From the
G4 nucleic acids are quadruplex structures involving
guanine-rich sequences that form in vitro under moderate
conditions. Experimental evidence exists supporting biological
functions for these elements; however, direct demonstration of G4
nucleic acids in vivo has not yet been achieved. Here we
purify and characterize a yeast protein, G4p2, which has a specific
affinity for G4 nucleic acids. G4p2 binds equivalently to RNA and DNA
in G4 form. The K
DNA and RNA containing guanine tracts will associate in vitro in the presence of salt into four-stranded
right-handed helices stabilized by a guanine base quartet (for review
see Refs. 1-5). Three major helical isoforms, characterized by
parallel or cis or trans antiparallel strand
orientations, have been identified by NMR and x-ray studies. These
quadruplex structures are called the G4 nucleic acids. G4 structures
anneal at moderate to low rates under physiological conditions and are
greatly stabilized by millimolar concentrations of potassium, which is
efficiently chelated by guanine carbonyl oxygens that line the axial
cavity of the helix.
Do G4 structures arise in vivo?
Presently, this question is unresolved, although there is tantalizing
evidence supporting roles for G4 structures at eukaryotic telomeres
(7, 8) and at retroviral dimer linkage sequences
(9, 10) . Also, Sep1/Dst2/Xrn1/Kem1 is a yeast enzyme
that catalyzes DNA strand exchange
(11, 12) , hydrolyzes
RNA
(13) , and possesses a nucleolytic activity specific for DNA
oligomers containing G4 domains
(14) . Null alleles of this gene
lead to mitotic loss of chromosomes and meiotic arrest at pachytene
(15) . These defects may be due to an inability of the mutant
cell to properly process G4 structures that may arise during the course
of chromosomal replication, recombination, or alignment. Further, a G4
DNA-annealing activity capable of accelerating the conversion of
oligomers into G4 structures by a factor of 10
To explore the possible biological
functions of G4 nucleic acids, we have probed extracts for activities
specific for G4 DNA. We have identified and characterized a yeast
protein, G4p2, that displays an avid and specific binding activity for
G4 nucleic acids. G4p2 slightly prefers substrates containing
antiparallel G4 domains over those containing the parallel form and
strongly prefers substrates containing multiple G4 domains. G4p2 binds
equivalently to DNA and RNA in G4 form. The protein was purified, and
the gene encoding G4p2 was isolated and sequenced. The G4P2 gene is identical to MPT4 and STO1, genes that
appear to function in a protein kinase-controlled signal transduction
pathway and in cell cycle progression, respectively.
SDS-PAGE was carried out by
standard procedures
(18) . 10-µl aliquots of G4p2 fractions
were loaded on 10% SDS-PAGE gels, and protein bands were visualized by
staining twice with a silver staining reagent (Bio-Rad).
Half of the partially purified G4p2 from
the previous step (6 ml) was concentrated with Microcon-10
microconcentrators (Amicon) to 0.15 ml, washed twice with 7 volumes of
buffer B (20 m
M NaHepes, pH 7.3, 1 m
M EDTA, 50 m
M KCl, 0.05% Triton X-100, 5 m
M BME, 10% glycerol), and
recovered in a volume of 0.5 ml. The sample was made 100 m
M NaOH for 5 min and neutralized with HCl.
A 1-ml G4 DNA affinity
bed was prepared as follows. Oligomer GL (see Fig. 1) with biotin
incorporated next to the 3` end (Biotin-ON phosphoramidite, Clontech)
was synthesized at a 1-µmol scale. The unpurified oligomer was
converted into parallel G4 form under conditions specified by Sen and
Gilbert
(17) . An aliquot was labeled with
[
Partially purified G4p2 from the previous
step (0.5 ml) was loaded at 2.3 ml/h on the G4 DNA affinity column and
washed with 5 ml of buffer B with 100 m
M NaCl, and bound
proteins were eluted with a 5-ml convex exponential gradient of
increasing NaCl concentration (100 m
M to 1
M). Fifty
0.1-ml fractions were collected and assayed by mobility shift assay for
G4p2 and by SDS-PAGE (see Fig. 3). Fractions 21-27 (0.7 ml)
containing largely pure G4p2 were pooled.
