the transcription ⁄ translation processes Within the limits of error the full expression level could be re-established by adding dT7 in approximately equimolar concentration to TmCSP (Fig 6), indicating that TmCSP is removed from its binding sites on nucleic acids by dT7 At very high concentrations of the heptanucleotide the expression level again decreases somewhat, probably because of unspecific effects of the oligonucleotide with components of the transcription ⁄ translation machinery Discussion The expression efficiency of various genes under the control of a strong promotor is influenced by many 4695 Influence of CSPs on transcription and translation different factors One of the most crucial factors next to codon usage is the occurrence of intramolecular RNA base pairings on mRNA encoding the protein of interest These secondary structures are able to mask regulatory sequences on the mRNA, for example the Shine–Dalgarno sequence [6], so that translation factors cannot bind easily to mRNA Another reason for low expression levels is the increase of energy necessary for dissolving secondary structures, e.g hairpin loops which has to be provided by the translation machinery [12] Consequently, minimization of mRNA secondary structures leads to a higher expression rate It was reported that CSPs function as RNA chaperones [2] These are defined as RNA binding proteins able to prevent the formation of RNA secondary structures [1] and therefore highly increasing the accessibility of the RNA to ribonucleases [2] On the other hand, it is reported that the translation activity is increased when CSPs are present [10,18,19] More recently it was shown on E coli that its translational apparatus undergoes significant modifications during cold shock, especially concerning the ribosomes [3] CspA, the major CSP from E coli was found to act as an activating factor after binding to specific mRNAs [20] The starting point for our experiments was the idea to use CSPs from different organisms in cell free expression systems in order to optimize the efficiency of our combined transcription ⁄ translation system, which is based upon E coli S-30 cell-free extracts In these systems mRNA accumulates due to the higher processivity of the widely used viral T7 RNA polymerase in contrast to the slower E coli ribosomes [13] This excessive pool of transcripts leads to an enriched level of RNA secondary structure formation Thereby we could check the hypothesis of [6] that CSPs should act on protein expression at high concentrations in an inhibiting manner As we wanted to study the effect of the addition of CSPs to our extracts we prepared our extracts from E coli BL 21 carefully under conditions where cold shock conditions did not prevail As a model system for characterizing the influence of CSPs on expression, H-Ras was chosen as it is available in the wild-type coding sequence and in a synthetic version [14] This synthetic version has been optimized via silent mutations in terms of codon usage and minimization of possible RNA secondary structures We verified the difference in the thermodynamics of the RNA secondary structures by predicting the free energy of the RNA foldings using the mfold program [16] This prediction results in a thermodynamic stabilization of the wild-type gene of 245 kJỈmol)1 with respect to the syn4696 R Hofweber et al thetic gene sequence The comparison of the energy dot blots reveals that the wild-type RNA sequence can form a large number of intramolecular base pairs at the 5¢-UTR of the sequence, which can be a reason for reduced translation initiation In agreement is the fact that silent mutations in the synthetic sequence result in a dramatic increase in expression rate compared with the wild-type sequence, as depicted in Fig The effect of cold shock proteins on the combined transcription and translation To test the CSPs’ function as RNA chaperones we used CSPs from T maritima, from B caldolyticus and from B subtilis Their respective optimal growth temperatures were 80, 60 and 35 °C (Table 1) The temperatures for cold shock response of these organisms differ from 10 to 60 °C (Table 1) and therefore different nucleotide binding affinities have to be expected for these highly homologous CSPs For comparable results we used the standardized temperature of 37 °C At this temperature, where the E coli system works optimally, three different scenarios are present: BsCSP experiences physiological conditions, BcCSP undergoes cold shock conditions (20 °C below optimal growth temperature) and TmCSP is even below cold shock conditions (50 °C below optimal growth temperature) As an easily quantifiable reporter gene assay