The Leucine Zipper Domains of the Transcription Factors
GCN4 and c-Jun Have Ribonuclease Activity
Yaroslav Nikolaev
1,2.
, Christine Deillon
1.
, Stefan R. K. Hoffmann
1.
, Laurent Bigler
3
, Sebastian Friess
4
,
Renato Zenobi
4
, Konstantin Pervushin
2
, Peter Hunziker
5
, Bernd Gutte
1
*
1 Biochemisches Institut der Universita¨tZu¨rich, Zu¨rich, Switzerland, 2 Biozentrum der Universita¨t Basel, Basel, Switzerland, 3 Organisch-Chemisches Institut der Universita¨t
Zu¨rich, Zu¨rich, Switzerland, 4 Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, Zu¨rich, Switzerland, 5 Functional Genomics Center
Zu¨rich, Zu¨rich, Switzerland
Abstract
Basic-region leucine zipper (bZIP) proteins are one of the largest transcription factor families that regulate a wide range of
cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ) domains of bZIP factors are widely
employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the
retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease
activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease
activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves
the breakage of 39,59-phosphodiester bonds with formation of 29,39-cyclic phosphates as the final products as
demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are
catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the
peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown
to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-
Jun to the pool of proteins with multiple functions (also known as moonlighting proteins). If expressed in vivo, the
endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation
and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list
of functional retro-peptides.
Citation: Nikolaev Y, Deillon C, Hoffmann SRK, Bigler L, Friess S, et al. (2010) The Leucine Zipper Domains of the Transcription Factors GCN4 and c-Jun Have
Ribonuclease Activity. PLoS ONE 5(5): e10765. doi:10.1371/journal.pone.0010765
Editor: Thomas Preiss, Victor Chang Cardiac Research Institute (VCCRI), Australia
Received September 25, 2009; Accepted April 26, 2010; Published May 21, 2010
Copyright:  2010 Nikolaev et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Kanton of Zu¨rich, the Mesta Foundation, Swiss National Science Foundation (grant 3100A0-118381 to K. Pervushin)
and the Stiftung fu¨r wissenschaftliche Forschung an der Universita¨tZu¨rich. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: gutte@bioc.unizh.ch
. These authors contributed equally to this work.
Introduction
Leucine zippers [1] are parallel alpha-helical coiled coil motifs
and as such one of the most common mediators of protein-protein
interactions [2]. The most widely known leucine zipper (LZ)
proteins are the basic-region leucine zippers (bZIP) [1], which
account for more than 51 unique members in Homo sapiens [3],
comprising the second-largest family of dimerizing transcription
factors in humans after bHLH proteins [4].
Simplicity of the coiled coil fold together with high stability of
the LZ motifs facilitated their extensive adoption in protein
engineering studies [5,6,7,8]. In one such study, a GCN4 (yeast
transcription activator) retro-leucine zipper [Fig. 1(I c)] seemed to
be most suitable as dimerization module for an artificial HIV
(Human Immunodeficiency Virus) enhancer-binding peptide. To
ensure dimerization of this retro-leucine zipper it was extended at
the N-terminus by the tripeptide sequence Cys-Gly-Gly [Fig. 1(I d)]
which allowed formation of a disulfide bond. Molecular weight
studies in the ultracentrifuge and the crystal structure revealed that
the disulfide peptide formed a noncovalent dimer or four-helix
bundle and that neighbouring bundles were bridged by histidine
side chains (Fig. 2C in [9]). The observed juxtaposition of
histidines was vaguely reminiscent of the active-site structure of
ribonuclease A (RNase A) [10] and prompted us to test the retro-
leucine zipper for ribonuclease activity. Surprisingly, weak
ribonuclease activity distinct from that of RNase A was not only
found for the GCN4 retro-leucine zipper but also for the authentic
leucine zipper domains of GCN4 and oncoprotein c-Jun
(component of transcription factor AP-1).
There are natural polypeptides and proteins reported to possess
‘‘moonlighting’’ endoribonuclease activity [11]. Examples are the
homodimer of a 30-residue single zinc finger motif of the human
male-associated ZFY transcription factor [12], the 278-residue
zinc-alpha 2-glycoprotein [13] and conserved, approximately 100-
residue DYW domains [14] of land plant pentatricopeptide repeat
(PPR) proteins [15,16]. The ZFY zinc finger cleaves single-
stranded RNA with k
cat
= 0.025 per min, comparable to the
catalytic efficiency of group II intron ribozymes (k
cat
= 0.03 per
min) [17]. The zinc-alpha 2-glycoprotein also cleaves single-
stranded RNA. The DYW domains of the PPR proteins have been
implicated in catalyzing RNA editing in plant organelles; in
addition, several recombinant DYW domains were found to
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possess weak endoribonuclease activity. It remains to be seen
whether the ribonuclease activity of the ZFY zinc finger and the
PPR DYW domains is preserved in their parent proteins and,
eventually, is of biological importance.
RNA is also cleaved by diverse artificial catalysts, for example
imidazole and guanidinium compounds, transition metal com-
plexes, and designed peptides. Both the GCN4 and c-Jun leucine
zipper peptides contain histidine and arginine residues. These
residues are essential for the RNase A-catalyzed digestion of RNA
and for specific protein-RNA binding, respectively. Therefore
the presence of catalytically active imidazole- and guanidinium-
containing compounds in the ribonuclease assays of the leucine
zipper peptides had to be avoided. It had been shown that
imidazole is active as 2-aminobenzimidazole (with a guanidinium
group in disguise) [18] or when bound to beta-cyclodextrin [19],
polyamines [20] or antisense oligonucleotides [21,22]. The
guanidinium group has ribonuclease activity as a bridged dimer
[23]. However, the presence in our experiments of these
imidazole and guanidinium derivatives and of biogenic amines
(spermine, spermidine) [24] which also have weak ribonuclease
activity could be excluded. This applied also to active complexes
of transition metals such as Cu, Zn, and lanthanides [22,25]
because leucine zipper sequences do not contain metal-chelating
motifs.
