In general the protein synthesis of bulk proteins becomes inhibited whereas expression of cold shock proteins CSPs increases rapidly [1].. The data are in line with a hypo-thesis that CS
Trang 1translation studied in cell-free model systems
Roland Hofweber1, Gudrun Horn1, Thomas Langmann2, Jochen Balbach3, Werner Kremer1,
Gerd Schmitz2and Hans R Kalbitzer1
1 Institut fu¨r Biophysik und Physikalische Biochemie, Universita¨t Regensburg, Germany
2 Institut fu¨r Klinische Chemie und Laboratoriumsmedizin, Klinikum der Universita¨t Regensburg, Germany
3 Laboratorium fu¨r Biochemie, Universita¨t Bayreuth, Germany
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
synthe-sis and degradation In the case of cold adaptation
many bacteria respond to a decrease in temperature
with the reorganization of their protein expression pat-tern 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 accli-mation phase after a downshift in temperature [2]
Keywords
cold shock protein; in vitro translation;
RNA chaperone
Correspondence
H R Kalbitzer, Universita¨t Regensburg,
Institut fu¨r Biophysik und Physikalische
Biochemie, Universita¨tsstraße 31,
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 7 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 sub-tilis, 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 expres-sion in in vitro translation systems obsolete Protein expresexpres-sion was completely inhibited at CSP concentrations present under cold shock con-ditions 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 heptanu-cleotide that competitively binds to CSP The data are in line with a hypo-thesis that CSPs act on bulk protein expression not as RNA chaperones but inhibit their transcription and translation by rather unspecific nucleic acid binding
Abbreviations
Bc, Bacillus caldolyticus; Bs, Bacillus subtilis; CAT, chloramphenicol acetyltransferase; CSP, cold shock protein; dT7, hepta-2’-desoxy-thymidylate; IF, initiation factor; Tm, Thermotoga maritima.
Trang 2Many 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 10C [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
fam-ily 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
stabil-ity, 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-regula-ted during the reorganization period (adaptation
phase) following cold shock Here a transient
inhibi-tion 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
regula-tion of these processes However, it has also been
sug-gested 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
dif-ferent mechanisms of inhibition in an unequivocal
manner
Cold shock proteins have the putative function as
‘RNA chaperones’ [2,10,11] and are reported to
gener-ally inhibit RNA secondary structure formation that
stabilize RNA by intramolecular base pairings These
secondary structures, especially hairpin loops, are
sup-posed 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
investigated the influence of different CSPs on the cell-free protein synthesis with E coli cell 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
80C, Bacillus caldolyticus at 60 C and Bacillus subtilis
at 35C 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 com-plement data from whole cells by a more quantitative analysis of the direct action of CSPs on protein synthe-sis It is especially well-suited for testing the RNA chaperone function of the CSPs because RNA secon-dary 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 com-bined 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 enco-ded 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
Trang 3the 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 kJÆmol)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 1 The
synthetic sequence of pK7Ras results in a prominent
protein band on the SDS PAGE whereas the two
wild-type sequences pET14bRasc¢ and pET14bRasfl do 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
colori-metric 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⁄
poly-acrylamide gel (Fig 1) With the addition of the CSP
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 1 In vitro transcription ⁄ translation of different Ras-constructs.
The reaction was performed for 1 h at 37 C, 10 lL of each
reac-tion 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.
Fig 2 Inhibition of Ras expression by TmCSP investigated by western blot with a-Ras Cell-free expression of H-Ras encoded on pET14brasc¢ in 1 h batch reaction under the influence of different concentrations of TmCSP as indicated Ten microlitres of each reac-tion were acetone precipitated and subjected to SDS ⁄ PAGE fol-lowed by western blotting with a-Ras.