we used the expression of CAT in our standard cell-free expression system We applied different concentrations of CSPs to this experiment Whether a very small increase in CAT expression does exist for BcCSP and BsCSP, but not for TmCSP, at low CSP concentrations (Fig 3) cannot be decided from our data because it is clearly not significant with respect to the inherent experimental errors of our assay However, the expression of CAT under the influence of any of the three used CSPs resulted in a significant, dramatic decrease of protein synthesis rate (Fig 3) in contrast to the expected increase of expression rate by addition of CSPs following the RNA chaperone theory The same inhibitory effect was visible when TmCSP was added to the cell-free batch expression of H-Ras The three different CSPs exhibit different concentration ranges for the inhibitory effect described above The CSP from T maritima inhibits the expression process most effectively followed by the CSP from B caldolyticus The protein that needs the highest concentration for an efficient inhibition was the CSP from B subtilis Our observed concentration levels necessary for inhibition of gene expression are in a physiological range FEBS Journal 272 (2005) 4691–4702 ª 2005 FEBS R Hofweber et al as confirmed by different studies on E coli [10,11] It was shown that the homologous protein CspA from E coli reaches concentrations of up to several per cent of the total soluble protein during temperature downshift Assuming a total soluble protein concentration of 200–300 mgỈmL)1 [21,22], the concentration of CspA in the cytosol can reach concentrations in the millimolar range during cold shock The effects described in this work are detectable at concentrations < 800 lm Therefore the effects described here not only exist in our in vitro system but can also occur in the original organisms suffering of too low temperatures for optimal growth The inhibition of protein expression by CSPs could be due to an interaction with other proteins or with nucleic acids If the observed effect is caused by binding to nucleic acids it should be influenced by the competition of suitable oligonucleotides for the CSP binding sites For the CSP from T maritima binding data studies to ssDNA are available The oligodesoxyribonucleotide dT7 showed maximum affinity to TmCSP with a KD value of (4.0 ± 0.2) · 10)3 lm at 30 °C and a KD value of (0.44 ± 0.02) lm at 50 °C [21] For smaller oligonucleotides or other homopentanucleotides tested the affinity was substantially smaller Our data show that at concentrations which are high enough for saturating the CSP in the assay the inhibitory effect of the CSPs on transcription and translation could be reversed (Fig 6) This means that for the suppression of protein expression an interaction of CSP with DNA and ⁄ or RNA is required and that protein– protein interaction or increased nuclease activity can be excluded as the main inhibitory factors for the expression process For reversing the inhibition dT7 concentrations of approximately 150 lm are necessary analogous to the TmCSP concentration used in this assay This implies that the DNA or RNA interaction sites with CSP in our assay have comparable or lower affinities for CSP than dT7 The effect of cold shock proteins on transcription In the isolated system with only polymerase, nucleotides, template DNA and CSPs without cell lysates and RNases the effect of CSPs on transcription alone become observable as no other cellular component of the heterologous expression system is present In the absence of CSPs, a prominent band of transcripts was present together with a continuous distribution of smaller transcripts resulting of earlier transcription termination These shorter transcripts represent only a very small part of all mRNA generated in this process as visible on the autoradiogram, especially with regard FEBS Journal 272 (2005) 4691–4702 ª 2005 FEBS Influence of CSPs on transcription and translation to the fact that equal amounts of radioactivity was applied to each lane of the polyacrylamide gel When CSPs are added in concentrations which lead to an inhibition of the protein synthesis in the combined expression experiment (Table 1), no RNA is detectable any more As no RNase activity is present, a degradation of mRNA can be neglected, so that we can conclude an inhibition of the transcription process After incubation of plasmid DNA with CSPs a complete digestion of the plasmid with the DNase RQ1 cannot be observed (data not shown) indicating that CSP binding nearly completely protects the DNA As CSPs are known to bind to single-stranded nucleic acids it seems reasonable to assume that the CSPs