Selected phage DNA was pooled and used as template for
thermal cycle sequencing with the CircumVent (New England Biolabs)
sequencing reagents. To obtain access to the G4p2 gene, 1.2 pmol of a
128-fold degenerate oligomer (49A,
5`-AA(C/T)CA(A/G)CA(A/G)GC(A/G/C/T)AA(C/T)AA(C/T)CA-3`) derived from
reverse translated NT-49 was labeled with
[
No other polypeptide sequences
with significant homologies to G4p2 have been identified thus far.
Searches of the PROSITE 8.0 data base failed to identify peptide motifs
of biological interest in the G4p2 amino acid sequence.
We have described a protein, G4p2, isolated from yeast, which
displays a selective affinity for nucleic acids in the G4 form. The
specific requirement for this structure was demonstrated by mobility
shift binding experiments with a parallel G4 DNA probe and with
competing single-stranded DNAs, double-stranded DNAs, and various DNAs
harboring G4 structure. We find G4p2 binds G4 domains in both RNA and
DNA with equivalent affinity and strongly prefers substrates containing
more than one G4 region. G4p2 has an unusual amino acid composition
generally abundant in alanine, asparagine, and lysine and also
possesses an unusual amino acid distribution containing regions rich in
proline, in alanine, in serine and threonine, in glutamine and
asparagine, and in asparagine, arginine, and glycine. The explicit
function of G4p2 is not yet known.
The identification of G4p2 with
MPT4 and STO1 provides the beginnings of a genetic
analysis of function. MPT4 was isolated as a multicopy
suppressor of staurosporine sensitivity in POP2 cells,
Associated with POP2 is an enhanced sensitivity to staurosporine,
STO1 is a multicopy suppressor of TOM1, which is an as yet
molecularly uncharacterized mutation that leads to cell cycle arrest at
the G2/M boundary in yeast.
We presently can only speculate as to the
function of G4p2. It is likely, in light of the suppression of
staurosporine sensitivity in POP2 cells by MPT4, that
G4p2 functions in a signal transduction pathway controlled by protein
kinases somewhere downstream of Pop2. Pop2 appears to regulate the
expression of genes involved in carbon source utilization
(6) .
Therefore, our results demonstrating a G4 nucleic acid binding property
for G4p2 open up the possibility that G4p2 may execute an effector
function in gene regulation by binding to G4 domains in DNA or RNA.
G4p2 may modulate gene expression by association with chromatin.
Snf/Swi and Spt/Sin proteins are moderately abundant
chromatin-associated proteins identified by their effects on the
regulation of specific genes
(29) . Another chromatin-associated
protein, Rap1, which binds to telomere sequences and silencer loci
throughout the yeast genome, has been shown to possess G4 nucleic acid
binding properties
(30) . G4 structures have been proposed to
arise at chromosome sites that have undergone local unwinding due to
superhelical stresses. A study of the human insulin gene inserted in a
plasmid demonstrates supercoil-dependent G4 structures at guanine
tracts in a polymorphic region directly upstream from the promoter
(31) . This region has been implicated in the negative
regulation of insulin expression
(32) .
As a possible
alternative to a DNA binding regulatory factor, G4p2 may have a role in
gene regulation through an interaction with RNA. G4p2 may modulate the
translation of a set of mRNAs by binding to signature G4 domains
contained within these molecules. This is analogous to the situation
for ribosomal proteins whose mRNAs are regulated at the translational
level by trans acting factors that interact with a
polypyrimidine tract located in the 5`-terminal regions
(33) .
Perhaps G4p2 acts by binding to an RNA component of the translation
appara-tus and thus indirectly modulates the expression of a set of
mRNAs. Experiments have already demonstrated the ability of E. coli 5 S rRNA to form parallel G4 structures in vitro (34) . An examination of the 26 S rRNA sequence of yeast
reveals multiple guanine tracts in regions that have no assigned
structure
(35) with the strong potential to form G4 structures.
