Allosteric Modulation of the Faecalibacterium prausnitzii Hepatitis Delta Virus-like Ribozyme by Glucosamine 6-Phosphate: The Substrate of the Adjacent Gene Product

Article abstract of DOI:10.1021/acs.biochem.7b00879

Self-cleaving ribozymes were discovered 30 years ago and have been found throughout nature, from bacteria to animals, but little is known about their biological functions and regulation, particularly how cofactors and metabolites alter their activity. A hepatitis delta virus-like self-cleaving ribozyme maps upstream of a phosphoglucosamine mutase (glmM) open reading frame in the genome of the human gut bacterium Faecalibacterium prausnitzii. The presence of a ribozyme in the untranslated region of glmM suggests a regulation mechanism of gene expression. In the bacterial hexosamine biosynthesis pathway, the enzyme glmM catalyzes the isomerization of glucosamine 6-phosphate into glucosamine 1-phosphate. In this study, we investigated the effect of these metabolites on the co-transcriptional self-cleavage rate of the ribozyme. Our results suggest that glucosamine 6-phosphate, but not glucosamine 1-phosphate, is an allosteric ligand that increases the self-cleavage rate of drz-Fpra-1, providing the first known example of allosteric modulation of a self-cleaving ribozyme by the substrate of the adjacent gene product. Given that the ribozyme is activated by the glmM substrate, but not the product, this allosteric modulation may represent a potential feed-forward mechanism of gene expression regulation in bacteria.

Full text of DOI:10.1021/acs.biochem.7b00879

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Article
Allosteric modulation of F. prausnitzii HDV–like ribozyme by glucosamine
6–phosphate ¬¬— the substrate of the adjacent gene product
Luiz F. M. Passalacqua, Randi M. Jimenez, Jennifer Y Fong, and Andrej Luptak
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00879 • Publication Date (Web): 18 Oct 2017
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Allosteric modulation of F. prausnitzii HDV–like ribozyme by glucosa-
mine 6–phosphate — the substrate of the adjacent gene product
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‡1
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1,2,3
Luiz F. M. Passalacqua , Randi M. Jimenez , Jennifer Y. Fong , Andrej Lupták*
1
Department of Pharmaceutical Sciences, University of California, Irvine, California 92697, USA
Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA
Department of Chemistry, University of California, Irvine, California 92697, USA
2
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6-21
and mechanism of self–scission
. HDV–like self–cleaving ribo-
ABSTRACT: Self–cleaving ribozymes were discovered 30 years
ago and have been found throughout nature, from bacteria to
animals, but little is known about their biological functions
and regulation, particularly how cofactors and metabolites
zymes exhibit great sequence diversity, but fold into a conserved
secondary structure that includes a nested double–pseudoknot
. HDV–like ribozyme have been found in many eukaryotes, in-
22
cluding humans, as well as in Chilo iridescent virus, several bacte-
alter their activity. An HDV–like self–cleaving ribozyme maps
ria, and most recently in several microbial metagenomic datasets
upstream of a phosphoglucosamine mutase (glmM) open
reading frame (ORF) in the genome of the human gut bacte-
rium Faecalibacterium prausnitzii. The presence of a ribozyme
in the untranslated region of glmM suggests a regulation
mechanism of gene expression. In the bacterial hexosamine
biosynthesis pathway, the enzyme glmM catalyzes the isom-
14, 22-27
.
These self–cleaving RNAs likely play a number of distinct
22-27
roles in biology
, but little is known about their regulation,
particularly with regard to the role ligands or metabolites may
have in modulating HDV–like ribozyme self–cleavage as either
allosteric effectors or cofactors.
