From the Department of Biology, University of
Michigan, Ann Arbor, Michigan 48109-1048 and the
§ Department of Biochemistry, Stanford University School of
Medicine, Stanford, California 94305-5307
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
We describe the isolation of Hsp15, a new, very
abundant heat shock protein that binds to DNA and RNA. Hsp15 is well
conserved and related to a number of RNA-binding proteins, including
ribosomal protein S4, RNA pseudouridine synthase, and tyrosyl-tRNA
synthetase. The region shared between these proteins appears to
represent a common, but previously unrecognized, RNA binding motif.
Filter binding studies showed that Hsp15 binds to a 17-mer
single-stranded RNA with a dissociation constant of 9 µM in 22.5 mM Hepes, pH 7.0, 5 mM MgCl2. A role of Hsp15 in binding nucleic
acids puts this protein into a different functional category from that
of many other heat shock proteins that act as molecular chaperones or
proteases on protein substrates.
Temperature upshifts and a number of other stress conditions
result in the rapid production of a number of proteins termed heat
shock proteins (Hsps).1
Although heat shock proteins were initially studied from a regulatory viewpoint, in recent years more and more attention has focused on
determining the function of these proteins. All four major classes of
heat shock proteins (Hsp90, Hsp70, GroE, and small heat shock proteins)
are known to function as molecular chaperones, proteins that help other
proteins adopt a biologically active conformation, without themselves
becoming part of the final structure (1). In addition, a number of heat
shock proteins are proteases, which hydrolyze irreversibly damaged
proteins (2). Thus, heat shock proteins appear to be generally involved
in the folding and degradation of proteins.
Sensitive RNA hybridization techniques and the availability of an
ordered, sequenced library of Escherichia coli clones have made possible the discovery of 26 new heat shock proteins in E. coli (3, 4). This represents a largely untapped resource of novel
heat shock proteins. In this paper, we report that one of these
proteins, Hsp15, is an abundant, well conserved nucleic acid-binding
protein, a function distinct from that of other previously described
heat shock proteins.
Cloning of Hsp15--
The BamHI fragment encoding the
Hsp15 gene, yrfH (hslR), was isolated from Purification of Hsp15--
The strain BL21(DE3) (Novagen)
harboring the plasmid pTHZ25 was induced at an
A578 of 0.8-1.0 with 1 mM
isopropyl-1-thio- Heat Shock Induction at the RNA Level--
hslR PCR
product (0.1 pmol) was fixed to a Hybond (Amersham Pharmacia Biotech)
nylon membrane and probed with labeled coding DNA as prepared by
reverse transcription of 20 µg of total RNA (isolated with Trizol
reagent; Life Technologies, Inc.) from log phase cultures grown at
37 °C or from the same culture heat-shocked for 10 min at
46 °C.
Determination of Cellular Abundance--
Cultures of E. coli AB1157 or MC4100 were grown in LB (6) at 29 °C until an
A546 = 1.0 (50 ml of culture in 250-ml
Erlenmeyer flasks, 300 rpm air shaker, doubling time 40-50 min).
Subsequent heat shock was achieved by transferring to a 42 °C
shaking water bath, the new temperature in the culture being reached
after <3 min. For the heat shock kinetic, aliquots were withdrawn,
cooled in ice-water, and centrifuged (1 min, 7900 × g,
4 °C), and the cells were washed in cold 150 mM NaCl and
re-centrifuged. The cell pellet was lysed in 2.4% (w/v) SDS, 50 mM Tris-HCl, pH 8.0, 8.0% glycerol by heating to 95 °C
for 10-30 min. The protein content of the lysate was determined by a
modified Lowry assay using BSA as a standard (9). The amount of Hsp15
present was determined by Western immunoblotting analysis according to
the protocol of the ECL system (Amersham Pharmacia Biotech). The films
were scanned with an Elscript 400 AT densitometer (Hirschmann, Neuried,
Germany) and analyzed using the PEAKFIT program (Jandel Scientific).
Known quantities of purified Hsp15 were treated on the same blots for calibration. Both strains gave consistent results. Polyclonal rabbit
antiserum against purified Hsp15 was a gift of Michael Ehrmann
(Universität Konstanz, Konstanz, Germany).
Determination of Association State of Hsp15--
The
sedimentation analysis data were collected with a Beckman model E
analytical ultracentrifuge in an AN-D rotor and double sector cells;
one sector contained Hsp15 at 11.6 µM in buffer B, and
the reference sector contained only buffer B. The molecular weight was
determined by the meniscus depletion method (10), and the evaluation of
the corresponding high speed (20,000 rpm) sedimentation equilibrium
data (ln c versus r2) was
done by a computer program developed by Dr. G. Boehm, Regensburg, Germany. The specific volume of the protein was assumed to be 0.735 ml/g (11). The temperature at equilibrium was 19 °C. Analytical gel
filtration was done on a Superdex 75 analytical grade HR10/30 (Amersham
Pharmacia Biotech) in 10 mM Hepes, 150 mM NaCl,
pH 7.0. The additional salt in the running buffer was necessary to
prevent Hsp15 from sticking to the column material. 5-14 µg of Hsp15
were loaded at a concentration of 150 µM in buffer B.