bind with high affinity to single-stranded DNA as present during transcription and consequently block the transcription process The effect of cold shock proteins on translation As shown above CSP inhibits transcription in the used concentration range but it could also interfere with protein translation For the investigation of translation we subjected mRNA encoding for CAT to a S30 lysate In this system we could analyze the influence of CSPs on the translation process The translation of CAT is clearly inhibited when CSPs are added to the system This effect can be interpreted as a binding of CSP directly to RNA due to the absence of DNA This binding property can result in an increased accessibility of ribonucleases to the mRNA or of masking of regulatory sequences The described translation inhibition of a non-cold shock mRNA can be combined with the results of [3] They found an increase in the translation of cold shock mRNAs and of cold tolerant mRNAs with the addition of CspA to a translation assay Thus translation of non-CSPs would be suppressed and that of cold shock-related proteins enhanced In their putative function as RNA chaperones, CSPs should lead to a higher overall protein synthesis rate as the formation of RNA secondary structures should be inhibited or melted [23] This general function described by several groups [2,24] cannot be confirmed with our results However, the RNA chaperone function could be limited to a number of genes whose mRNA shows motifs like cold shock boxes [7] or cold shock cut boxes [25] The preferential binding to these sequence motifs prevents these specific mRNAs from folding to stable secondary structures so that CSPs act as RNA chaperones in these proposed cases Therefore we can confirm the hypothesis of [6] that high concentrations of CSPs lead to an inhibition of general 4697 Influence of CSPs on transcription and translation protein expression Here we bring first evidence for this theory on three different CSPs in a cell-free system without regulatory events of a living cell and therefore focussed on direct nucleic acid binding In the light of our results the reduced expression of bulk proteins visible after the high level expression of CSPs could be a combined effect of the inhibitory properties on transcription and translation of CSPs at these high concentrations where even binding motifs of lower affinity are occupied The sense of this breakdown in protein synthesis might be to give the cell time to rearrange the protein expression pattern to the new environmental conditions Apparent dissociation constants for CSP As we have shown that direct binding of CSPs to nucleic acid influences the expression and not specific RNA chaperone activity, we can analyze the binding properties of the CSPs used in this study For a quantitative evaluation of the in vitro transcription ⁄ translation curve different models for the description of the system are plausible The simplest model assumes a direct suppression of the steady state protein expression by binding of CSP to DNA or mRNA when the mRNA would be the limiting factor As mentioned, it already fits well the data except the constant part at low CSP concentrations The slopes of the curves in Fig (and thus the apparent binding constants) are different for the three used CSPs The steepest descent can be observed with the addition of TmCSP, followed by BcCSP and BsCSP indicating the highest nucleic acid binding affinity for TmCSP followed by BcCSP as resulting out of the optimal growth temperatures of their organisms as indicated above The apparent dissociation constants are also obtained in a more elaborate model which assumes the existence of a limiting component different to mRNA as it could be for example the number of translation-active ribosomes Only when the available mRNA concentration would drop below this value would an effect on protein expression be observed The minimum length of DNA or RNA for optimal binding to BsCSP (and probably for all CSPs) is six to seven bases From the tested sequences the heptanucleotide (dT)7 has the highest affinity to CSP of B subtilis [7] and T maritima The extrapolated affinities are about one order of magnitude higher than those obtained from the fit of the data (Table 1) However, they are known to drop substantially for shorter stretches of thymine nucleotides and for sequences including other nucleotides In pK7CAT the sequence (T)7 occurs once, but outside coding sequences However, in the 4698 R Hofweber et al Ras gene the largest stretch of poly(T) has a length of four and occurs twice, in the CAT gene the largest stretch comprises six nucleotides and occurs once These results