One particular case of interest is the drz–Fpra–1 HDV–like ri-
erization of glucosamine 6–phosphate into glucosamine 1
phosphate. In this study, we investigated the effect of these
metabolites on the co–transcriptional self cleavage rate of
the ribozyme. Our results suggest that glucosamine 6
phosphate, but not glucosamine 1–phosphate, is an allosteric
ligand that increases the self cleavage rate of drz Fpra 1,
providing the first known example of an allosteric modulation
of a self cleaving ribozyme by the substrate of the adjacent
–
bozyme, found in the human gut bacterium Faecalibacterium
22
prausnitzii . F. prausnitzii is a Gram positive Firmicute that rep-
–
resents more than 5 % of the total bacterial population in the
fecal microbiota of a healthy human and is suggested to be nega-
–
tively correlated with certain pathologies, such as Crohn’s dis-
28-30
–
–
–
ease and ulcerative colitis
. The ribozyme cleavage site maps
1
06 nucleotides upstream of phosphoglucosamine mutase
22
–
(glmM) open reading frame (ORF)
(Figure 1A). The enzyme
gene product. Given that the ribozyme is activated by the
glmM substrate, but not the product, this allosteric modula-
tion may represent a potential feed–forward mechanism of
gene expression regulation in bacteria.
glmM is a component of the hexosamine biosynthesis pathway,
catalyzing the transformation of glucosamine 6–phosphate
31
(GlcN6P) into glucosamine 1–phosphate (GlcN1P) (Figure 1B).
The final product of the hexosamine biosynthesis pathway is
uridine diphosphate N–acetyl–glucosamine (UDP–GlcNAc), a key
32
substrate used for cell wall biosynthesis . The secondary struc-
ture of the ribozyme is shown in Figure 1C.
INTRODUCTION
In bacteria, a common mode of regulation of gene expression
involves sensing a metabolite related to the adjacent gene prod-
Ribozymes are RNA molecules that can catalyze a chemical
1
-3
33
transformation in the absence of a protein cofactor . Self–
cleaving ribozymes comprise a group of RNA molecules that
promote a site–specific self–scission reaction. In all known self–
cleaving ribozymes, the cleavage reaction is a transesterification
that involves a nucleophilic attack by a 2′ oxygen on the adjacent
phosphodiester bond, producing a 2′–3′ cyclic phosphate and a
uct through an RNA regulatory element called a riboswitch . In
3
4
the case of the glmS, a GlcN6P–sensing riboswitch , the RNA is
also a self–cleaving ribozyme that resides at the 5′ untranslated
region (UTR) of the transcript encoding glutamine fructose–6–
phosphate amidotransferase, which catalyzes the transformation
11
of fructose–6–phosphate into GlcN6P . The metabolite GlcN6P
4
-14
5
′–hydroxyl products
. To date, nine self–cleaving ribozyme
acts as a co–factor of the glmS ribozyme, accelerating its self–
1
0
11, 35-39
families have been discovered, comprising the hairpin , ham-
scission nearly one–million–fold
, and promoting the deg-
8,
9
6, 7
merhead
, hepatitis delta virus (HDV)
, glucosamine–6–
radation of the adjacent ORF through an RNase J–dependent
11
40
phosphate synthase (glmS) , Neurospora Varkud satellite (VS)
mechanism . Thus, this feedback system senses the amount of
12
13
14
,
twister , twister sister (TS), pistol, and hatchet motifs . First
GlcN6P in the cell and represses gene expression through the
ribozyme–dependent activity. This metabolite–responsive regu-
lation system has been well–characterized, by structural, bio-
6, 7, 15
characterized 30 years ago
has been extensively studied, with elucidated crystal structures
, the HDV self–cleaving ribozyme
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Figure 1. HDV–like ribozymes in F. prausnitzii. (A) Genome locus of F. prausnitzii showing the glmM ORF, the upstream drz–Fpra–1 ribozyme
and the newly discovered drz–Fpra–2 ribozyme. (B) The isomeration reaction promoted by the glmM enzyme. (C) A schematic representation of
the secondary structure of HDV–like ribozymes (left), and predicted secondary structures of drz–Fpra–1 and drz–Fpra–2 ribozymes, including
hairpins in the 5′ leader sequence. Red arrowheads mark the cleavage sites.