Gel Retardation Assays--
Single-stranded, circular M13mp18
DNA (7249 bases; U. S. Biochemical Corp.) was heated to 80 °C for 2 min prior to use. The plasmid pGEM-3Z (2743 base pairs; Invitrogen) was
prepared with the kit Nucleobond AX 500 (Macherey-Nagel, Düren,
Germany). The commercially available RNA preparation (yeast RNA type
III; Sigma, catalog no. R7125) is reported to consist mainly of tRNA
(12). Protein, nucleic acid, buffer, and salt were mixed in a final volume of 18 µl, and complex formation was allowed to proceed for at
least 30 min at room temperature. To assure equilibrium, up to 1 week
of incubation time was tested with no change in the apparent band
patterns compared with the 30-min incubation time. Immediately prior to
loading 2 µl of 10× loading buffer (50% glycerol, 0.25% bromphenol
blue, and 0.25% xylene cyanol) was added. Electrophoresis was
performed in 1% agarose (Biozyme) gels using TAE buffer (6) at 120 V
for 3-4 h. Afterwards, the gels were stained with ethidium bromide
(6).
Quantitative Zonal Affinity Chromatography--
The quantitative
zonal affinity chromatography was performed as described in Jenuwine
and Shaner (13), except that instead of the high performance liquid
chromatography we used a FPLC system with the HR 5/5 column (Amersham
Pharmacia Biotech). The elution buffer was 1 mM sodium
phosphate, pH 7.7, 0.1 mM EDTA, with NaCl added to the
designated concentration (13). The protein concentrations and volumes
loaded on the column were as follows: RNase A (Sigma), 6.6 mg/ml in
H2O, 10 µl loaded; Hsp15, 2.39 mg/ml in buffer B, 6 µl
loaded; cro repressor (a gift from Katrin Ramm,
Universität Regensburg, Regensburg, Germany), 1.6 mg/ml in 50 mM Tris-HCl, pH 7.0, 50 mM NaCl, 0.1 mM EDTA, 10 µl loaded; lac repressor (a gift
from Sonya Melcher, University of Wisconsin, Madison, WI), 4.18 mg/ml
in 10 mM Hepes, pH 7.55, 100 mM KCl, 0.1 mM 1,4-dithio-DL-threitol, 30% glycerol, 5 µl loaded. To calculate the affinities, the concentration of DNA in
nucleotides was used. This was 1.26-2.73 mM accessible nucleotides depending on the DNA-cellulose preparation, determined as
in Ref. 13. The data were consistent, independent of the batch of
DNA-cellulose. The data were plotted and analyzed with the SigmaPlot
program (Sigma).
Filter Binding Experiments--
A DEAE filter (NA45
DEAE-cellulose, Schleicher & Schuell) was soaked for 10 min in 10 mM EDTA, pH 8.0, then 10 min in 0.5 M NaOH with
shaking at room temperature, washed to neutrality with water, and
incubated for >1 h in the wash buffer (22.5 mM Hepes, pH
7.0, 5 mM MgCl2, 0.75 mM EDTA). The
nitrocelluose-filter (Protran nitrocellulose, Schleicher & Schuell) was
treated as the DEAE-cellulose but without the EDTA step. Immediately
prior to application of the samples, the two filters were mounted in a
48-slot blotting device (Hoefer), the nitrocellulose filter directly on
top of the DEAE filter. Only a very gentle vacuum was applied to
prevent the filters from drying out. For the same reason, all samples
were added as fast as possible, which usually did not take more than 5 min in total. The volume of the binding reaction was 10 µl; the
buffer was the same as the wash buffer plus additional 1.0 µM BSA (New England Biolabs), 8 mM
1,4-dithio-DL-threitol, and 3.2 units of RNAsin (Promega).
Binding reactions were incubated at room temperature (23-25 °C) for
about 20 min. Addition of BSA prior to the addition of Hsp15 was
important to prevent Hsp15-RNA-complexes from sticking to tube walls.
The Hsp15 preparation was RNase-free, but the RNase inhibitor RNAsin
was always added when using BSA. When the liquid of the samples had
completely passed the filters, the slot was immediately washed with 200 µl of wash buffer.