Fig 3 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 differ-ent 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 proce-dures) The obtained apparent dissociation constants Kapp of CSP (Kappgiven in Table 1, the other fit parameters for TmCSP, BcCSP, and BsCSP, respectively, are c DNAtotal¼ 335 ± 244 l M , N ¼ 4.01 ± 1.35 l M , Kribo¼ 0 32 ± 1.3 n M , and cribototal 0.69 ± 0.05 l M
Trang 4In detail, the CSP of the hyperthermophilic
organ-ism T maritima has no significant effect up to a
con-centration of £ 100 lm, then the expression rate
decreases rapidly so that at concentrations ‡ 230 lm
the expression of CAT is fully suppressed The
addi-tion of the CSP from the thermophilic bacterium
B caldolyticusshows no significant effect at
concentra-tions£ 200 lm The expression rate also decreases until
it is not detectable at BcCSP concentrations‡ 420 lm
Furthermore, the CSP CspB from the mesophilic
bac-terium 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 1
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
sys-tem (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 2 in
Experimen-tal procedures) the data are well described and
appar-ent dissociation constants can be determined (Table 1)
Note that the values obtained do 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 His6 -tagged 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 Bio-analyzer In the absence of CSPs and in the presence
of the equivalent amount of BSA in the according buf-fer 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 auto-radiogram is shown in Fig 4 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 sup-pression of protein exsup-pression in the combined tran-scription⁄ translation assay (Table 1)
Inhibition of translation by CSPs The effect of CSPs on the translatory process was inves-tigated by in vitro translation of mRNA encoding chlo-ramphenicol 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 transla-tion 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 lgÆmL)1CAT
in 1 h using an mRNA concentration of 33 lgÆmL)1 When CSPs were present in concentrations where complete inhibition of the combined in vitro
Table 1 Inhibition of CAT-expression by CSPs from different
micro-organisms.
Source
Topt
(K) a
c50 (l M ) b
c99 (l M ) c
Kapp (l M ) d
KD (l M )(dT)7
KD (l M )(dA)7
T maritima 353 139.5 230 0.59 0.02 e 8.0 f
B caldolyticus 333 215.0 420 2.33 – g – g
B subtilis 308 451.8 800 4.51 0.37h –g
a In vitro transcription-translation assay was performed at 310 K.
b
Optimal growth temperature.cCSP concentration for 50%
inhibi-tion d CSP concentration for virtually complete inhibition (> 99%).
e Apparent K D from the fit of the in vitro data using Eqn (2) (Fig 3).
The obtained apparent dissociation constants are largely
indepen-dent of the other free fit parameters used on Eqn (2) f KDfor
bind-ing of (dT)xor (dA)7to TmCSP interpolated to 310 K from the data
of M Zeeb (Universita¨t Bayreuth, Germany; personal
communica-tion) using the relation lnK ¼ –DG 0 ⁄ RT g No data available h KDfor
binding of (dT)7to BsCSP interpolated to 310 K from the data of
[17].
Trang 5translation⁄ transcription was observed (Table 1),
trans-lation 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
transcription and⁄ or translation by interaction with proteins involved in this process or an increased degra-dation of nucleic acids by an increased nuclease activ-ity It was shown for TmCSP (M Zeeb, Universita¨t Bayreuth, Germany; personal communication) that it binds tightly to the oligodesoxynucleotide hepta-2’-des-oxy-thymidylate (dT7) Therefore, we tested if binding
of this nucleotide to TmCSP can interfere with the sug-gested DNA or RNA interaction in the combined transcription⁄ translation assay At a TmCSP concen-tration of 139.5 lm (the TmCSP concenconcen-tration for 50% inhibition of the protein expression), the protein expression was tested in dependence on different con-centrations 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 transcrip-tion⁄ translation processes Within the limits of error the full expression level could be re-established by add-ing 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 expres-sion level again decreases somewhat, probably because
of unspecific effects of the oligonucleotide with compo-nents of the transcription⁄ translation machinery
Discussion
The expression efficiency of various genes under the control of a strong promotor is influenced by many
Fig 5 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 8 mgÆmL)1BSA (control), 230 l M
TmCSP, 420 l M BsCSP or 800 l M BcCSP The insert displays a
western blot of these samples Samples (5 lL) were acetone
preci-pitated and subjected to SDS ⁄ PAGE [41] following
immunodetec-tion of the His 6 -tagged CAT by His-Probe.
Fig 4 Influence of CSPs on the in vitro transcription of CAT.
In vitro transcription was performed in the presence of
[ 32 P]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:
tran-scription experiment under the influence of buffer A (pH 6.5) with
2 mgÆmL)1 BSA as control protein; lane C: transcription
experi-ment under the influence of buffer B (pH 7.8) with 8 mgÆmL)1
BSA as control protein; lane D: addition of 230 l M TmCSP in
buf-fer A; lane E: addition of 420 l M BcCSP in buffer B; lane F:
addition of 800 l M BsCSP in buffer B.