Designed peptides with RNA-hydrolyzing activity include Ni-
chelating tripeptides [26], metal-free tri- and tetrapeptides
containing Arg, His, Lys and Glu [27], and helical polypeptides
containing basic and hydrophobic amino acid residues (Lys and
Leu; [28]). However, peptides of this kind could not form during
the synthesis of the leucine zipper sequences.
Figure 1. Synthetic peptides and RNA employed in the study. (I) Sequences of leucine zippers and RNA18. The a- and d-positions of the
repeating heptads of the GCN4 and c-Jun leucine zippers are shown in bold print. In the retro-sequences, a- and d-positions are reversed. In the wild-
type sequences (a, b) leucine residues in d-positions are highlighted grey; anionic, cationic and polar residues in the proposed active sites are
highlighted red, blue and orange, respectively. a) c-Jun LZ36 (residues 280-315); b) GCN4 LZ35 (residues 247–281), asterisk (*) indicates positionsof
point mutations; c) GCN4 rLZ35, retro-sequence of LZ35; d) GCN4 rLZ38, obtained through N-terminal extension of rLZ35 by CysGlyGly; e) rLZ67,
obtained through fusion of rLZ38 with shortened HIV enhancer-binding peptide R42; f) R42, artificial HIV enhancer-binding peptide; g) RNA18. (II)
Topological arrangement of side chains in the proposed catalytic sites of c-Jun LZ36 (PDB: 1JUN) and GCN4 LZ35 (PDB: 2ZTA). Both monomer and
coiled coil active site arrangements are shown. Residue color code corresponds to that of c-Jun and GCN4 sequences in (I).
doi:10.1371/journal.pone.0010765.g001
Figure 2. HPLC fractionation of RNA degradation products. (A) Cleavage products of 85 mM RNA18 formed within 24 hours in presence of
28 mM GCN4 rLZ38, (B)56mM GCN4 rLZ35, and (D)28mMrLZ67. (C) The mixture of R42 (14 mM) and RNA18 (48 mM) served as negative control.
Reactions were performed in 20 mM Tris-HCl/80 mM KCl at pH 7.2 and 37uC. Retention time of uncleaved RNA18 was 50 min.
doi:10.1371/journal.pone.0010765.g002
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Catalytic hydrolysis of RNA is an essential part of RNA
turnover and decay and thus a major mechanism for gene
expression control [29,30]. If the bZIP leucine zipper portions of
transcription factors GCN4 and c-Jun were active in vivo, they may
contribute to the ribonuclease activities controlling gene expres-
sion and RNA biogenesis in the nucleus, the major site of RNA
turnover [31,32,33]. Transcription factors containing leucine
zipper or zinc finger motifs would thus combine transcription
activation with slow RNA degradation.
In this work we have focused on the ribonuclease activity of the
authentic leucine zippers of the bZIP regions of yeast GCN4 and
human c-Jun. We show that the activity of these leucine zippers is
not affected by a specific inhibitor of the RNase A family but by
mutations in the amino acid sequence, thus attributing the
observed RNA cleavage to the synthetic peptides. In addition,
our experimental data show that this activity is preserved in full-
length c-Jun.
Results
Highest priority was paid to the purity of all materials used in
the ribonuclease activity assays to exclude contamination by
ribonuclease-active compounds at all experimental stages. The
RNase activities reported were observed for several different
syntheses of the peptides, prepared in our laboratory and by
external suppliers. The water used for the ribonuclease assay
buffers and LC column eluents was ribonuclease-free (commercial
RNase/DNase-free water or ultrafiltered water assayed for the
lack of RNA hydrolytic activity). The peptides had been repeatedly
exposed to basic (20% piperidine) and acidic (95% trifluoroacetic
acid) conditions during solid phase synthesis and work-up and
were shown by amino acid analysis, HPLC, and mass spectrom-
etry to be homogeneous. Cleavage of the RNA substrates by the
leucine zipper peptides was assayed by monitoring the time-
dependent depletion of the substrates using HPLC. Prior to each
activity assay a substrate control was run through the HPLC
column; even after 96-h incubations in the same reaction buffer
only a single sharp peak eluted at the position of the intact RNA
substrate. This indicated that substrate, buffers and HPLC system
were not contaminated by ribonucleases. Handling of the
enzymatically active and inactive peptides during synthesis and
purification was identical. In control experiments, R42 (a synthetic
HIV-1 enhancer-binding peptide similar in size to the leucine
zippers but strongly cationic), bovine serum albumin (BSA),
glyceraldehyde-3-phosphate dehydrogenase and glucagon were
inactive in 2-h to 48-h incubations with RNA substrate.
Furthermore, the ribonuclease activity of the GCN4 and c-Jun
leucine zippers was not affected by the ribonuclease A family-
specific inhibitor RNasin. Finally, the strongest evidence for the
intrinsic ribonuclease activity of the GCN4 leucine zipper peptide
comes from the fact that several of its alanine mutants were
inactive albeit their synthesis, purification and assaying procedures
were identical to those of the wild-type peptide.
Assays of the ribonuclease activity of wild-type and
mutant GCN4 leucine zipper peptides
The first assays were performed with GCN4 rLZ38, the parallel
four-helix bundle [9] with possible ribonuclease activity, and a
commercial octadecaribonucleotide (RNA18) [Fig. 1 (I g)] as
substrate. Based on the positive result of these assays [Fig. 2 (A)],
single-chain rLZ35 ([Fig. 1(I c)], net charge +2) and tetrameric
rLZ67 ([Fig. 1(I e)], net charge +11 per chain), the fusion peptide
of rLZ38 with a shortened version of R42, a designed HIV
enhancer-binding peptide, were tested for ribonuclease activity
and were also found to cleave RNA18 [Fig. 2(B, D)]. R42 itself
[Fig. 1(I f)] [34,35,36], a 42-residue strongly HIV enhancer-
binding peptide (net charge +14), was inactive [Fig. 2(C)]. This
allowed to attribute the ribonuclease activity of rLZ67 to the retro-
leucine zipper component of the fusion peptide and it showed that
the activity of the GCN4 retro-leucine zipper was preserved in a
larger, heterologous sequence context. The sequence of rLZ67
[Fig. 1(I e)] comprised rLZ38 [Fig. 1(I d)] followed by the positively
charged flexible linker KRAR and the C-terminal 25 residues of
R42 [Fig. 1(I f)].