Also, EF-3 is a yeast translational elongation factor with an affinity
for poly(dG) in vitro, which binds to a guanine tract in 18 S
rRNA
(36) . Whether this guanine tract is involved in a G4
structure is not yet known. We are presently using genetic,
cytological, and biochemical approaches to answer these questions
surrounding the function of G4p2.
Counts were
obtained by exposing a dried mobility shift gel, on which aliquots of
each fraction were analyzed, to an imaging plate (Fuji). SA, specific
activity (counts/mg).
We thank Dipankar Sen, Zhiping Liu, and other members
of the Gilbert Lab for very useful discussion and Nancie Munroe and
Lloyd Schoenbach for invaluable administrative and computer assistance.
We also thank Michael Snyder for the yeast genomic library and Akira
Sakai and Yoshiku Kikuchi for sharing unpublished data.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
for G4p2 binding to a G4 DNA
oligomer is 2.2
10
M
under near physiological conditions. We have cloned and sequenced
the gene encoding G4p2 and have shown it to be identical to MPT4 and STO1. MPT4 was isolated in a screen for
multicopy suppressors of staurosporine sensitivity in POP2 cells. Pop2 is a complex regulatory factor that participates, in
part, in the repression of certain genes in the absence of glucose
(Sakai, A., Chibazakura, T., Shimizu, Y., and Hishinuma, F. (1992)
Nucleic Acids Res. 20, 6227-6233). STO1 was
isolated as a multicopy suppressor of TOM1, an uncharacterized
mutation that leads to temperature-sensitive cell cycle arrest at the
G2/M boundary. Suppression of these mutations by G4p2 indicate this G4
nucleic acid binding protein may function in signal transduction
pathways regulated by protein kinases, which control carbon source
utilization, and in cell cycle progression.
was
identified in a subunit of a Oxytricha telomere binding
complex, suggesting that G4 structures do exist, perhaps transiently,
at chromosome ends
(16) .
Preparation of G4 Oligomers
DNA and RNA
oligomers (see Fig. 1) were synthesized on an ABI model 392 DNA
synthesizer, and full-length products were purified on 8
M urea/polyacrylamide gels. G4 structures were annealed and purified
on native polyacrylamide gels containing KCl under conditions specified
by Sen and Gilbert
(17) and stored in 10 m
M Tris, pH
7.6, 1 m
M EDTA, 100 m
M KCl at 4 °C. Yields were
determined by absorbance at 260 nm.
Mobility Shift Assays, Competitions, and
SDS-PAGE
GL(G) (see Fig. 1) was 5`-labeled
with [
-
P]ATP and T4 polynucleotide kinase
and precipitated twice with ethanol. 5000 counts per second of labeled
GL(G
) (about 8 fmol or 320 pg, unless otherwise indicated)
was added to extract or G4p2 fractions in binding buffer (20 m
M NaHepes, pH 7.3, 1 m
M EDTA, 50 m
M KCl, 5 m
M BME,
(
)
5 m
M NaPO
, 0.05%
Triton X-100, 2.5% glycerol, 1 µg of bovine serum albumin, 2.5
µg of double-stranded salmon sperm DNA, tracking dyes) in a final
volume of 10 µl and incubated at room temperature for 10 min. The
reactions were loaded on 5% polyacrylamide gels and run at room
temperature in 50 m
M TBE (50 m
M Tris borate, 1 m
M EDTA, pH 8.2) with 1 m
M KCl at 12.5 V/cm. The gels were
dried and exposed for several hours to x-ray film or to an imaging
plate (Fuji) for 15 min.
Figure 1:
G4
oligomers used in mobility shift and competition experiments. Guanines
in boldface are those that participate in quartet formation.
G (p) indicates tetrameric parallel
structures, and G`
(ap) indicates dimeric
antiparallel structures (nomenclature according to Sen and Gilbert
(2)).
Competition experiments were carried out as
above, with saturating amounts of GL(G) probe. 10-fold
serial dilutions of competitor nucleic acids were premixed with extract
or G4p2 fractions in binding buffer just prior to adding the probe.
Relative mass amounts of competitor to probe ranged from 0.1 to 1000
and were determined by absorbance at 260 nm. Single-stranded
competitors were fully denatured by treatment with 100 m
M NaOH
at 100 °C just prior to use.