11, 35-39, 41-44
chemical and mechanistic studies
. Other metabolites,
scription assay with the following modifications: 4.5 mM MgCl ;
2
32
such as glucose 6–phosphate (Glc6P), may compete with GlcN6P
and thus upregulate the gene expression by inhibiting the self–
1.25 mM of each GTP, UTP, CTP; 250 μM ATP; 4.5 μCi [α– P]–
ATP (Perkin Elmer); and 40 mM HEPES pH 7.4. A 10 μL transcrip-
tion reaction was initiated by the addition of DNA and incubated
at 24 ˚C for 10 min. A 1.0 μL aliquot of the reaction was with-
drawn and its transcription and self–scission terminated by the
addition of urea loading buffer. The remaining 4.0 μL volume was
diluted 25–fold (100 µL final volume) into a physiological–like
buffer: 50 mM HEPES pH 7.4, 10 mM NaCl, 140 mM KCl, and the
3
9, 41
cleavage of the RNA
. Surprisingly, only 3 mutations in the
active site are necessary to convert the ribozyme into a coen-
zyme–independent self–cleaving ribozyme, revealed by an in
vitro selection of a GlcN6P–insensitive glmS ribozyme that used a
divalent cation for catalysis without changing the overall fold of
42
.
the ribozyme
desired concentration of MgCl and metabolite. Control experi-
Due to the proximity of the HDV–like ribozyme and the glmM
gene, we hypothesized that the ribozyme drz–Fpra–1 and the
metabolites involved in the hexosamine biosynthesis pathway
may contribute to gene expression regulation through modula-
tion of the ribozyme activity. During the course of this work, we
found another HDV–like ribozyme, named drz–Fpra–2 (Figure
2
ments showed that this dilution efficiently prevented any new
RNA synthesis; therefore, the kinetics of transcription did not
need to be accounted for in our kinetic analysis, contrasting with
the previously described analysis of co–transcriptional cleavage
45
by Long and Uhlenbeck . For conditions requiring consistent
ionic strength, the buffer and metabolite stocks were pH–
1
C), downstream of the glmM gene and we chose to study the
+
adjusted by the addition of KOH and the contribution of K and
two ribozymes in parallel. To explore the mechanism of this pu-
tative regulation, we studied the in vitro ribozyme self–cleavage
kinetics in the presence of several metabolites. The kinetic and
structural probing of the drz–Fpra–1 in the presence of the me-
tabolites shows an allosteric modulation of the ribozyme by the
substrate, but not the product, of the adjacent gene product. The
effect on drz–Fpra–2 was largely the opposite.
+
Na from the metabolite stocks was tracked. The concentration
+
of K was adjusted by the addition of KCl for a final reaction con-
centration of 140 mM. 5 μL aliquots were collected at the indi-
cated times following the dilution of the transcription reaction
into the physiological–like buffer at 37 ˚C and self–scission was
terminated by adding 5 μL volume of stop buffer containing 20
mM EDTA, 5 mM Tris pH 7.4, 8 M urea, with xylene cyanol and
bromophenol blue loading dyes. The denaturing PAGE gel of
cleavage products was exposed to phosphorimage screens and
analyzed using Typhoon phosphorimager and ImageQuant soft-
ware (GE Healthcare). The band intensities were analyzed by
creating line profiles of each lane using ImageQuant, exporting
the data to Microsoft Excel. Self–cleavage data were fit to a
mono–exponential decay function (Equation 1)
MATERIALS AND METHODS
In vitro RNA transcription. RNA was transcribed at 37 ˚C for
one hour in a 20 μL volume containing 10 mM DTT, 2 mM sper-
midine, 1.25 mM each rNTP, 7.5 mM MgCl , 1 unit of T7 RNA
2
polymerase, and 0.5 pmole of DNA template. The transcripts
were purified by 10 % polyacrylamide gel electrophoresis (PAGE)
under denaturing conditions (7M urea). RNA was eluted from the
gel into 300 μL of 300 mM KCl and precipitated by adding 700 μL
of 100% ethanol at –20 °C.
ꢀꢁꢂꢃ
ꢄ ꢅ ∙ ꢆꢇꢈꢉꢊꢋ ꢌ ꢍ
(Equation 1)
In vitro co–transcriptional cleavage kinetics. In vitro transcrip-
tion was performed similarly to the above–described RNA tran-
where A and C represent the relative fraction of the ribozyme
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Biochemistry
population cleaving with a rate constant k or remain uncleaved,
respectively. The model was fit to the data using a linear least–
squares analysis and the Solver module of Microsoft Excel.