The digestion patterns produced by the retro-leucine zippers
[Fig. 2(A, D)] and the authentic leucine zipper of GCN4 [Fig. 3(C)]
in 24-h reactions using RNA18 as substrate were remarkably
similar. Results of the cleavage of RNA18 by wild-type and mutant
35-residue leucine zipper peptides are summarized in Table 1.
Full-length RNA18 is predicted to form a double-stranded
structure with 50% of its sequence being involved in the base-
pairing interactions (see below and [Fig. 3 inset]). To avoid
uncertainties about the proportions of oligomeric states of the
leucine zipper peptides and RNA in the degradation experiments
of this study, the concentrations given for the peptides and RNA
are uniformly those of the monomeric species. The cleavage
percentages reflect the decrease in the integral area of the full-
length 18-mer RNA substrate peak. These percentages do not take
into account the subsequent secondary cleavages of smaller RNA
fragments during the reaction and are therefore underestimations
of the actual number of cleavages of individual phosphodiester
bonds.
In 20 mM Tris-HCl and 80 mM KCl, pH 7.2, wild-type GCN4
LZ35 [Fig. 1(I b)] (30 mM) cleaved 47% of RNA18 (50 mM)in
13 h at 37uC. Under the same conditions, the GCN4 LZ peptide
with Glu12 replaced by alanine was completely inactive. Low
activity (6 to 12% cleavage in 13 h) was retained by the Glu12Gln,
Glu13Ala, Ser16Ala, and Ser16Thr analogues whereas exchange
of Lys17 and Tyr19 for alanine did not affect the enzymatic
activity (47% and 40%, respectively). In the presence of the
His20Ala mutant the area of the RNA18 HPLC peak was
unchanged after a reaction time of 2 h but had decreased by
approximately 40% after 13 h without formation of detectable
cleavage products. This indicated that the His20Ala mutant
had very little if any activity and pointed to time-dependent
aggregation of His20Ala - RNA18 complex(es). The latter could be
reversed by dilution in 80 mM phosphate, carbonate, or Tris-HCl
and heating of the assay mixture to 65uC before application to the
HPLC column [supplementary material, Fig. S4(E,F)].
The activity maximum of the GCN4 leucine zipper peptides
was at approximately pH 7 and at KCl concentrations between 75
and 100 mM. The presence of 1 mM EDTA did not affect the
results of the activity assays.
The products of a 24-h digestion of RNA18 catalyzed by GCN4
LZ35 were separated and characterized using HPLC-electrospray
ionization mass spectrometry and were compared with those of a
3-h digestion by RNase A [Fig. 3(B,C)]. LZ35 cleaved the RNA18
substrate mainly at the 39-end of U and C and to a smaller extent
at the 39-end of A and G. Almost all products were obtained as the
29,39-cyclic phosphates [Fig. 3(C)]. In contrast, RNase A could not
cleave RNA at the 39-end of G and catalyzed the hydrolysis of the
intermediate 29,39-cyclic phosphates to give the final 39-phosphate-
containing products [Fig. 3(B)]. Even after 96 h no degradation of
RNA18 substrate was detected in the absence of LZ35 or RNase A
[Fig. 3(A)].
Based on the sequence composition, RNA18 is predicted to
form a stable double-stranded structure with 50% of nucleotides
being involved in the base-pairing interactions ([Fig. 3(A), inset];
Leucine Zipper RNase Activity
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Figure 3. Comparison of RNA18 cleavage by GCN4 LZ35 and RNase A. RNA18 concentration was 80 mM. Samples were incubated in 20 mM
Tris-HCl/80 mM KCl, pH 7.2, at 37uC. The cleavage products obtained were separated by HPLC and characterized by electrospray ionization mass
spectrometry. Retention time for uncleaved RNA18 was approximately 50 min. (A) RNA18 blank (incubation time, 24 h). (B) RNA18 + RNase A (90 nM;
incubation time, 3 h); the major products were AGG (956 Da) lacking phosphate at the 39-end, and the 39-phosphate-containing GAAU (19326 Da),
GGU (19013 Da), AC (651 Da), and GC (667 Da). (C) RNA18 + GCN4 LZ35 (50 mM; incubation time, 24 h); the major products were AGG (956 Da)
lacking phosphate at the 39-end, GAAUU. (19614 Da), GUCUG. or GGUCU. (19606 Da), and ACC. (938 Da); the composition of several of the
minor products indicated cleavage of RNA18 at the 39-end of guanosine phosphate or adenosine phosphate residues, for example: AAUU. or
AUUA., UACCAG.,GA. or AG., UCUG. or GUCU.,GG., G, UCU., and G. (‘‘.’’ represents 29,39-cyclic phosphate group). The largely dimeric
structure of RNA18 (DG=27.3 kcal/mol) was established using MFOLD software [37]. Cleavage efficiencies at particular RNA bonds are shown as
arrows above the RNA18 sequence. Size of arrows reflects the concentration of oligonucleotides with the corresponding 39- and 59-termini relative to
the combined concentration of all reaction products. Product concentrations are calculated as absorbance peak integrals normalized by extinction
coefficients of the corresponding oligonucleotides.
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DG=27.3 kcal/mol) [37]. The corresponding K
d
of ,7 mMat
37uC establishes that RNA18 is largely double-stranded under the
initial conditions of the ribonuclease assays. Analysis of RNA18
degradation products suggests that GCN4 LZ35 avoids duplex
structures, preferably targeting the flexible single-stranded regions
of RNA (red arrows in [Fig. 3(C), inset]).
The ribonuclease activity of GCN4 LZ35 was confirmed using
commercial RNA (baker’s yeast; Sigma) as substrate [Fig. S5]. In
this experiment the concentrations of RNA, leucine zipper
peptide, and the BSA control were 22 mM (0.5 mg/mL), 30 mM
(0.13 mg/mL), and 7.35 mM (0.5 mg/mL), respectively, in 50 mM
sodium acetate, pH 5; the reaction time was 24 h and the
temperature 25uC. The results of this experiment were unequiv-
ocal: GCN4 LZ35 cleaved baker’s yeast RNA whereas BSA was
inactive.