Large Scale Preparation of Yeast Extract
Diploid
yeast (Saccharomyces cerevisiae, strain SK1) was
cultured in 50 liters of YPD (1% yeast extract, 2% peptone, 1%
dextrose), harvested at midlog phase, and lysed in liquid nitrogen
according to published procedures
(19) . The extract was
prepared at 4 °C. Proteins were extracted from lysed cells in
extraction buffer (20 m
M NaHepes, pH 7.3, 1 m
M EDTA,
10 m
M MgCl, 420 m
M KCl, 0.05% Triton
X-100, 5 m
M BME, 10% glycerol, 1 m
M phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A) for 1 h at
0.5 g of cell paste/ml. The extract was cleared by centrifugation at
25,000
g for 15 min and dialyzed (Spectra/Por 7;
molecular weight cutoff, 3500) with one change for 48 h against 10
volumes of dialysis buffer (20 m
M NaHepes, pH 7.3, 1 m
M EDTA, 45 m
M KCl, 0.05% Triton X-100, 5 m
M BME,
10% glycerol). The dialysate was cleared again by centrifugation at
90,000
g for 1 h and frozen at -100 °C. The
protein concentration and yield were determined to be 25 mg/ml and 0.07
g of protein/g of cell paste, respectively, using a Bradford dye
binding assay (Bio-Rad).
Purification of G4p2
All extract and G4p2 fraction
manipulations were done at 4 °C. Thawed yeast extract (12.5 ml) was
dialyzed (Spectra/Por 7; molecular weight cutoff, 3500) against 1250 ml
of buffer A (20 m
M sodium bicarbonate, pH 11, 1 m
M EDTA, 50 m
M KCl, 100 m
M NaCl, 0.05% Triton
X-100, 5 m
M BME, 10% glycerol) for 12 h and cleared by
centrifugation at 10,000 g for 10 min. The
supernatant (240 mg in 12.5 ml) was loaded at 25 ml/h on a 25-ml
S-Sepharose FF (Pharmacia) bed, which had been equilibrated in buffer A
and packed in a 2.5
10-cm EconoColumn (Bio-Rad). The bed was
washed with 125 ml of buffer A, and proteins were eluted on
simultaneous 50-ml linear gradients of decreasing NaCl concentration
(100-0 m
M) and increasing NaPO
pH 11
concentration (0-100 m
M). Forty 1.25-ml fractions were
collected and analyzed by mobility shift assay for G4p2 and by
SDS-PAGE. Fractions 17-25 (11.25 ml) containing partially
purified G4p2 were pooled.
-
P]ATP and T4 polynucleotide kinase and
shown by gel electrophoresis to be composed of parallel G4 DNA and
higher order G4 structures
(20) . The G4 DNA (3.95 mg) was bound
to 2 ml of 50% streptavidin-agarose (Sigma) in 10 m
M Tris, pH
7.6, 1 m
M EDTA, 100 m
M KCl by gentle rocking for 30
min. The affinity matrix was washed twice in buffer B with 100 m
M NaCl, packed in a 0.7
5-cm EconoColumn (Bio-Rad), and
equilibrated at 4 °C.
Figure 3:
Purification of G4p2. A,
fractions from the G4 DNA affinity column were analyzed by mobility
shift assay with 320 pg of probe as described under ``Experimental
Procedures.'' 1 µl of a 100-fold dilution of each fraction was
analyzed. The input fraction ( IN) is pooled, and concentrated
fractions 17 through 25 were obtained from the S-Sepharose FF pH 11
column. G4p2 is indicated. B, SDS-PAGE of the same fractions
as in A. In each case, 10 µl of undiluted fraction was
analyzed. G4p2 and molecular weights of markers are indicated. The gel
was stained twice with silver.
Determination of the K
Mobility shift experiments as described
above were carried out with 2.7 fm of GL(G for G4p2
Binding to G4 DNA
) probe (Fig. 1)
and with 4-fold serial dilutions of G4p2 (1350, 338, 84.4, 21.1, and
5.27 fm) obtained from the G4 DNA affinity column (prep 2; see
``Results''). Stock G4p2 was quantitated by titrating a
dilution of G4p2 with increasing amounts of precisely quantitated
GL(G
) DNA probe until binding to G4p2 was saturated. At
saturation, molarity of the bound probe is equivalent to that of G4p2.