The sequences of the various RNA substrates were: GMP as monomer, CUG
as trimer, CUCG as tetramer, CCCUC(dT) as hexamer, GGGAACGUC as
nonamer, and GGGAACGUCGUCGUCGC as heptadecamer (17-mer). All substrates
were 5'-32P-labeled as described in Ref. 14. About 2000 cpm/slot were used. The dried filters were quantitated and analyzed
with the PhosphorImager System (Molecular Dynamics). The curve fit in
Fig. 7 was done with the program Kaleidagraph (Macintosh).
Enzyme Assays--
The protease assay was done according to
Twining (15); the chaperone assay was performed as described by Jakob
et al. (16). Dialysis buffer was used as negative control.
Positive controls were trypsin (Sigma) and Hsp90 (a gift from U. Jakob).
Characterization of New Heat Shock Proteins in E. coli
Twenty-six new heat shock genes were discovered in E. coli by global transcription analysis and mapped to specific Hsp15 Is Highly Conserved in Prokaryotes
The Hsp15 sequence was used to search the nonredundant data base
and the data base of unfinished microbial genomes maintained at
National Center for Biotechnology Information (NCBI). Significant matches were found for sequences present in a wide variety of prokaryotic organisms including both Gram-negative and Gram-positive bacteria (Fig. 1). All of the homologues
are very basic proteins, with pI values ranging from 9.9 to 10.3. We
conclude that Hsp15 is a member of a previously undescribed family of
well conserved, highly basic proteins. The molecular mass values of the
Hsp15 homologues in Gram-negative organisms are in the range of
15.1-15.5 kDa. The proteins in the Gram-positive organisms
Streptococcus and Bacillus are 5 kDa smaller in
size due to a shortened C terminus. We decided to further investigate
the function of this protein because its high degree of conservation
implies that it plays an important role. We reasoned that, because it
is not related to any known heat shock protein, this role may turn out
to be new.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
clone 621 (4) and ligated into pUC19 (New England Biolabs) giving the
plasmid pTHZ13. The hslR gene was isolated via PCR using
pTHZ13 as the template, primer Hsp15-P1 (5'-TGAAGGAGACCATATGAAAGAGAAA-3'), primer Hsp15-P2
(5'-TCTTGCAGGATCCAGTTATTCACT-3'), and Taq polymerase
(Stratagene). The PCR product was purified with the QIAEX kit (QIAGEN,
Valencia, CA), digested with BamHI and NdeI, and
ligated into the overexpression vector pET11a (Ref. 5; Novagen) to
generate the plasmid pTHZ25. The sequence of this construct was
confirmed by sequencing the entire insert region on both strands using
an ABI automated sequencer (Perkin-Elmer).
-D-galactopyranoside for 4 h and
harvested by centrifugation (15 min, 5000 × g,
4 °C). All further steps were performed at 4 °C. The cell pellet
was resuspended in cold buffer A (30 mM sodium borate, 1 mM EDTA, pH 8.5) and lysis was performed by two passes
through a French pressure cell (1 inch in diameter, American
Instrument) at 18,000 p.s.i. The lysate was centrifuged (25 min,
39,100 × g) and the supernatant immediately loaded
onto a tandem ion-exchange chromatography system consisting of 50 ml of
Q-Sepharose and subsequent 25 ml of SP-Sepharose (Amersham Pharmacia
Biotech) both equilibrated with buffer A. After washing to base line at
a flow rate of 1 ml/min and removing the Q-Sepharose column, the
SP-Sepharose column was eluted with 10 column volumes (250 ml) of a
linear KCl-gradient (0.0-1.0 M KCl in buffer A).
Hsp15-containing fractions as assayed by SDS-polyacrylamide gel
electrophoresis were pooled and dialyzed over night against a
>500-fold volume of buffer B (30 mM Hepes, 1 mM EDTA, pH 7.0). During this dialysis a precipitate formed that was removed by centrifugation (15 min, 34,800 × g, 4 °C). The supernatant was brought to 1.5 M ammonium sulfate in buffer B and loaded onto a 25-ml
Phenyl-Sepharose (Amersham Pharmacia Biotech) column equilibrated with
the same buffer. After washing to base line, the protein was eluted
with 250 ml of a linear gradient with decreasing ammonium sulfate
concentration (1.5-0.0 M ammonium sulfate in buffer B).
Fractions that contained Hsp15 only were pooled and concentrated by
ultrafiltration (180-ml Amicon cell; 3-kDa exclusion volume membrane
(Millipore)). To test for coelution of Hsp15 with nucleic acids, the
Q-Sepharose column was eluted separately with a linear KCl gradient
(500 ml, 0.0-1.0 M KCl in buffer A). Aliquots of the
fractions were concentrated by ethanol precipitation (6) and assayed
for nucleic acid content with agarose gels (6). Using the method of
Edelhoch (7) and the variations of Pace (8) the molar extinction
coefficient was determined as 15,524 M
1
cm
1, which is in good agreement with the calculated value
of 15,470 M
1 cm
1 (8), and was
used for determination of Hsp15 concentrations.