Fig 6 Suppression of the CSP inhibition of CAT-expression by ssDNA Titration of poly dT7 to the combined transcription ⁄ transla-tion of pK7CAT in the presence of TmCSP Poly(dT 7 ) of different concentrations (final concentrations in expression as indicated) was incubated with TmCSP (final concentration was 139.5 l M ) for
20 min at 25 C and then subjected to CAT expression.
Trang 6different 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
fac-tors 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
trans-lational 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
poly-merase 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
muta-tions in terms of codon usage and minimization of
possible RNA secondary structures We verified the
difference in the thermodynamics of the RNA
secon-dary structures by predicting the free energy of the
RNA foldings using the mfold program [16] This
pre-diction results in a thermodynamic stabilization of the
wild-type gene of 245 kJÆmol)1with respect to the
syn-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 1
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 tem-peratures were 80, 60 and 35 C (Table 1)
The temperatures for cold shock response of these organisms differ from 10 to 60C (Table 1) and there-fore different nucleotide binding affinities have to be expected for these highly homologous CSPs For com-parable results we used the standardized temperature
of 37C At this temperature, where the E coli system works optimally, three different scenarios are present: BsCSP experiences physiological conditions, BcCSP undergoes cold shock conditions (20C below optimal growth temperature) and TmCSP is even below cold shock conditions (50C below optimal growth tem-perature)
As an easily quantifiable reporter gene assay we used the expression of CAT in our standard cell-free expres-sion 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 expres-sion 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 concentra-tion 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 con-centration for an efficient inhibition was the CSP from
B subtilis
Our observed concentration levels necessary for inhi-bition of gene expression are in a physiological range
Trang 7as confirmed by different studies on E coli [10,11] It
was shown that the homologous protein CspA from
E colireaches concentrations of up to several per cent
of the total soluble protein during temperature
down-shift Assuming a total soluble protein concentration
of 200–300 mgÆmL)1 [21,22], the concentration of
CspA in the cytosol can reach concentrations in the
millimolar range during cold shock The effects
des-cribed in this work are detectable at concentrations
< 800 lm Therefore the effects described here do not
only exist in our in vitro system but can also occur in
the original organisms suffering of too low
tempera-tures 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
bind-ing 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
oligodesoxy-ribonucleotide dT7 showed maximum affinity to
TmCSP with a KD value of (4.0 ± 0.2)· 10)3lm at
30C and a KD value of (0.44 ± 0.02) lm at 50C
[21] For smaller oligonucleotides or other
homopenta-nucleotides tested the affinity was substantially smaller
Our data show that at concentrations which are high
enough for saturating the CSP in the assay the
inhibi-tory effect of the CSPs on transcription and translation
could be reversed (Fig 6) This means that for the
sup-pression of protein exsup-pression 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,
nucleo-tides, 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
ter-mination These shorter transcripts represent only a
very small part of all mRNA generated in this process
as visible on the autoradiogram, especially with regard
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 degrada-tion of mRNA can be neglected, so that we can con-clude 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 acces-sibility of ribonucleases to the mRNA or of masking
of regulatory sequences The described translation inhi-bition 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 sup-pressed 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 des-cribed by several groups [2,24] cannot be confirmed with our results However, the RNA chaperone func-tion 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 concen-trations of CSPs lead to an inhibition of general
Trang 8protein 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
break-down 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
quan-titative evaluation of the in vitro transcription⁄
transla-tion curve different models for the descriptransla-tion of the
system are plausible The simplest model assumes a
direct suppression of the steady state protein
expres-sion 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 3 (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
dis-sociation constants are also obtained in a more
elabor-ate 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
heptanucleo-tide (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
stret-ches of thymine nucleotides and for sequences including
other nucleotides In pK7CAT the sequence (T)7occurs
once, but outside coding sequences However, in the