By this method, prep 2 contains 1.35 µ
M G4p2. The dried
gels were exposed to an imaging plate (Fuji), and the amounts of free
probe at each G4p2 dilution were measured. K
was
estimated from a plot of log(free G4p2 concentration) versus log(bound probe = (total probe - free probe)/free
probe)
(21) . The free G4p2 concentration was estimated by
subtracting the concentration of bound probe at each dilution, which
equals the concentration of bound G4p2, from that of input G4p2.
Peptide Sequencing and DNA Hybridization
Probes
Partially purified G4p2 (2.7 ml) obtained from the
S-Sepharose column was precipitated with 12.5% trichloroacetic acid and
0.5 mg/ml deoxycholate and fractionated on a 10% SDS-PAGE gel. The
proteins were electrotransferred to a nitrocellulose membrane and
visualized by staining with 0.1% Ponceau S in 5% acetic acid. G4p2 was
excised and sequenced (Harvard Microchemistry Facility). The sequences
of two internal tryptic peptides (NT-49, TAQLSLQDYLNQQANNQFNKVPEAK and
NT-40, EAQADAAAEIAEDAAEAEDAGKPK) were obtained. A unique oligomer (49C,
5`-CAAGATTACTTGAACCAACAAGCTAACAACCAATTCAACAAGGTTCCAGAAGCTAA-3`)
incorporating most frequent codon usage information for yeast
(22) was synthesized based on reverse translated NT-49. This
hybridization probe was tested on Southern blots of yeast genomic DNA
and was found suitable for library screening.
Isolation, Sequencing, and Subcloning of the G4p2
Gene
Oligomer 49C labeled with
[-
P]ATP and T4 polynucleotide kinase was
used to screen 6
10
plaques of recombinant
EMBL3a containing a yeast genomic library
(23) . Five
positive plaques were pooled and rescreened at low density. Three
positive plaques from the second screen were selected for further
analysis.
-
P]ATP and T4 polynucleotide kinase and
was used as a primer on 400 ng of template DNA. The nucleotide sequence
obtained permitted the synthesis of unique primers to extend similarly
and to finish both strands of the DNA sequence encoding G4p2. A
5-kilobase EcoRI fragment from a
EMBL3a clone containing
the intact G4p2 coding sequence was inserted into pUC19 using standard
methods, generating the subclone pUC19G4p2.
Preparation of Recombinant G4p2
A bacterially
expressed G4p2 fusion to glutathione S-transferase was
produced with the pGEX-3X (Pharmacia) vector system. Two complementary
oligomers (top, 5`-GATCTCCAACCCATTTGATTTGTTAGGTAACGACGTCGTCG-3` and
bottom, 5`-GATCCGACGACGTCGTTACCTAACAAATCAAATGGGTTGGA-3`) containing
N-terminal G4p2 coding sequence were annealed and inserted into
BamHI-cleaved pGEX-3X. A suitable clone containing the
properly oriented insert was cleaved with AatII (at the site
in the oligomer sequence) and EcoRI, and a 2.1-kilobase DNA
fragment derived from pUC19G4p2 cleaved with EcoRI and
partially with AatII was inserted. The desired clone was
identified by restriction site analysis and by sequencing. This clone
should direct the synthesis of a protein identical to G4p2 in an amino
acid sequence after removal of the glutathione S-transferase
domain, except the initiator methionine is replaced by
glycine-isoleucine. A large scale preparation of recombinant protein
was prepared in the Escherichia coli host strain LE392
according to recommended procedure
(24) . A yield of 0.75 mg of
recombinant G4p2/liter of culture was obtained after proteolytic
removal of the glutathione S-transferase domain with factor
Xa.
Data Base Searches and Sequence and Image Analysis
Programs
Non-redundant data base searches were carried out at
the National Center for Biotechnology Information with the BLAST
network service. Sequence manipulation and analysis was done with the
GCG Sequence Analysis software package 7.2. Secondary structure
prediction was done with PHD 1.0
(25) via E-mail server.