RESULTS
clones in the ordered Kohara library (3, 4). Taking advantage of the
estimated molecular weights of the corresponding heat shock proteins
and the recently released sequence of the complete E. coli
genome, we have been able to assign most of these "heat shock loci"
(hsl) genes to sequenced open reading frames.2 We
consider these genes a largely unexplored resource of heat shock genes
unrelated to previously studied heat shock genes. One of these,
yrfH (hslR) encodes a 15,496-Da protein with a
calculated isoelectric point (pI) of 9.94. Based on the molecular
weight and the heat inducibility of the product of the yrfH
(hslR) gene, we have assigned it the name Hsp15.
View larger version (52K):
[in a new window]
Fig. 1.
Multiple alignment of the newly identified
Hsp15 family. Sequence homology searches using GAPPED BLAST (17)
against the GenBank nonredundant data base as well as searches in the
incomplete microbial genome data base maintained at NCBI performed on
May 22, 1998 revealed 14 members of the Hsp15 family. Sequence
alignments were performed with CLUSTALW (18). Amino acid residues
identical in 50 or more percent of the sequences are highlighted in
black; related amino acids were shaded gray using
the display program BOXSHADE. The source abbreviations and Swiss
Protein or accession numbers, when available, are as follows: E. coli, Escherichia coli, YRFH_ECOLI; Yersin,
Yersinia pestis Sanger|Contig1280; H.influ,
Hemophilus influenzae YRFH_HAEIN; Actinob,
Actinobacillus actinomycetemcomitans
OUACGT A.actin_Contig585; Vibrio, Vibrio
cholerae TIGR|GVCDC26R; Aeromo, Aeromonas
salmonicida, accession no. X96968; Pseudom,
Pseudomonas aeruginosa gnl|PAGP|Contig246;
Neisseria, Neisseria gonorrhoeae
OUACGT|Contig222; Streptoc, Streptococcus
pneumoniae TIGR stp_4125; Bacillus, Bacillus
subtilis YABO_BACSU. Because the genomes of Yersinia,
Actinobacillus, Vibrio, Pseudomonas,
Neisseria, and Streptococcus are not yet
complete, the sequences from these organisms should be regarded as
preliminary.
Hsp15 Shows Homology to RNA-binding Proteins
The search of the nonredundant data base with E. coli Hsp15 was iterated using the sensitive PSI-BLAST program, which includes profile construction (17). This search method automatically combines the significant alignments produced by BLAST into a matrix of position-specific scores. Searching the data base using this matrix in many cases allows the discovery of faint, but biologically relevant sequence similarities. Using this approach, a number of proteins that interact with RNA were found to contain a motif in common with each other and Hsp15 (Fig. 2). These include ribosomal protein S4, RNA pseudouridine synthase, and tyrosyl-tRNA synthetase. A 31-amino acid motif that usually begins with the sequence RLD and ends with the sequence NG is shared among Hsp15, these families of RNA-binding proteins, and HlyA. HlyA is homologous to FtsJ, a RNA methylase,3 suggesting that members of the HlyA family may bind RNA as well.
|
Purification of Hsp15
A purification strategy based on the high isoelectric point of Hsp15 was devised. It involved a tandem ion-exchange chromatography system at pH 8.5 consisting of an anion exchange "pre-clearing" column, immediately followed by a subsequent cation exchange "trapping" column. Of the contaminating proteins co-eluting with Hsp15 from the cation exchange matrix, nearly all precipitated during the following dialysis, which therefore served as an effective purification step. A hydrophobic chromatography step separated Hsp15 from a small amount of degradation product. Hsp15 was assayed for purity by SDS-polyacrylamide gel electrophoresis and subsequent silver staining and was estimated by densitometry (data not shown) to be of greater than 99.7% purity. The identity of the purified protein was confirmed by N-terminal sequencing of the first nine amino acids; the initiation methionine was still present.
Although Hsp15 is expected to still be positively charged at pH 8.5, elution of the anion exchange column yielded some Hsp15-containing fractions. These fractions also showed the presence of nucleic acids in ethidium bromide-stained agarose gels (data not shown). This co-elution suggested to us that Hsp15 bound to the anion exchange matrix via nucleic acids, suggesting a tight interaction between Hsp15 and nucleic acids.