are in accordance with earlier findings that during the acclimation phase of cold shock the protein expression pattern of the organism drastically changes and that the synthesis of bulk proteins is temporarily dramatically reduced [1] Our in vitro data indicate that CSPs are directly involved in this downregulation of protein expression by binding to elements with intermediate specificity which occur in most genes According to our data CSP would inhibit the new transcription of the majority of genes and also inhibit the translation of still existing RNA as already postulated [6] Most probably the affinities are adapted during evolution to their specific temperature ranges as necessary for the different growth conditions of their source organisms At least the affinities of CSP for poly(T) at a given temperature are positively correlated with the optimal growth temperature (Table 1) The above mechanism does not exclude a second mechanism were the activity of specific regulatory elements in DNA or RNA leads to an increased expression of some proteins in the cell The visible high levels of CSPs during cold shock can result of many different mechanisms, like differential increase in its mRNA stability [26] and preferential translation of cold shock mRNAs resulting out of an increase of the three translation initiation factors [3,11] Furthermore it is known for E coli, that the 159 nucleotides of the 5¢-UTR of the cspA mRNA plays a critical role in the cold shock adaptation In many mRNAs encoding for CSPs and for cold tolerant proteins a special sequence motif (the so-called cold shock box) was found to be responsible for their cold shock regulation [3,18,19] Thereby, the 5¢-UTR that is responsible for the autoregulation of the transient expression boosts the expression visible in the early acclimation phase [8,27] In this particular case, CSPs may function as RNA chaperones and therefore promote their own expression After reaching a distinct concentration level in the acclimation phase, one could argue that CSPs also bind to nucleotide sequences of lower specificity Therefore, regulatory sequences of other mRNAs are then silenced This leads to a general decrease of bulk protein synthesis as postulated [6] and demonstrated experimentally in this work Concluding remarks The observed inhibition of protein expression can be explained in principle on different levels of the FEBS Journal 272 (2005) 4691–4702 ª 2005 FEBS R Hofweber et al combined transcription ⁄ translation assay: (a) the inhibition of transcription; (b) the increased decay of transcribed RNA; (c) the decreased translation; and (d) the increased activity of proteolytic enzymes cleaving the target protein Other effects which can occur in whole cells (e.g the induction of new proteins interfering with the protein expression) cannot occur in our isolated in vitro system The in vitro system also has the advantage that the different steps of protein expression can be observed separately Therefore we can clearly determine that inhibition occurs on the level of transcription which can be explained by binding of the CSPs to single-stranded nucleic acids as shown by in vitro transcription As even the digestion of the plasmid is protected and the inhibitory effect can be reversed by a competing oligonucleotide we can exclude decay of the transcribed RNA In our cell-free system, we can also observe a decrease of translation in an isolated in vitro translation assay The effects described here cannot be mapped to a well-defined binding site for CSPs on the plasmid or on the mRNA as the effects are visible using different plasmids and different genes lacking defined recognition sequences in the regulatory regions The strongest binding of CSPs to desoxyoligonucleotides has been determined for dT7 As this motif can hardly be found in any coding sequences (the uncorrected statistical probability to find it in one gene coding for 100 amino acids is of the order of ⁄ 50), the CSPs are expected to bind to more unspecific sequence motifs with lower affinities This is consistent with the apparent KD values obtained here At the concentrations of CSP existing in vivo, the unspecific inhibition of protein expression observed in the living cell after a cold shock can be explained by a direct binding of CSP to RNA and ⁄ or DNA Besides this, in our fit, TmCSP shows the tightest binding, followed by BcCSP and then BsCSP These relative proportions correspond well to those expected from their organisms’ growth temperature and of their fully inhibiting concentrations Influence of CSPs on transcription and translation as described previously [28] To remove the bulk of the E coli