Silver-stained gels and autoradiographs were photographed and scanned
onto PhotoCD (Kodak). Images were converted to PICT files with
Photoshop 2.5.1 (Adobe), labeled with Canvas 2.1 (Deneba), and printed
with a Phaser II SDX dye sublimation printer (Tektronix).
Identification of a G4 Nucleic Acid Binding Protein,
G4p2, in Yeast
We have analyzed a yeast whole cell extract for
binding activities specific for G4 DNA. Fig. 2 A shows a
mobility shift assay with a parallel G4 DNA oligomer probe (
Fig. 1
lists the oligonucleotides used) and excess double-stranded
carrier DNA, demonstrating two major (G4p1 and G4p2) and several minor
components of yeast that bind to GL(G). (This paper deals
with the characterization of G4p2; the characterization of G4p1 will be
described elsewhere.(
)
) Protease and nuclease
treatment of the extract showed that G4p2 is a protein without a
nucleic acid component (data not shown). Fig. 2 B shows
competition binding experiments with specific DNAs in single-stranded
or parallel G4 form. Single-stranded DNAs do not compete, but DNAs
containing parallel G4 regions surrounded by either As or Ts compete
effectively. This demonstrates that the G4 domain of the probe is
required and sufficient for stable binding to G4p2. Mobility shift
experiments with extracts from several yeast strains showed that G4p2
is not restricted to strain SK1 (data not shown). We undertook the
purification of G4p2 in order to characterize it further.
Figure 2:
Identification of G4 nucleic acid-specific
binding proteins in a yeast extract. A, 5-fold serial
dilutions of yeast extract were analyzed by mobility shift assay with
320 pg of probe as described under ``Experimental
Procedures.'' Extract dilutions are indicated above the
lanes. 1 µl of undiluted extract (25 µg) was analyzed
in the left-hand lane. The G4 DNA probe and the two major G4
DNA binding proteins are indicated. B, the binding specificity
of G4p2 was analyzed by mobility shift competition assays with 320 pg
of probe and with 10-fold serial dilutions of unlabeled G4 and
single-stranded competitor DNAs (see Fig. 1) as described under
``Experimental Procedures.'' 1 µl of a 20-fold dilution
of extract (1.25 µg) was analyzed. Relative mass of competitor over
probe is indicated above the lanes. GL(SS) indicates single-stranded GL oligomer, and sal DNA(SS) indicates single-stranded salmon sperm
DNA.
Purification of G4p2
Small-scale batch experiments
revealed that G4p2 binds to S-Sepharose at pH 11 in a sodium
bicarbonate buffer and not in a sodium phosphate buffer. At pH 11,
relatively few proteins in the yeast extract bind S-Sepharose, and
fewer still are eluted by the buffer exchange. We used a G4 DNA
affinity column as a second step. Table I outlines the purification and
yields of G4p2. A decline in G4 DNA binding activity can be partially
reversed by treating G4p2-containing fractions with 100 m
M NaOH for several minutes just prior to loading G4p2-containing
fractions on the G4 DNA affinity column. The final preparation contains
9.4 µg of G4p2/ml. In a subsequent purification of G4p2 (prep 2),
we processed twice the amount of extract, and the concentration and
buffer exchange step (Amicon) was omitted in favor of dialysis with
improved yield (42 µg of G4p2/ml). Fig. 3 shows a SDS-PAGE gel of
G4p2 containing fractions from the G4 DNA affinity column (prep 1),
identifying a 32-kDa protein as G4p2. Quantitative estimates of
G4p2 by mobility shift assay are in agreement with quantitative
estimates of the
32-kDa protein by SDS-PAGE. The protein is
essentially purified in fraction 22. G4p2 comprises 0.017% of total
yeast protein by mass or about 35,000 molecules/cell and is therefore a
moderately abundant protein.