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RNA 3′–terminus labeling. RNA was in vitro transcribed and
PAGE–purified. The appropriate RNA species was excised, precip-
itated, and re–suspended in water. RNA was then ligated at 37 °C
for 3 hours in a volume of 10 μL, in RNA ligase buffer containing
50 mM Tris–HCl, 10 mM MgCl , 1 mM DTT and pH 7.5 (NEB), 2
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μCi [5′– P] cytidine 3′, 5′– bisphosphate (Perkin Elmer) and one
unit of T4 RNA ligase (NEB) and PAGE purified again.
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In–line probing. The 3′–end labeled RNA was incubated with
varying amounts of ligand for up to 2 days at 37 °C in a buffer
containing 140 mM KCl, 10 mM NaCl, 20 mM Tris chloride, pH
8.5, 1 mM MgCl , and 1 mM spermidine, based on the in–line
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probing technique of Soukup and Breaker . The partially hydro-
lyzed RNAs were resolved using denaturing PAGE, exposed to
phosphorimage screens (Molecular Dynamics/GE Healthcare),
and scanned by GE Typhoon phosphorimager. The sequences in
the degradation pattern were assigned by running α–
phosphorothioate nucleotide modified RNA cleaved by treat-
Figure 2. Influence of GlcN6P on the co–transcriptional self–scission
of drz–Fpra–1 and drz–Fpra–2. To study the effect of the metabolite
on the cleavage rate of the ribozymes, in vitro co–transcriptional
cleavage kinetics were performed in presence and absence of
GlcN6P. (A) PAGE analysis of the co–transcriptional self–scission of
the two ribozymes in the absence (top) and presence (bottom) of 20
mM GlcN6P. (B) Log–linear graphs of the ribozyme self–scission.
GlcN6P accelerates the self–cleavage rate of drz–Fpra–1 (left; no
GlcN6P, open circles; 20 mM GlcN6P, solid circles) but inhibits drz–
Fpra–2 (right). Early time points are shown in insets.
4
7, 48
ment with iodoethanol in parallel lanes
. The band intensities
were analyzed by creating line profiles of each lane using Im-
ageQuant and exporting the data to Microsoft Excel. The areas of
the fitted curves were used to measure intensity changes related
to the binding, divided by intensities of a control band. The re-
sulting ratios were plotted in Excel as a function of ligand con-
centration and modeled with a dissociation constant equation for
a single ligand:
RESULTS
Discovery of a second HDV–like ribozyme in the same locus
of F. prausnitzii genome. We decided to analyze the F. prausnitzii
genome in order to verify the mapping of the drz–Fpra–1 ribo-
zyme near the glmM gene. To our surprise, we found another
HDV–like ribozyme, drz–Fpra–2, with high sequence similarity to
drz–Fpra–1 in the same locus. This second ribozyme was found
ꢛꢜꢝꢞꢟꢠꢡꢢ
ꢇꢛꢜꢝꢞꢟꢠꢡꢢꢣꢤ
ꢚ
ꢥꢡꢖꢈꢖꢦꢧꢃꢨꢩꢪꢫꢨꢬ
ꢎꢏꢐꢑꢒꢓꢔꢕꢖꢗꢔꢘꢕꢙ ꢄ
(Equation 2)
ꢭꢧꢫꢮꢨ
5
58 nucleotides downstream of the glmM gene (Figure 1A). Alt-
The model was fit to the data using a linear least–squares analy-
sis and the Solver module of Microsoft Excel.
hough similar in structure and sequence, some differences near
the active site were observed in the proposed secondary struc-
tures (Figure 1C), as discussed below. One unique feature found
in both drz–Fpra ribozymes is an A–U base–pair in the top of the
Metabolites. All the metabolites used in this study were pur-
chased from Sigma Aldrich. To measure the concentration of free
phosphate, which may affect the ribozyme kinetics by forming an
2+
P1.1 region, where a G–C base–pair is usually found in other
insoluble complex with the Mg ions, we used a fluorescent
49
.