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 tem-porarily dramatically reduced [1] Our in vitro data indicate that CSPs are directly involved in this down-regulation 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 evo-lution 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 pro-teins 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 expres-sion 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
Trang 9combined 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
clea-ving the target protein Other effects which can occur
in whole cells (e.g the induction of new proteins
inter-fering 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
bind-ing of the CSPs to sbind-ingle-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
recogni-tion 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 1⁄ 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
tempera-ture and of their fully inhibiting concentrations
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 lgÆmL)1
ampicil-lin and 68 lgÆmL)1chloramphenicol at 37C to D600¼ 1
Protein expression was induced by adding 1 mm isopropyl
thio-b-d-galactoside, and bacterial growth was continued
at 37C for 3 h Purification of the protein was performed
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 80C for 30 min Pure TmCSP was obtained after hydrophobic interaction chromatography at pH 8.0 and size exclusion chromatography with Superdex 75 (Amer-sham, Freiburg, Germany) The total yield of TmCSP was about 15 mgÆL)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)1Æcm)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
37C in dYT medium containing 25 lgÆmL)1 chloram-phenicol and 300 lgÆmL)1 ampicillin to D600¼ 0.8 Pro-tein expression was induced by addition of 2 mm IPTG and carried out for 4 h at 37C 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 0 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 4 FF column (Amer-sham) 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 concentra-ted by ultrafiltration to a concentration of 5.9 mm From
1 L of cell culture, 5 mg of BsCSP could be prepared The molar extinction coefficient of BsCSP was
5800 m)1Æcm)1[32,34]
Expression and purification of B caldolyticus CSP
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 30C in dYT medium con-taining 25 lgÆmL)1 kanamycin and 300 lgÆmL)1 ampicillin
to D600¼ 0.8 Protein expression was induced by tempera-ture shift to 42C carried out for 4 h Cells were centri-fuged and lysed as described [31] After cell lysis and centrifugation the cell-free extract was heated to 65C for
40 min to precipitate most of the E coli proteins All fol-lowing steps were carried out at 4C according to the puri-fication of BsCSP described above The purified protein
Trang 10was concentrated by ultrafiltration to a concentration of
3.0 mm The used molar extinction coefficient of BcCSP
was 7300 m)1Æcm)1[35]
Template DNA for combined
transcription/trans-lation 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
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
accord-ing to [38] The system for cell-free transcription and
trans-lation 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
phos-phate (CP) (Sigma, St Louis, MO, USA), 250 lgÆmL)1
cre-atine kinase (CK) (Roche, Indianapolis, IN, USA), 4.0%
PEG 8000, 0.64 mm 3¢,5¢-cyclic AMP, 68 lm
l(3)-5-formyl-5,6,7,8-tetrahydrofolic acid, 170 lgÆmL)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 lgÆmL)1of
the respective plasmid DNA, 140 lgÆmL)1T7 RNA
polym-erase, 35.1% (v⁄ v) S30 extract in volume of 26 lL and
4 lL water to a final volume of 30 lL The reaction
mix-ture was incubated at 37C for 1 h at 500 r.p.m in a
microtiterplate on a rotary shaker When CSPs were
titra-ted to the combined transcription⁄ translation experiment,
the 4 lL of water was replaced by CSP of different
concen-trations
In vitro transcription
The in vitro transcription was performed using the
Ribo-probe 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 3 lL of the
reaction volume were either 50 mm NaH2PO4, 100 mm
NaCl, 1 mm EDTA, pH 6.5 (buffer A) with or without
TmCSP or 50 mm Tris⁄ HCl pH 7.8, 100 mm KCl (buffer B) with or without BsCSP or BcCSP
In vitro translation
For determining the influence of CSPs on the transcription apparatus the system for combined transcription⁄ transla-tion was used without T7 RNA polymerase and protease inhibitor cocktail mRNA from the transcription experi-ments was used as template instead of the plasmid DNA Concentrations of the CSPs necessary for virtually complete inhibition were used as indicated in Table 1 The reaction volume was 30 lL
Assay of the reaction products
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]
Prediction of RNA secondary structures
The calculation of mRNA secondary structure formation was performed with the program mfold based on the algo-rithm described by [16]
Fitting of the binding isotherms
For a direct comparison of CSP from different organisms, apparent dissociation constants were derived for the inhibi-tion 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
Aðctotal CSPÞ ¼ A0
cDNAtotal Kapp ctotal
CSP
þ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðctotal DNA Kapp ctotal
CSPÞ2þ 4Kappctotal
DNA
ð1Þ with A0c total
DNA as the protein expression (mgÆmL)1Æs)1) in the absence of CSP, c total
CSP , the total concentration of CSP,
c total DNA , the total concentration of the CSP binding sites on the plasmid and Kappthe 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 concentra-tion c bound
ribo , of ribosomal complexes bound to the mRNA, that is