Characterization of Purified G4p2
Fig. 4 shows a
quantitation of a series of competition experiments with the purified
protein. G4p2 possesses equivalent affinities for parallel G4 DNA and
RNA (compare GL(G) with rGL(G
)). This
establishes the possibility that, in vivo, this protein binds
DNA and/or RNA. G4p2 has the highest affinity for molecules containing
multiple G4 domains, with a 100-fold preference for parallel G4 DNA
bearing two G4 domains over a similar molecule with just one domain
(Fig. 4, compare TGT2(G
) with
TGT(G
)). Further, G4p2 displays a mild
(3-fold) preference for antiparallel G4 DNA over parallel G4 DNA
(Fig. 4, compare TGT2(G`
) with
TGT(G
)) and virtually no affinity for
single-stranded and double-stranded DNA competitor (Fig. 4,
compare GL(SS) and GL(DS) with
GL(G
)). The small amount of competition
observed with high concentrations of single-stranded competitor
(Fig. 4, GL(SS)) can be attributed to conversion of a
fraction of the competitor into G4 structures during the course of the
experiment.
Figure 4:
Binding specificity of purified G4p2 for
G4 nucleic acids. Purified G4p2 was analyzed by mobility shift
competition assays with 2 ng of probe and with serial dilutions of
unlabeled competitor nucleic acids (see Fig. 1) as described under
``Experimental Procedures.'' Bound probe was measured by
exposing dried gels to an imaging plate (Fuji). 1 µl of a 200-fold
dilution of purifed G4p2 (210 pg, prep 2) was analyzed. Competitor
nucleic acids are indicated. GL(SS) indicates a
single-stranded GL oligomer, and GL(DS) indicates GL oligomer
annealed to a complementary sequence.
Incubations of purified G4p2 with parallel G4 DNA in the
presence of various divalent cations and ATP indicate this protein, on
its own, does not possess other activities, such as cleaving or
unwinding activity, toward this substrate (data not shown). Fig. 5
shows that the equilibrium constant for G4p2 binding to parallel G4 DNA
(GL(G)) is 2.2
10
M
in 50 m
M KCl at pH 7.3 at
room temperature. This value is comparable with those for other
protein-DNA interactions, which involve proteins of moderate abundance
(21) .
Cloning and Sequencing of the G4p2 Gene
In order
to obtain the primary sequence of G4p2, we determined to isolate its
gene. We had sufficient material at hand to obtain a partial amino acid
sequence of G4p2. An aliquot of pooled fractions 21-27 obtained
from the S-Sepharose column was fractionated by SDS-PAGE and
electrotransferred to nitrocellulose. G4p2 was excised, and the
sequences of two internal peptides were obtained. A hybridization probe
based on a reverse translated peptide sequence was used to screen a
yeast genomic library in EMBL3a, and three strongly hybridizing
phage were isolated. A degenerate oligomer based on a reverse
translated peptide sequence was used to obtain access into the G4p2
gene, and primer walking from the access point provided the remaining
DNA sequence.
Analysis of the G4p2 Peptide Sequence
Fig. 6 shows
the DNA sequence encoding G4p2. It contains an open reading frame
potentially encoding a protein of 273 amino acids with a calculated
molecular mass of 29.9 kDa. The amino acid composition predicts a
hydrophilic protein with an unusually high pI of 10.41, due in part to
an abundance of lysine (11.4%). Five regions are characterized by
skewed amino acid compositions. A region rich in proline (Fig.
6 A, (A)) is located at residues 38-54, and a
region rich in serine and threonine (Fig. 6 A,
(B)) is located at residues 77-102. Residues
119-141 are rich in alanine (Fig. 6 A,
(C)), and residues 147-164 are rich in glutamine and
asparagine (Fig. 6 A, (D)). Finally, a region
containing mostly asparagine, arginine, and glycine
(Fig. 6 A, (E)) is located at residues
225-245. Regions rich in glutamine or asparagine, such as regions
(D) and (E) (Fig. 6 A), have been
implicated in protein-protein contacts
(26) , whereas some
arginine- and glycine-rich sequences, such as that in region (E) (Fig. 6 A), have been shown to bind RNA
(27) . The PHD secondary structure prediction algorithm
(25) predicts with an expected accuracy of 61% that G4p2
contains -helices at residues 114-140 (
-helix I) and
147-159 (
-helix II). Helix I harbors the alanine-rich region
(Fig. 6 A, (C)) described above. A segment of
helix I containing residues 125-138 is strongly amphipathic, with
one side of the helix carrying five acidic residues. Helix II, which
contains the glutamine- and asparagine-rich region
(Fig. 6 A, (D)), has an interesting
``zipper'' of alternating hydrophobic and glutamine or
asparagine (amido) residues.