Heat Shock Induction of Hsp15 and Steady State Expression Levels
To verify that Hsp15 is indeed a member of a new heat shock protein family, mRNA hybridization experiments were performed (Fig. 3, inset). A transient burst of heat shock RNA synthesis normally occurs in the 5-15 min following temperature shift. mRNA was prepared from log phase E. coli cultures grown at 37 °C or from the same culture after a 10-min heat shock treatment at 46 °C. As shown in the inset to Fig. 3 and characteristic of heat shock genes, Hsp15's mRNA is strongly up-regulated upon a temperature shift from 37 °C to 46 °C. This transient burst of heat shock mRNA synthesis has the effect of rapidly allowing the heat shock proteins to reach a new higher steady state level following temperature shift.
|
In order to quantitate the level of Hsp15 protein present during steady
state growth, we measured the intracellular abundance of Hsp15 in
exponentially growing cultures at 29 °C and various times following
a shift to 42 °C. Western immunoblot analysis showed that cultures
at 29 °C contained 1.29 w/w Hsp15 per total cell protein. This
fraction increased to 2.22
, 2.37
, and 2.04
after 11, 22.5, and
45 min of heat shock treatment (42 °C), respectively. Thus
12,000-22,000 molecules of Hsp15 are present per cell, as calculated
using a conversion factor of 2.34 ×10
13 g of
protein/cell (Ref. 19; Fig. 3). To calculate the intracellular concentration, the association state of Hsp15 needed to be determined. Hsp15 was found to be a monomer, in the absence of nucleic acid, by
both analytical ultracentrifugation (14.7 ± 1.3 kDa) and gel permeation chromatography (15.8 ± 2.4 kDa) (Fig.
4). According to an average volume of an
E. coli cell of 0.63 µm3 (Ref. 20; doubling
time of 40 min), the intracellular concentration of Hsp15 is 30-60
µM. This makes Hsp15 a very abundant protein in the cell
before and after heat shock, even more abundant than the GroEL 14-mer,
which has been estimated (using slightly different conversion factors)
to be present in 1580 copies or at a concentration of 2.6 µM at 37 °C (21). The observed ~2-fold increase in
Hsp15 at the protein level is very similar to the degree of increase in
protein level seen with the other characterized heat shock proteins in
E. coli. The average increase in steady state level following shift from 30 °C and 42 °C for the 11 heat shock
proteins that have been measured in E. coli is 1.97-fold
(22). Many show similar n-fold increases such as GroEL
(2.3-fold) or DnaK (2.0-fold); some, such as GrpE (1.3-fold) and lon
(1.1-fold), show less of an increase; one other, HtpG, shows a
substantially greater increase of 3.8-fold (22).
|
Nucleic Acid Binding
Gel Retardation Assays--
To directly show that Hsp15 binds
nucleic acids, we tested its ability to shift various kinds of nucleic
acids in gel retardation assays. Three different substrates were
tested: the plasmid pGEM-3Z as an example of dsDNA, the single-stranded
vector M13mp18 as an example of ssDNA, and a commercially available RNA
preparation that consists mainly of tRNA. Under the same conditions and
within the same gels, Hsp15 was compared with two well characterized nucleic acid-binding proteins: RNase A, as an example for a very weak
binder (23), and the phage cro repressor, which exhibits high binding affinities (24). The binding reaction was performed in
buffer B plus 150 mM KCl to mimic physiological ionic
strength to a first approximation. The highest protein concentration
was also tested in buffer B without additional KCl to give an
impression of binding in low salt.
Fig. 5a shows the gel retardation assay using the dsDNA substrate (pGEM-3Z). The migration behavior of the naked DNA is seen in lanes 1 and 17. As is typical for plasmid preparations, there are four major bands corresponding to the supercoiled and nicked form of the plasmid monomers and dimers (25). RNase A shows hardly any effect at the RNase A concentrations tested, not even in low salt (Fig. 5a, lanes 2-6). This indicates that the assay in this range of protein concentrations is not sensitive enough to detect the low binding affinity of RNase A to dsDNA. In contrast, the cro repressor clearly shifts all four bands of the plasmid preparation (Fig. 5a, lanes 12-16). The retardation becomes more pronounced with rising protein concentration arguing for an additive binding mode. A very similar behavior is seen for Hsp15 (Fig. 5a, lanes 7-11). This is clear evidence that Hsp15 is binding to dsDNA.
|
The extent of band shift for both Hsp15 and the cro repressor was increased at the low salt conditions (Fig. 5a, lanes 11 and 16). This confirms that the binding interaction is sensitive to ionic strength.
The same type of experiment was repeated using a single-stranded DNA substrate (Fig. 5b). Here, RNase A shows a slight shift at high concentration reflecting its higher affinity for ssDNA than dsDNA (23). Hsp15 retards ssDNA to a greater extent at lower concentration than the cro repressor, perhaps suggesting a preference of Hsp15 for single-stranded over double-stranded substrates (see Fig. 5b, lanes 10 and 11 versus 15 and 16).