Ribonuclease assays of GCN4 LZ35 in presence of RNase
A inhibitor
Five units of the recombinant RNase A inhibitor RNasin
(Promega) did not affect the RNA18-cleaving activity of 12.5 mLof
a50mM solution of GCN4 LZ35 whereas 150 units of the
inhibitor lowered the activity by approximately 60%. The RNA18
concentration at the start of the experiments was 50 mM,
incubation time was 13 h. In a control experiment, five units of
RNasin abolished the activity of 12.5 mLofa9pM solution of
RNase A completely. The effect of the inhibitor on RNA18
cleavage by GCN4 LZ35, RNase A, and RNase T
1
is illustrated in
electronic supplementary material, Fig. S3.
Ribonuclease assays of the leucine zipper of c-Jun [Fig. 1
(I a)] and full-length c-Jun
Fig. 4 shows the HPLC chromatograms of the cleavage
products of RNA18 produced by synthetic 36-residue leucine
zipper of c-Jun (LZ36) and full-length recombinant c-Jun. In
20 mM Tris-HCl/80 mM KCl, pH 7.2, at 37uC, 60 mM synthetic
c-Jun LZ36 degraded 28% of 170 mM RNA18 in 4 h [Fig. 4(A)]
and 63% in 24 h. Under the same conditions, 20 mM full-length
recombinant c-Jun (Promega) cleaved approximately 50% of
90 mM substrate in 48 h [Fig. 4(B,C,D)]. The prolonged reaction
time chosen for the latter assay was to compensate for the lower
concentration of recombinant c-Jun in these experiments (20 mM
c-Jun versus 60 mM c-Jun LZ36). The cleavage patterns obtained
showed similarities but were not identical [Fig. 4(A and B)]. The
experiments with recombinant c-Jun were preliminary and were
merely performed to see if the ribonuclease activity of the c-Jun
leucine zipper was preserved in the full-length protein. Impor-
tantly, under the same conditions used in the inhibition
experiments with GCN4 LZ35 (i.e., five units of RNasin in
12.5 mL of digestion mixture) the ribonuclease activity of
recombinant c-Jun and synthetic c-Jun LZ36 was not affected.
Activity assays of c-Jun LZ36 in presence of 150 units of the
inhibitor were not performed.
Kinetic analysis of RNA18 cleavage by the LZ peptides
Degradation rates of full-length 18-mer RNA substrate were
used to determine the lower boundaries for the second-
order catalytic rate constants (k
2
. k
2obs
= V
obs
/([LZ]N[RNA]),
M
21
min
21
) and turnover numbers (k
cat
. k
obs
= V
obs
/[LZ],
min
21
) of phosphodiester bond cleavage by LZ peptides. Based on
the ribonuclease activity assays, the minimal turnover numbers for
GCN4 LZ35, c-Jun LZ36 and full-length c-Jun were 0.0024,
0.0033 and 0.0008 min
21
, respectively (Table 2).
Mass spectral analysis of RNA18 - GCN4 LZ35 complex
formation
Matrix-assisted laser desorption/ionization (MALDI) mass
spectrometry [Fig. 5] showed signal groups starting at 119547
and 109015 Da indicating the presence of double-stranded
RNA18 and a 1:1 molar complex of monomeric 35-residue
peptide with single-stranded RNA18, respectively. The deviations
from the masses expected (119540 and 109008 Da) were below
0.1% and thus well within the range of accuracy of the instrument
and the associated mass calibration. In both groups, the spacing of
the signals with higher mass were m = 38 (dominant, exchange of
K for H) and m = 22 (minor, exchange of Na for H), respectively.
The alkali cationization of the phosphate backbone of RNA18
could not be suppressed completely despite the use of citrate in the
matrix. The lower mass range of the spectrum showed the signals
for the monomers of RNA18 (59771 Da) and LZ35 (49237 Da) as
well as signals for major cleavage products of RNA18 at 19906 Da
(AAUUAC.), 19624 Da (GGUCU), 19616 Da (GAAUU.), and
964 Da (AAU.) (where U.,C.,G., and A. refer to terminal
29,39-cyclic phosphates of U, C, G, and A).
Mass spectral analysis showed also that the leucine zipper
peptides emerged unaltered from the catalytic process; as true
enzymes, they were not oxidized or otherwise modified.
Discussion
This project started with the assumption that the synthetic 38-
residue retro-leucine zipper of GCN4 (GCN4 rLZ38) could have
ribonuclease activity. First assays showed that rLZ38, rLZ67
[Fig. 1(I d,e)](tetramers: dimers of disulfide dimers), and rLZ35
[Fig. 1(I c)](monomer), cleaved the synthetic octadecaribonucleo-
tide RNA18 [Fig. 1(I g)] [Fig. 2(A,B,D)] whereas the HIV
enhancer-binding peptide R42, partly contained in rLZ67, did not
have ribonuclease activity; after a 24-h incubation with R42,
RNA18 eluted unchanged from the HPLC column [Fig. 2(C)].
Expectations that the presence of a sequence-specific nucleic acid-
binding component may increase the RNA-cleaving activity of the
retro-leucine zipper domain of fusion peptide rLZ67 did not prove
to be true. Unexpectedly, the normal, ‘‘forward’’ sequence of the
GCN4 leucine zipper (GCN4 LZ35) and the c-Jun leucine zipper
(c-Jun LZ36) [Fig. 1(I b,a)] also cleaved the RNA18 substrate
Table 1. RNA18 cleavage efficiency by Wild-Type (wt) and
Mutant GCN4 LZ35
a
.