Figure 6:
Nucleotide sequence of the G4P2 gene and its deduced amino acid sequence. A, the
underlined regions are segments of the protein that
demonstrate skewed amino acid composition as described in the text.
-Helices I and II, identified by secondary structure prediction
programs, are enclosed in curved brackets. B, a
partial restriction map of the G4p2 gene.
Results of Data Base Searches
A TBLASTN search of
the non-redundant nucleotide sequence data base at the National Center
for Biotechnology Information located two entries, MPT4 and
STO1, potentially encoding gene products identical to G4p2.
MPT4 (accession number D26183) was isolated with a screen
designed to identify multicopy suppressors of staurosporine sensitivity
associated with the POP2 mutation in yeast.(
)
The POP2 mutation itself was originally identified
in a screen for genes required for the repression of gene expression in
the absence of glucose
(6) . STO1 (accession number
D32208) was isolated as a multicopy suppressor of TOM1, a
molecularly uncharacterized mutation in yeast that leads to
temperature-sensitive cell cycle arrest at the G2/M
boundary.
(
)
Analysis of Recombinant G4p2
As proof that we had
identified the correct protein and gene, we prepared a recombinant
version of G4p2. Bacterially synthesized G4p2 was produced as a fusion
to glutathione S-transferase. The protein was purified from a
bacterial extract with a glutathione-agarose matrix and released from
the glutathione S-transferase domain by proteolytic cleavage
with factor Xa. SDS-PAGE of purified and factor Xa-treated recombinant
G4p2 revealed a homogeneous band at 32 kDa (data not shown). Fig.
7 shows a mobility shift assay demonstrating that recombinant G4p2
retains the G4 DNA binding property of the natural protein. During the
preparation of recombinant G4p2, we observed that the protein was
unstable in bacteria. A survey of common E. coli host
strains then identified LE392 as permitting the highest yields of
intact G4p2.
which are described as containing a mutation in a gene required
for glucose derepression of gene expression
(6) . POP2 was selected as a recessive mutation that leads to the expression
of
-amylase linked to the phosphoglycerate kinase promoter at low
glucose levels. Usually, this promoter is only active at glucose
concentrations high enough to activate catabolite repression. The
mutant is also defective at low glucose levels in full derepression of
invertase and isocitrate lyase, enzymes used in the metabolism of
carbon sources other than glucose. These defects suggest that Pop2 is a
factor that inhibits the expression of glucose-utilizing enzymes while
stimulating the expression of enzymes converting alternative carbon
sources. Cells lacking Pop2 are also unable to sporulate and
demonstrate temperature-sensitive growth, suggesting that this protein
has additional functions in the cell.
a
specific inhibitor of certain protein kinases, including the
phospholipid/calcium-dependent, cAMP-dependent, and cGMP-dependent
protein kinases
(28) . This phenotype is believed to arise when
a target protein kinase acquires a greater affinity for staurosporine
through mutation. The specific cause of staurosporine sensitivity in
POP2 cells, however, has not yet been established.
Molecularly, POP2 is a nonsense mutation producing a protein
truncated at the 62nd amino acid
(6) . Thus, it is not clear how
staurosporine sensitivity could arise in cells lacking most of the
POP2 gene product. Perhaps Pop2 is complexed with a
staurosporine binding protein and reduces the affinity of this protein
for the inhibitor. Excess MPT4 gene product provided by
expression from a multicopy plasmid suppresses the staurosporine
sensitivity in POP2 mutants. We do not yet know if the other
defects in POP2 cells are similarly suppressed.
The mutant was fortuitously
identified during a procedure intended to disrupt an unrelated gene.
Identifying the nature of the cell cycle defect in TOM1 cells
is under investigation.
-mercaptoethanol; PAGE,
polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.