Fig. 5c shows that both Hsp15 and the cro repressor clearly retard the migration of a mainly tRNA substrate. The abundance of the shifted complex appears to be higher with Hsp15.
Zonal Quantitative Affinity Chromatography on DNA Cellulose-- In order to assess the interaction of Hsp15 with dsDNA in a more quantitative way, we employed zonal quantitative affinity chromatography. This method provides a rapid and convenient means to quantitatively compare a protein of interest with standard proteins in its nucleic acid binding affinity as well as the salt dependence of this affinity (13).
Proteins with affinity for DNA will cyclically bind and unbind as they pass through a DNA cellulose column. The time it takes a protein to elute from such a column compared with the elution time under high salt conditions, where no binding occurs, is directly correlated to the affinity constant KB, if chromatography is performed in the linear regime allowing rapid determinations of KB values (13).
The range of KB values for which the method is
applicable is limited to the window of 102 to
104 M1. Higher affinities will
generate prolonged elution times, diffusion of the protein peak, and
subsequent detection problems. However, most nonspecific protein-DNA
interaction equilibrium constants can be shifted into the
102 to 104 M
1 window
by adjusting the salt concentration since the cation concentration is
the major determinant for the affinity of a nonspecific protein-DNA interaction (26).
Using this method, Hsp15 was compared in its elution properties at varying salt concentrations to three proteins whose nonspecific binding affinities to DNA are well studied: the lac repressor, RNase A, and the cro repressor (Fig. 6, a-c).
|
The binding affinity of Hsp15 to the dsDNA matrix was found to be
virtually the same as that of the lac repressor (Fig.
6a). Linear regressions for the data sets of Hsp15 and the
lac repressor are in good agreement. Further, the measured
values for the lac repressor meet very well previously
published data for the nonspecific binding of the lac
repressor to dsDNA (Fig. 6b; Refs. 27-30; corrected for pH
according to Ref. 31). A linear regression for the combined sets of
data from the literature and our measurements for the lac
repressor (log(KB/M1) =
2.92-9.61 log([NaCl]/M); r2 = 0.941) resembles very much the salt dependence of the affinity constant
for the lac repressor as published by Jenuwine and Shaner (Ref. 13; log(KB/M
1) =
2.83-9.76 log([NaCl]/M); r2 = 0.975). For the other two tested proteins, RNase A and the cro repressor (Fig. 6c), measurements of binding
constants at varying salt concentrations also agreed well with data
from the literature (23, 24, 27). As already suggested by the gel retardation experiments, Hsp15 binds with much higher affinity to dsDNA
than RNase A and with somewhat lower affinity than the cro
repressor does. For each protein, the salt dependence of
KB appeared to be linear, as would be expected
for the absence of significant specific ion binding effects associated
with the proteins (26).
Filter Binding-- The binding of Hsp15 to ssRNA was assayed by filter binding experiments. Labeled RNA substrate was incubated with increasing amounts of Hsp15 and analyzed by washing over a nitrocellulose and subsequent DEAE-cellulose filter. Protein-RNA complexes are retained on the first filter and free RNA on the second filter. The ratio of the amount of RNA on the nitrocellulose filter to the sum of the RNA on both filters gives a measurement of the fraction of protein-RNA complexes in the binding reaction. Because the RNA is present only in trace amounts, the free protein concentration can be assumed to be equal to the total protein concentration.
The binding curve in Fig. 7 shows a sigmoidal shape. A curve fit assuming a single site binding curve does not fit the data well (not shown). This speaks for more than one Hsp15 molecule bound to the 17-mer RNA substrate. A curve fit to a Hill-equation allowing the Hill constant to vary (see figure legend) agrees well with the experiment and gives a dissociation constant of 8.9 ± 3.0 µM and a Hill constant of 2.6 ± 0.4.
|
Substrate Length Effects-- The filter binding curve suggested that 2-3 Hsp15 molecules could bind to a 17-mer RNA substrate, which would amount to a binding site size between 6 and 8 nucleotides. To further approximate the binding site size, RNA substrates of decreasing length were tested for binding to Hsp15 at two different concentrations (Fig. 8). Substrates with 1, 3, or 4 nucleotides were not significantly bound by Hsp15, as tested by filter binding. The onset of retention on the nitrocellulose was with the 6-mer RNA substrate, although only at the high concentration of Hsp15 and only to a small extent. The 9-mer was well bound by Hsp15 at both concentrations but still to a lesser extent than the 17-mer. Six nucleotides appeared to be the minimal required length for binding under these conditions consistent with the Hill constant of the binding curve for the 17-mer (Fig. 7), which suggested that 2-3 molecules of Hsp15 could be bound per 17-mer.