proteins without significant coprecipitation of TmCSP, the supernatant was diluted fivefold and heated to 80 °C for 30 Pure TmCSP was obtained after hydrophobic interaction chromatography at pH 8.0 and size exclusion chromatography with Superdex 75 (Amersham, Freiburg, Germany) The total yield of TmCSP was about 15 mgỈL)1 cell culture The purified protein was concentrated by ultrafiltration to a final concentration of 3.3 mm The used molar extinction coefficient of TmCSP was 12660 m)1Ỉcm)1 Expression and purification of B subtilis CSP A gene encoding BsCSP B was overexpressed using the T7 RNA polymerase promotor system as described [31] The plasmid containing the gene for BsCSP was transformed into E coli BL21 (DE3) pLysS The cells were grown at 37 °C in dYT medium containing 25 lgỈmL)1 chloramphenicol and 300 lgỈmL)1 ampicillin to D600 ¼ 0.8 Protein expression was induced by addition of mm IPTG and carried out for h at 37 °C Cells were harvested and lysed as described [31] and the supernatant was applied to a DEAE anion exchange column (Amersham) equilibrated with 50 mm Tris ⁄ HCl pH 7.8 [32] Bound protein was eluted with a linear NaCl gradient from to 600 mm BsCSP eluted at approximately 100 mm NaCl Fractions containing BsCSP were adjusted to 50% (w ⁄ v) ammonium sulfate, bound to a butyl-sepharose FF column (Amersham) and washed with 50 mm Tris ⁄ HCl, 50% ammonium sulfate pH 7.6 to remove bound nucleic acids Elution with 50 mm Tris ⁄ HCl pH 7.6 yielded > 95% pure BsCSP, as judged from SDS ⁄ polyacrylamide gels [33] After size exclusion chromatography (HiLoad SuperdexTM 75 prep grade column; Amersham) in 50 mm Tris ⁄ HCl, 100 mm KCl pH 7.8, fractions free of nucleic acids were concentrated by ultrafiltration to a concentration of 5.9 mm From L of cell culture, mg of BsCSP could be prepared The molar extinction coefficient of BsCSP was 5800 m)1Ỉcm)1 [32,34] Expression and purification of B caldolyticus CSP Experimental procedures Expression and purification of T maritima CSP Protein expression of TmCSP was performed as described [28,29] The plasmid coding for TmCSP was transformed into E coli Rosetta (DE3) pLysS The cells were grown in Luria–Bertani medium [30] containing 50 lgỈmL)1 ampicillin and 68 lgỈmL)1 chloramphenicol at 37 °C to D600 ¼ Protein expression was induced by adding mm isopropyl thio-b-d-galactoside, and bacterial growth was continued at 37 °C for h Purification of the protein was performed FEBS Journal 272 (2005) 4691–4702 ª 2005 FEBS BcCSP was overexpressed in E coli K38 pGP1-2 containing the plasmid pBluescriptII SK with the coding sequence for BcCSP The cells were grown at 30 °C in dYT medium containing 25 lgặmL)1 kanamycin and 300 lgặmL)1 ampicillin to D600 ẳ 0.8 Protein expression was induced by temperature shift to 42 °C carried out for h Cells were centrifuged and lysed as described [31] After cell lysis and centrifugation the cell-free extract was heated to 65 °C for 40 to precipitate most of the E coli proteins All following steps were carried out at °C according to the purification of BsCSP described above The purified protein 4699 Influence of CSPs on transcription and translation was concentrated by ultrafiltration to a concentration of 3.0 mm The used molar extinction coefficient of BcCSP was 7300 m)1Ỉcm)1 [35] R Hofweber et al TmCSP or 50 mm Tris ⁄ HCl pH 7.8, 100 mm KCl (buffer B) with or without BsCSP or BcCSP In vitro translation Template DNA for combined transcription/translation and in vitro transcription As template DNA the plasmid pK7CAT [36], pET14b CAT, pKRAS and pET14bRas fl and pET14bRas c¢ were used The plasmids were purified from E coli TG1 using the QIAfilter Plasmid Maxi Kit (Qiagen, Hilden, Germany) Reaction conditions for the batch system of the combined in vitro transcription/translation For determining the influence of CSPs on the transcription apparatus the system for combined transcription ⁄ translation was used without T7 RNA polymerase and protease inhibitor cocktail mRNA from the transcription experiments was used as template instead of the plasmid DNA Concentrations of the CSPs necessary for virtually complete inhibition were used as indicated in Table The reaction volume was 30 lL Assay of the reaction products The E coli S30 cell extract used for the cell-free protein synthesis was prepared according to [37] from E coli strain BL21 (Amersham) due to the lack of proteases and T7 RNA polymerase The T7 RNA polymerase was added in defined amounts and its preparation was performed according to [38] The system for cell-free transcription and translation was adopted from [39] with minor modifications The standard system without CSPs consisted of 58 mm