LZ peptide % cleaved (2 h) % cleaved (13 h)
wt 17 47
E12A 0 2
E12Q 2 6
E13A n.d.
b
12
S16A ,19
S16T , 1
K17A 4 47
Y19A 13 40
H20A
c
a
Percent RNA cleaved after 2 h and 13 h, respectively, based on the amount
present at the beginning of the experiment (50 mM) and determined by HPLC at
254 nm. The concentration of the GCN4 leucine zippers was 30 mM. Cleavages
were performed in 20 mM Tris-HCl/80 mM KCl, pH 7.2, at 37uC.
b
Not determined.
c
Assay commented in Results.
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(Fig. 3 and Fig. 4). The ribonuclease activity of both normal and
retro-leucine zipper of GCN4 could be the result of their close
structural similarity [9] coupled with the flexibility of enzyme
active sites [38]. As the ribonuclease activity of the GCN4 and c-
Jun leucine zippers may be of cell biological relevance, all further
studies were performed with the normal leucine zippers.
Figure 4. Comparison of chromatograms illustrating RNA18 cleavage by synthetic c-Jun LZ36 and full-length 40 kDa recombinant
c-Jun. Reactions were performed in 20 mM Tris-HCl/80 mM KCl, pH 7.2, at 37uC. (A) c-Jun LZ36 (60 mM) + RNA18 (170 mM); reaction time, 4 h;
column, 3.14 mL Nucleosil C-18 300-5 (Macherey & Nagel). (B) Recombinant c-Jun (20 mM) + RNA18 (90 mM); reaction time, 48 h; column, 2.5 mL
Eclipse XDB C-18 (Agilent). (C) c-Jun + RNA18 (as in B) in the presence of 1 U of the ribonuclease A inhibitor RNasin. (D) RNA18; incubation time, 48 h.
Resolution of the digestion products by the two different HPLC columns required a different gradient set-up (30-mL gradient in panel A versus 13-mL
gradient in panels B, C and D). The elution profiles are presented relative to the percentage of acetonitrile in the two gradients (dashed blue lines).
Asterisk (*) marks the position of intact RNA18.
doi:10.1371/journal.pone.0010765.g004
Table 2. Minimal turnover numbers and second-order rate constants of the LZ-catalyzed RNA18 degradation
a
.
peptide RNA18 cleavage time V
obs
k
obs
(#k
cat
) k
2obs
(#k
2
)
mM mM %hnM min
21
min
21
M
21
min
21
GCN4 LZ35 30 50 17 2 70.8 0.0024 47
cJun LZ36 60 170 28 4 198 0.0033 19
cJun 20 90 50 48 15.6 0.0008 9
a
Based on the degradation rates of full-length 18-mer RNA substrate in 20 mM Tris-HCl, 80 mM KCl, pH 7.2, 37uC.
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Substrate specificity
First it was shown that the nuclease activity of the GCN4
leucine zippers was RNA-specific. Besides the synthetic RNA18
[Fig. 2(A,B,D), Fig. 3(C) and Supplementary Fig. S3] they cleaved
yeast RNA extract [Fig. S5(B)) and poly(C/U/A/G) (analyzed by
thin-layer chromatography, data not shown). They did not cleave
DNA and were also inactive in chymotrypsin and trypsin activity
assays. Although potential substrates, the dinucleoside phosphates
CpG and UpG were not split by LZ35 as shown by HPLC; most
likely, their binding to the leucine zipper was too weak.
Products of catalysis
To determine the nature of the ribonuclease activity of GCN4
LZ35, the cleavage products of RNA18 were separated by HPLC
and then characterized by electrospray ionization mass spectrom-
etry. Fig. 3(C) shows that the nucleolytic activity of LZ35 was
unspecific. The observation that in 24-h reactions almost all
products were obtained as 39-terminal 29,39-cyclic phosphates and
that LZ35 was completely inactive in 6 M urea, made contam-
ination by RNase A unlikely. Furthermore, a number of products
was formed through cleavage of G-A, G-C, G-G, and G-U 39,59-
phosphodiester bonds ([Fig. 3(C)], from right to left: AAUU.,
GGUCUG., GUCU., UCUG., UACCAG., UCUGC.,
GG., the terminal G, UCU.,G.) providing additional
evidence that the ribonuclease activity of LZ35 was intrinsic
because contamination by a G-specific ribonuclease such as RNase
T
1
could be excluded. For comparison, digestion of RNA18 by
RNase A gave the expected hydrolysis products [Fig. 3(B)] lacking
completely fragments formed through cleavage after G.
The location of cleavage sites in context of the double-
stranded RNA18 structure ([Fig. 3(C), inset], predicted using
MFOLD software [37]), suggests that LZ35 is more catalyti-
cally active towards the unstructured regions of the RNA18
molecule.
Negative controls
In the absence of LZ35 or RNase A, RNA18 was not cleaved
[Fig. 3(A)]. RNA18 was also unaffected in assays with R42 (the
synthetic 42-residue artificial HIV enhancer-binding peptide, net
charge +14) [Fig. 2(C)], bovine serum albumin [Fig. S3(G,H)],
glyceraldehyde-3-phosphate dehydrogenase and glucagon (not
shown). The ribonuclease activity of GCN4 LZ35 and complete
c-Jun protein was not affected by RNasin [Fig. S3(A,B) and
Fig. 4(B,C)], a specific inhibitor of the ribonuclease A enzyme
family. However, at very high concentration (30-fold above the
recommended values) RNasin impaired the activity of GCN4
LZ35 by 60%, most likely caused by unspecific interactions
between the leucine zipper and the hydrophobic inhibitor [39].
Most importantly, the ribonuclease activity of GCN4 LZ35 was
affected by stepwise replacement of several amino acid residues of
the leucine zipper with alanine, unequivocally attributing the
observed activity to the GCN4 leucine zipper and excluding
possible contamination. Fig. 1(II), lower panels, shows that those
residues of GCN4 LZ35 that are also found in ribonuclease active
sites (Glu, Ser, Lys, Tyr, His) are aligned on one side of the GCN4
leucine zipper [40] possibly representing a binding site of the
RNA18 substrate. Table 1 summarizes the effect of replacement of
these residues mainly by alanine. The Glu12Ala analogue was
completely inactive compared to the wild-type sequence. The
ribonuclease activity of the Glu12Gln, Glu13Ala, Ser16Ala and
Ser16Thr analogues was strongly reduced as compared with that
of wild-type LZ35. The activity assays of the His20Ala mutant
showed a time-dependent decrease of the area of the RNA18
substrate peak without detectable formation of cleavage products,
suggesting reversible aggregation of the His20Ala-RNA18 com-
plex(es) under the conditions employed (see Results and [Fig.