|
![]() |
DISCUSSION |
---|
Our results define a newly recognized, well conserved, abundant heat shock protein that binds nucleic acids. Hsp15 was found to have a domain homologous to a number of RNA-binding proteins (Fig. 2) including ribosomal protein S4, RNA pseudouridine synthase, and tyrosyl-tRNA synthetase. The RNA binding domain on ribosomal protein S4 has been mapped to residues 48-177 (32), a region that includes the residues 97-127 that show homology to Hsp15. The region responsible for tRNA binding in the tyrosyl-tRNA synthetases is near the C terminus (33). The Arg-19 in the motif shown in Fig. 2 is at the homologous position to Arg-371 of the Bacillus stearothermophilus tyrosyl-tRNA synthetase. This arginine is directly implicated in tRNA binding (34). Since the only sequence shared by all these proteins is this motif and the only function they have in common is RNA binding, we propose that this RLD motif represents a previously unrecognized RNA binding motif.
This newly recognized potential RNA binding motif is surprisingly commonly found; eight proteins in E. coli contain this motif (Fig. 2 and data not shown).
The very basic properties of Hsp15 (pI of 9.94) and co-elution with nucleic acids from an anion exchange column during the purification further suggested a possible nucleic acid binding activity. The nonspecific nucleic acid binding properties were investigated by gel mobility shift assays, zonal quantitative affinity chromatography, and filter binding experiments.
In gel retardation experiments, Hsp15 was shown to bind to double-stranded plasmid DNA, to single-stranded phage m13 DNA, and to a yeast RNA preparation that mainly consisted of tRNA (Fig. 5, a-c). The more Hsp15 was added, the more retarded the nucleic acid band became, speaking for multiple binding sites on the substrates, which is typical for nonspecific protein-nucleic acid interactions (35). Also as expected was the sensitivity of the nucleic acid interaction to the salt concentration (26). This behavior was compared with the nonspecific nucleic acid binding properties of RNase A and the cro repressor in the same assay. Whereas RNase A showed hardly any binding in this system, the cro repressor clearly shifted the nucleic acid substrate, as expected for a protein with high nonspecific DNA binding activity (24). Hsp15 was shown to retard all three nucleic acid substrates. Compared under the same buffer conditions and protein concentrations to the cro repressor, the extent of the change in the migration position of the nucleic acid band in the gel was somewhat less for the dsDNA substrate, but similar if not greater with the ssDNA and the tRNA substrates. For both proteins the shifted bands had a smeared appearance, consistent with an intermediately cooperative binding mode (36).
The nonspecific binding affinity of Hsp15 to dsDNA was assessed via zonal quantitative affinity chromatography (13). Here, Hsp15 behaved virtually identically to the lac repressor on the DNA cellulose matrix in absolute terms as well as in the dependence of the affinity constant to the salt conditions (Fig. 6a). In order to confirm the applicability of the measurements with our column system, we compared our data for the lac repressor with the extensive data in the literature (Refs. 27-30; Fig. 6b). The measurements of the lac repressor on our column system met very well with the literature data. The published data stem from different methods, and there is a considerable amount of scatter among them. An agreement of the data within 0.2 log units is considered to be good (28). We also tested RNase A and the cro repressor (Fig. 6c), and both gave binding constants very similar to those in the literature, further corroborating the precision of this method (23, 24, 27). Data for the cro repressor are obtained at different conditions: pH 7.3 versus 7.7 and KCl versus NaCl, but the nonspecific DNA binding affinity of the cro repressor is insensitive to these changes.4
As Hsp15 behaved very similarly to the lac repressor, we
used the literature data for the lac repressor to estimate
Hsp15's affinity constant under conditions that, in first
approximation, mimic physiological ionic strength (150 mM
KCl). This affinity constant cannot be measured directly with the
affinity column because Hsp15 binds too tightly (see "Results" and
Ref. 13). By correction for pH according to the relationship
(log(KB/M
1))/
pH =
2.1 (31), we extract from Refs. 28 and 29 a range of binding
affinities of the lac repressor at 150 mM NaCl,
pH 7.7, of 25-4.7 104 M
1. These
correspond to dissociation constants of 4-20 µM. Thus, we conclude that Hsp15 binds to dsDNA in the micromolar range at
conditions that mimic physiological salt concentration to a first
approximation. The close resemblance of the salt dependence of Hsp15
KB to that of the lac repressor in
both the slope and y axis intercept also shows that this
interaction for Hsp15 is like that of lac repressor mainly driven by
the electrostatic contribution, i.e. counterion release
(26).