Hepes ⁄ KOH pH 7.5, 1.7 mm dithiothreitol, 1.2 mm ATP, 0.9 mm each of CTP, GTP and UTP, 81 mm creatine phosphate (CP) (Sigma, St Louis, MO, USA), 250 lgỈmL)1 creatine kinase (CK) (Roche, Indianapolis, IN, USA), 4.0% PEG 8000, 0.64 mm 3¢,5¢-cyclic AMP, 68 lm l(3)-5-formyl5,6,7,8-tetrahydrofolic acid, 170 lgỈmL)1 E coli tRNA from MRE 600 (Roche), 203 mm potassium glutamate, 27.7 mm ammonium acetate, 4.0 mm magnesium acetate, protease inhibitor cocktail 1· Complete (Roche), 0.5 U anti-RNAse (Ambion, Austin, TX, USA), 1.0 mm tyrosine, 0.3 mm of each of the other 19 amino acids, 33 lgỈmL)1 of the respective plasmid DNA, 140 lgỈmL)1 T7 RNA polymerase, 35.1% (v ⁄ v) S30 extract in volume of 26 lL and lL water to a final volume of 30 lL The reaction mixture was incubated at 37 °C for h at 500 r.p.m in a microtiterplate on a rotary shaker When CSPs were titrated to the combined transcription ⁄ translation experiment, the lL of water was replaced by CSP of different concentrations The amount of synthesized CAT protein was quantified by a colorimetric assay as described by [40] The proteins were also analyzed by SDS ⁄ PAGE [41] and western blotting after acetone precipitation The transcription products were detected with an AGILENT 2100 Bioanalyzer (Palo Alto, CA, USA) and polyacrylamide gel electrophoresis under denaturing conditions after transcription in the presence of [32P]CTP[aP] In vitro transcription total with A0 cDNA as the protein expression (mgỈmL)1Ỉs)1) in the absence of CSP, cCSPtotal , the total concentration of CSP, total cDNA , the total concentration of the CSP binding sites on the plasmid and Kapp the apparent dissociation constant In a somewhat more complex description the availability of the ribosomal translation system is incorporated The protein expression is assumed to be proportional to the concentration cribobound , of ribosomal complexes bound to the mRNA, that is The in vitro transcription was performed using the Riboprobe kit (Promega, Madison, WI, USA) and the plasmid pK7CAT The kit was used for the generation of template RNA for in vitro transcription assays and for analysis of the influence of CSPs at the transcriptory level In the latter case the reaction volume was 20 lL, where lL of the reaction volume were either 50 mm NaH2PO4, 100 mm NaCl, mm EDTA, pH 6.5 (buffer A) with or without 4700 Prediction of RNA secondary structures The calculation of mRNA secondary structure formation was performed with the program mfold based on the algorithm described by [16] Fitting of the binding isotherms For a direct comparison of CSP from different organisms, apparent dissociation constants were derived for the inhibition of the protein expression by CSP using two models In the most simple model the inhibition is predominantly due to the binding of CSP to one component of the system (e.g a regulatory element of the plasmid) and switches off its activity responsible for protein expression The activity A (protein expression) is then given by À total total total AcCSP ị ẳ A0 cDNA Kapp cCSP q total total total ỵ cDNA Kapp cCSP ị2 ỵ 4Kapp cDNA 1ị FEBS Journal 272 (2005) 4691–4702 ª 2005 FEBS R Hofweber et al À total bound total total AcCSP ị ẳ A1 cribo ẳ A1 Kribo ỵ NcRNA ỵ cribo q total total total total Kribo ỵ NcRNA ỵ cribo ị2 4Ncribo cRNA Influence of CSPs on transcription and translation ð2Þ where A1 is an appropriate normalization constant, Kribo the apparent dissociation constant of the transcription complex from the RNA, cRNAtotal the total steady state concentration of mRNA, cribobound the concentration of bound ribosomes and cribototal the total concentration of bound total ribosomes The RNA concentration cRNA can be calculatotal as the RNA conted with Eqn (1) interpreting A0 cDNA centration in the absence of CSP Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft 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chloramphenicol-resistant bacteria Methods Enzymol 43, 737–755 41 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685 FEBS Journal 272 (2005) 4691–4702 ª 2005 FEBS ... on the transcriptional or on the translational level, the system of combined transcription ⁄ translation was decoupled The in? ??uence on the transcriptional level was investigated by in vitro transcription. .. dissociation constants were derived for the inhibition of the protein expression by CSP using two models In the most simple model the inhibition is predominantly due to the binding of CSP to one component... non -cold shock mRNA can be combined with the results of [3] They found an increase in the translation of cold shock mRNAs and of cold tolerant mRNAs with the addition of CspA to a translation