S4(E,F)]). The same aggregation takes place at high concentrations
of wild-type GCN4 LZ35 [Fig. S4(B,C)] and at high concentra-
tions of catalytically active aminobenzimidazole [41].
Figure 5. MALDI-MS analysis of LZ35–RNA18 complex formation. The 1 to 14 kDa portion of the MALDI mass spectrum of a mixture of GCN4
LZ35 (69 mM) and RNA18 (68 mM) recorded in negative ion mode using 6-aza-2-thiothymine/citrate as matrix. The signals obtained are explained in
Results.
doi:10.1371/journal.pone.0010765.g005
Leucine Zipper RNase Activity
PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10765

LZ-RNA complex formation
Complex formation between GCN4 LZ35 and RNA18 was
demonstrated by MALDI mass spectrometry [Fig. 5]. MALDI
data of noncovalent complexes, however, have to be interpreted
with caution because sample preparation and laser action are
generally disruptive; in addition, nonspecific clusters of sample
constituents can be produced in a dense MALDI plume.
Disruption of the LZ35 - RNA18 complex by the MALDI process
may have been responsible for the strong signals at 49237 and
59771 Da (monomeric 35-residue peptide and single-stranded
RNA18, respectively). The intense signal groups starting at 109015
and 119547 Da [Fig. 5, inset] indicated the presence of specific
noncovalent complexes (monomeric 35-residue peptide - single-
stranded RNA18 and double-stranded RNA18, respectively)
whereas the exponentially decreasing signal intensity around
89483 Da was typical for the presence of nonspecific clusters.
Catalytic conformations of the GCN4 and c-Jun leucine
zipper peptides
At present the nature of the catalytically active conformation
(coiled coil versus monomer) of the native leucine zippers remains
unclear. The leucine zipper of GCN4 exhibits an equilibrium
dissociation constant (K
d
)of8nM [42] and therefore 99% of the
peptide should be in the dimeric coiled coil state in the conditions
of the ribonuclease assays. However, the results of MALDI-MS
indicate that monomeric GCN4 LZ35 can also form a 1:1 molar
complex with the RNA18 substrate. Similarly, under the
conditions employed disulfide-crosslinked rLZ38 and rLZ67
[Fig. 1(I d,e)] form stable tetramers while non-crosslinked
rLZ35 [Fig. 1(I c)] is monomeric [43]. Nevertheless, all three
retro-peptides exhibit comparable ribonuclease activity [Fig. 2].
The leucine zipper of c-Jun has a K
d
of 448 mM [44] and
therefore only 18–24% of the peptide shall be in the dimeric state
under the conditions employed [Fig. 4] (60–90 mM peptide
concentration).
Catalytic mechanism
Based on the substrate specificity [Fig. 3(C)], the cleavage of
RNA by GCN4 LZ and rLZ may involve elements of an RNase
T
1
-like mechanism. In RNase T
1
, Glu58/His92 were found to act
as the active-site base/acid couple with His40 participating in the
electrostatic stabilization of the transition state [45]. Later it was
shown that RNase T
1
was still active when both histidines were
replaced by aspartate [46]. It is conceivable that two of the four
acidic residues which are located within approximately 5.5 A
˚
on
the same face of the GCN4 leucine zipper (Glu8, Asp9, Glu12,
Glu13) formed an active site, giving rise to the ribonuclease
activity of LZ35 and rLZ35. It is also of interest that rLZ35 was
active despite being undoubtedly monomeric in the conditions of
the assay [43]. All this shows that at present suggestions for a
mechanism of the cleavage of RNA by GCN4 leucine zippers must
remain speculative. This applies also to the RNA cleavage by c-
Jun LZ36. Whether the partial sequence identity of the GCN4 and
c-Jun leucine zippers (KVEEL versus LEEKV) [Fig. 1(I b and a)]
hints to a common active site can not be answered and also raises
the question if maximum ribonuclease activity is confined strictly
to the sequences of the GCN4 and c-Jun leucine zippers. Most
likely, the two leucine zippers, although structurally and
functionally related, belong to those ‘‘enzyme’’ families in which
active site residues are not conserved [38]. In Fig. 1(II), the
structure of the region of GCN4 LZ35 in which residues were
mutated (lower panels) was compared to that of the corresponding
region of c-Jun LZ36 (upper panels). It showed that GCN4 and c-
Jun have a reversed polarity of charge distribution in these regions
of their leucine zippers.
Kinetics of the ribonuclease reaction of GCN4 LZ35 and c-
Jun LZ36
The cleavage of the RNA18 substrate by the leucine zipper
peptides was accompanied by a decrease of the area of the RNA18
peak in the HPLC chromatogram which allowed calculation of
rate constants and turnover. Considering the conformational
dynamics [47] and the relative lack of substrate specificity of these
leucine zipper peptides, the resulting numbers were most likely
mean values of several primary, kinetically equivalent cleavages
(strong arrows in [Fig. 3(C)]). Chromatographic analysis showed
also that initially produced fragments were cut further, demon-
strated by the formation of internal cleavage products of RNA18
such as ACC. and GAAUU. [Fig. 3(C)]. The complexity of
these secondary cleavages, however, did not allow estimation of
their individual kinetic constants.
Based on the degradation rate of full-length RNA18 (decrease of
the area of the RNA18 peak with time) the lower boundaries of
the second-order rate constants and turnover numbers were
k
2obs
=47 M
21
min
21
and k
obs
= 0.0024 min
21
for GCN4 LZ35
and k
2obs
=19 M
21
min
21
and k
obs
= 0.0033 min
21
for c-Jun
LZ36. The turnover numbers are one order of magnitude lower
than those of the zinc finger domain of transcription factor ZFY
(k
cat
= 0.023 min
21
) [12] and group II intron ribozymes
(k
cat
= 0.03 min
21
) [17].