The nonspecific binding of Hsp15 to ssRNA at 5 mM MgCl2 was measured by filter binding experiments. A binding curve (Fig. 7) has a sigmoidal shape, suggesting, as seen with the gel retardation experiments, that more than one Hsp15 molecule bound to the nucleic acid substrate. Hsp15 is a monomer in the absence of nucleic acid at concentrations between 12 and 150 µM independent of the presence of 150 mM NaCl (analytical ultracentrifugation; see also Fig. 4). Both the gel retardation experiments and the binding curve with the 17-mer ssRNA show that Hsp15 seems to multimerize in a cooperative way on nucleic acid substrates that are long enough to accommodate several Hsp15 molecules. Whether the multimerization on the 17-mer ssRNA affected significantly the retention on the nitrocellulose and therefore enhanced the impression of cooperativity cannot be ruled out at this stage. The filter binding data are fitted well with the Hill equation that is based on a model of cooperative binding. The dissociation constant derived from the fit was 8.9 ± 3.0 µM. The Hill constant was fitted to be 2.6 ± 0.4. It is a measure for the cooperativity of the interaction and gives also an estimate for the number of molecules bound, in this case speaking for 2-3 Hsp15 molecules being bound to a 17-mer ssRNA substrate. The binding site size therefore appears to cover between 6 and 8 nucleotides. This was confirmed by filter binding experiments with ssRNA substrates of decreasing length (Fig. 8). A 6-mer substrate is just on the verge of being significantly bound by Hsp15. We estimate the minimal length required for strong binding under the condition tested to be about 6 nucleotides.
The intracellular concentration of Hsp15 was determined to be 30 µM constitutively and 60 µM after heat shock (Fig. 3). In this concentration range of Hsp15, all the substrates in the gel retardation experiments (Fig. 6, a-c) and also the 17-mer ssRNA in the filter binding assay (Fig. 7) were tightly bound. The estimated KD values for both DNA and RNA substrates are well below the determined intracellular concentration of Hsp15. Thus, it is very likely that the function of Hsp15 lies in binding of a nucleic acid substrate in vivo, too.
What is the in vivo substrate for Hsp15? The main clues so far are the high abundance of Hsp15 and its motif homology to four RNA-binding proteins. At 29 °C about 12,000 molecules of Hsp15 are present per cell and roughly 2-fold more (22,000) are expressed after heat shock. This abundance makes Hsp15 unlikely to recognize a specific DNA site. Nonetheless, a nonspecific DNA binding function is possible. The histone-like proteins HU, H-NS, and IHF are present in E. coli at about the same abundance (30,000, 18,900, and 15,000 molecules/cell, respectively, at 37 °C; Refs. 22 and 37).
The motif homology to RNA-binding proteins, however, strongly suggests that Hsp15 may recognize a RNA substrate in vivo, rather than a DNA substrate. If the in vivo function involves RNA binding, a specific binding site is certainly possible since tRNAs as well as rRNAs are present in high copy numbers in the cell (7000-70,000 rRNA molecules per cell and about 200,000 tRNA molecules; Refs. 19 and 38).
Of the heat shock proteins in E. coli characterized so far,
13 of 21 exhibit either molecular chaperone or protease function (2).
Hsp15 was tested for molecular chaperone and protease activity but gave
negative results (data not shown). Besides the heat-inducible sigma
factors (70,
32,
E) that
take part in the regulation of the heat shock response, only the
lysyl-tRNA synthetase LysU is known so far as a nucleic acid-binding
heat shock protein (2). Our isolation of the very abundant, nucleic
acid-binding, heat shock protein Hsp15 suggests that the heat shock
response may comprise not only functions acting on the protein but also
on the nucleic acid level.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Tom Record and Sandy Shaner for helpful advice on the zonal affinity chromatography and insightful comments on the manuscript. We also thank Ursula Jakob for useful discussions. We thank Wilson Muse for the RNA hybridization experiments and help with the figures and Michael Ehrman for generating antibodies to Hsp15. We especially thank Rainer Jaenicke for conducting the analytical ultracentrifuge experiment and for all the support he provided.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the National Institutes of Health, Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, and the Pew Charitable Trusts.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to Tim Hering on the occasion of his 70th birthday and in honor of his encouraging example.
¶ To whom correspondence should be addressed. Tel.: 734-764-8028; Fax: 734-647-0884; E-mail: jbardwel{at}umich.edu.
2 W. B. M. Muse, T. Zander, and J. C. A. Bardwell, unpublished data.
3 H. Buegl, U. Jakob, and J. C. A. Bardwell, unpublished data.
4 Y. Takeda, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Hsp, heat shock protein; PCR, polymerase chain reaction; BSA, bovine serum albumin; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; ssRNA, single-stranded RNA..
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|