It must be noted that the kinetic data of the two leucine zippers
for the cleavage of RNA18 were obtained in test-tube conditions.
If active in vivo, binding of GCN4 and c-Jun to their DNA targets
may affect the catalytic rates of their bZIP leucine zippers. In any
case, in vivo nuclease activity of GCN4 and c-Jun for the fine
regulation of transcription would have to be low, otherwise the
result would be energetically costly futile cycles of mRNA synthesis
and degradation.
Summary and Outlook
We have shown in test-tube experiments that the leucine zipper
domains of c-Jun and GCN4 have intrinsic ribonuclease activity
and that this activity is preserved in full-length 40 kD c-Jun, a
component of the transcription activation complex AP-1 [Fig. 4(B,
C)], and in the artificial sequence context of the rLZ67 fusion
protein [Fig. 2(D)]. Based on an analysis of the UniProt database
(release 15.1, November 2009), c-Jun and GCN4 are the first
known examples of specialized transcription factors possessing
ribonuclease activity.
However, it may be difficult to demonstrate that the GCN4 and
c-Jun leucine zippers catalyze slow RNA degradation in vivo. If this
were the case, other bZIP- as well as zinc finger-containing
transcription factors [12] may show a similar coupling of
transcription activation with slow RNA processing/degradation.
Other non-enzyme proteins possessing weak catalytic activities
are scarce and mainly comprise DnaK, the Escherichia coli
Hsp70 molecular chaperone that catalyzes the isomerization of
specific peptide bonds [48], and antibodies that convert O
2
to
H
2
O
2
[49] and catalyze ozone formation in bacterial killing and
inflammation [50]. In contrast, the number of proteins found to
be bi- or multifunctional with more or less equally strong
activities is growing rapidly, striking examples being fumarate
hydratase, a citric acid cycle enzyme which also acts as tumor
suppressor [11], and ERK2, an externally regulated (or MAP)
kinase which is also a transcriptional repressor of interferon
signaling [51].
Leucine Zipper RNase Activity
PLoS ONE | www.plosone.org 9 May 2010 | Volume 5 | Issue 5 | e10765

Materials and Methods
Peptide synthesis and purification
All leucine zipper peptides were synthesized by the solid phase
method [52,53] on an Applied Biosystems 433A Peptide
Synthesizer using Fmoc (fluorenylmethyloxycarbonyl) chemistry
[54] and were purified by reversed-phase HPLC. The purity of the
peptides was verified by amino acid analysis and mass spectrom-
etry. The amino acid analysis after acid hydrolysis of synthetic
GCN4 LZ35 was representative for the purity of the synthetic
peptides used in this study and gave the following amino acid
ratios (theoretical numbers are in parentheses): Asp 3.1 (3), Glu 8.0
(8), Ser 1.0 (1), Gly 1.1 (1), Ala 1.0 (1), Val 3.0 (3), Met 0.9 (1), Leu
7.0 (7), Tyr 0.9 (1), His 1.0 (1), Lys 4.9 (5), Arg 3.0 (3). The mass
spectra of the synthetic wild-type peptide and the Glu12Ala,
Ser16Ala, and His20Ala analogues are shown in Fig. S1 and Fig.
S2 of the electronic supplementary material. Cysteine-containing
peptides [Fig. 1(I d,e)] were air-oxidized before purification in
20 mM Tris-HCl, pH 7.2, at 5 to 7uC; oxidation was shown to be
complete after 6 to 8 hours using Ellman’s reagent.
Ribonuclease assays of leucine zipper peptides of GCN4
and c-Jun
Ribonuclease activities and kinetic data of the leucine zipper-
catalyzed RNA degradation were calculated from the time-
dependent decrease of the area of the RNA18 peak compared to
the RNA18 peak in peptide-free control samples using HPLC.
All reagents, buffers and column eluents employed in the assays
were prepared using ribonuclease-free water (commercial RNase/
DNase-free water, DEPC-treated water or ultrafiltered water
assayed for the lack of RNA-hydrolyzing activity). In all tests,
contaminating ribonuclease activity of plastic ware, reagents and
buffers was excluded. The stability of the RNA substrate in the
reaction buffer for 48–96 hours was imperative for the validity of
all ribonuclease assays.
Synthetic wild-type and mutant GCN4 leucine zippers (30 mM,
based on the relative molecular mass of the wild-type LZ35
monomer: 4238.5 Da) and RNA18 (50 mM) were incubated in
12.5 mLof20mM Tris-HCl and 80 mM KCl, pH 7.2, at 37uC
for 2 to 24 h. Then 100 mLof20mM ammonium acetate, with
35 mM bromophenol blue as internal reference, was added and
100 mL of the resulting mixture was fractionated on a Nucleosil C-
18 300-5 HPLC column (Macherey & Nagel) using a solvent
gradient (first solvent: 20 mM ammonium acetate, second solvent:
97% acetonitrile). Uncleaved RNA18 and the cleavage products
were detected at 254 nm. Digestions in 50 mM potassium
phosphate and 50 mM KCl gave very similar product patterns.
The assays were repeated in the presence of two concentrations
of the recombinant RNase A inhibitor RNasin (5 U and 150 U
per 12.5 mL assay solution). The GCN4 LZ35 and RNA18
concentrations were 25 mM and 50 mM, respectively. RNase A
(9.35 pM) was used as control. Incubation time was 13 h.
The peptide and RNA concentrations in the ribonuclease
activity assays of synthetic c-Jun LZ36 [Fig. 4(A)] were 60 mM and
170 mM, respectively, in the Tris-HCl buffer; reaction time was 2
to 24 h.
To detect a possible ribonuclease activity of full-length c-Jun,
the commercial recombinant protein (8 mM) was submitted to
ultrafiltration on a Millipore Ultrafree-0.5 membrane to increase
its concentration to 50 mM and to change the buffer to 20 mM
Tris-HCl, 85 mM KCl, pH 7.2. The final c-Jun and RNA18
concentrations in the assays were 20 mM and 90 mM, respectively,
in 10 mL of the Tris-HCl buffer. The assays were performed in
presence or absence of 1 U of RNasin. After 48 h at 37uC the
reaction mixtures were analyzed by gradient elution from an
Eclipse XDB-C18 RP-HPLC column (Agilent, 0.46 cm615 cm;
solvent: 20 mM ammonium acetate, pH 7.8/acetonitrile; elution
was recorded between volume ratios 98:2 and 85:15 at 254 nm;
gradient volume: 16 mL). The results are shown in Fig. 4(B,C,D).
Mass spectral analysis of RNA18 - GCN4 LZ35 complex
formation and RNA18 cleavage by LZ35
Complex formation was shown by MALDI mass spectrometry
using 6-aza-2-thiothymine/citrate as matrix. Spectra were record-
ed in negative ion, linear mode on a time-of-flight instrument
(AXIMA CFR, Shimadzu/Kratos) equipped with a nitrogen laser
(l= 337 nm, 3 ns pulse width). Both RNA18 and LZ35
concentrations were 69 mM in 20 mM ammonium acetate,
80 mM KCl, pH 6.5. The volume applied was 1 mL.
The cleavage of RNA18 by GCN4 leucine zippers and retro-
leucine zippers was analyzed by HPLC-electrospray ionization
mass spectrometry ([M-H]
2
). The cleavage experiments were
performed in 20 mM Tris-HCl/80 mM KCl at pH 7.2 and 37uC.
Samples (5 mL each) were applied on a Nucleosil 100-3 C-18 HD
column (Macherey & Nagel) and eluted using a stepwise gradient
from 100% 20 mM ammonium acetate to 97% acetonitrile. The
masses of the separated RNA18 cleavage products were
determined on a Bruker ESQUIRE-LC quadrupole ion-trap
instrument.
Supporting Information
Figure S1 Mass spectrum of synthetic 35-residue leucine zipper
of GCN4 representing the purity of the peptides used in this study.
Experimental and theoretical compound mass of LZ35 were
identical (49237.9 Da). Spectra were recorded on an API III+
instrument (Sciex, Toronto) and compound masses were calculat-
ed using the MacSpec software (Sciex).
Found at: doi:10.1371/journal.pone.0010765.s001 (0.14 MB TIF)
Figure S2 Mass spectra of synthetic mutants of GCN4 LZ35.
Glu12Ala (A), Ser16Ala (B), His20Ala (C). Theoretical compound
masses are 49179.9 Da (Glu12Ala), 49221.9 Da (Ser16Ala) and
49171.9 Da (His20Ala).
Found at: doi:10.1371/journal.pone.0010765.s002 (0.50 MB TIF)
Figure S3 Effects of RNasin on ribonuclease activity of LZ35,
RNase A and RNase T1. Effects of 0.5 U/mL RNasin on the
cleavage of 34 mM RNA18 by 50 mM GCN4 LZ35 (A,B), 1 nM
RNase A (C,D), 300 nM RNase T1 (E,F), and 150 mM BSA
control (G,H). Reactions were performed for 1.5 h (RNase A),
13 h (LZ35), 23 h (RNase T1), and 36 h (BSA) at 37uCin20mM
Tris-HCl, 85 mM KCl, pH 7.2. Uncleaved RNA18 elutes after
approximately 40 min.
Found at: doi:10.1371/journal.pone.0010765.s003 (0.55 MB TIF)
Figure S4 Aggregation of RNA18-LZ complexes in the presence
of His20Ala mutant and at high concentrations of wild-type LZ-
GCN4. (A) Digestion of 34 mM RNA18 by 50 mM wild-type LZ-
GCN4. (B) Aggregation of RNA in the presence of high (500 mM)
concentrations of LZ-GCN4. (C) Resolubilization of the RNA
pellet obtained at high LZ-GCN4 concentration (panel B). (D)
RNA18 was not digested by 50 mM Glu12Ala mutant. (E)
Aggregation of RNA18 in presence of 50 mM His20Ala mutant.
(F) Resolubilization of the RNA pellet obtained in the presence of
50 mM of the His20Ala mutant (panel E). Reactions were run for
13 h at 37uC in 20 mM Tris-HCl, 85 mM KCl, pH 7.2.
Resolubilization in (C) and (F) was performed by 10-fold dilution
of the sample in 80 mM sodium phosphate, pH 7.4, followed by 2-
min incubation at 65uC prior to HPLC fractionation. The
Leucine Zipper RNase Activity
PLoS ONE | www.plosone.org 10 May 2010 | Volume 5 | Issue 5 | e10765

difference in substrate retention times between panels A–C
(RNA18 eluted after ,45 min) and D–F (RNA18 eluted after
,40 min) are caused by shortening the column equilibration time
to optimize the LC analysis time experiments D–F.
Found at: doi:10.1371/journal.pone.0010765.s004 (0.40 MB TIF)
Figure S5 Chromatographic analysis of the cleavage of baker’s
yeast RNA by the GCN4 LZ35 peptide. Incubations were in
50 mM sodium acetate, pH 5, at 25uC. After 24 h, the samples
were applied on a Nucleosil C-18 300-5 column (Macherey and
Nagel) and eluted using a stepwise gradient from 20 mM
ammonium acetate to 97% acetonitrile in 60 min. The starting
concentration of RNA in (A), (B), and (C) was ,22 mM (0.5 mg/
mL). (A) RNA blank. (B) RNA + GCN4 leucine zipper (30 mM).
(C) RNA + BSA (7.35 mM).
Found at: doi:10.1371/journal.pone.0010765.s005 (0.55 MB TIF)
Acknowledgments
We thank Bianca Bergamaschi and Vishal Agrawal for excellent
experimental assistance.
Author Contributions
Conceived and designed the experiments: YN CD SH LB SF RZ KP PH
BG. Performed the experiments: YN CD SH LB SF PH. Analyzed the
data: YN CD SH LB SF RZ KP PH BG. Contributed reagents/materials/
analysis tools: LB RZ KP PH BG. Wrote the paper: YN BG. Initiated the
project: BG.
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