HDV–like ribozymes
phosphate sensor based on the bacterial phosphate–sensing
protein (Thermo Fisher). The molar ratio of free phosphate in the
GlcN1P, GlcN6P, and GlcP was found to be 0.0001, 0.002, and
Self–scission kinetics of drz–Fpra ribozymes in the presence
of metabolites. To study the effect of the metabolite on the
cleavage rate of the ribozymes, we started with an in vitro co–
transcriptional cleavage kinetics performed in the presence or
0
.0004.
2+
absence of 20 mM GlcN6P at 5 mM Mg . Surprisingly, the effect
2+
2+
Figure 3. Effect of GlcN6P and Mg on the drz–Fpra ribozymes. (A) Drz–Fpra–1 self–scission dose–response to GlcN6P at constant (5 mM) Mg ,
2+
normalized to the no–metabolite control. (B) Mg dependence of drz–Fpra–1 self–scission in presence of 20 mM GlcN6P (open squares) or no
2+
metabolite (open circles). (C) Mg dependence of drz–Fpra–1 (open circles) and drz–Fpra–2 (open triangules) self–scission in 20 mM GlcN6P
normalized to a no–metabolite control.
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of the metabolite on each ribozyme was different. GlcN6P accel-
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erates the self–scission of the drz–Fpra–1 ribozyme, whereasit
decreases the self–cleavage rate of the drz–Fpra–2 ribozyme
(Figure 2). The low amplitude of the effect of the metabolite on
the two ribozymes indicated that GlcN6P is an allosteric effector
(modulator) and not a co–factor or co–enzyme that directly par-
ticipates in catalysis. To further investigate the ligand effect on
the activity of drz–Fpra–1, we performed a titration of GlcN6P,
keeping the ionic strength of the solution constant and [Mg ]
fixed at 5 mM. Self–cleavage kinetics under conditions approxi-
mating co–transcriptional self–scission showed a dose–response
between the cleavage rate constant and the metabolite, increas-
ing the kGlcN6P more than 2–fold at higher concentrations of
GlcN6P, when compared to no–metabolite kcontrol (Figure 3A). At
low concentrations of GlcN6P, the cleavage rate was similar to
the no–metabolite control, indicating that the metabolite may
not be affecting the catalytic mechanism of the ribozyme.
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To investigate the origin of this dose–response, we varied the
2+
concentration of Mg , the divalent metal ion used by the ribo-
17, 18, 50-52
zyme for catalysis
, in the presence of 20 mM GlcN6P. At
2+
low concentrations of Mg , both k
and kcontrol behave simi-
GlcN6P
larly, with a strong overlap between both data sets (Figure 3B).
2+
This trend changes at about physiological Mg , where the cleav-
age rate increases in the presence of GlcN6P. At higher concen-
trations of the divalent metal ion, kcontrol decreases, possibly due
to misfolding. In contrast, this effect is not seen for kGlcN6P, which
continues to gradually increase. This result is consistent with a
model, in which the metabolite interacts with the ribozyme, but
does not act directly in the catalytic step of the reaction (Figure
2
+
3
B). We also performed the same titration of Mg in the pres-
ence of 20 mM of GlcN6P for drz–Fpra–2. As expected, an oppo-
site effect was observed, where a decrease in the cleavage rate
was found in the same conditions when compared to a no–
metabolite control (Figure 3C).
Figure 4. Structural probing of GlcN6P binding of the drz–Fpra–1
ribozyme. (A) In–line probing of the 3′–labeled drz–Fpra–1 ribozyme
in the presence of GlcN6P. The band intensities that change with
GlcN6P concentration are indicated in the figure by the nucleotide
identity. The sequence of the RNA was determined using iodoetha-
nol cleavage of ribozymes with phosphorothioate–modified back-
bone at positions indicated above each lane. The intensities of the
control band (G69) from the same experiment are shown below the
rest of the gel. (B) Graph of band intensities for the regions P3/L3
(open circles), J1.1/4 (open triangles), L4 (open squares), and A60
To investigate the specificity of the ribozyme–metabolite in-
teraction, we decided to test compounds related to GlcN6P. The
ribozyme self–cleavage kinetics were tested in the presence of
the following molecules: GlcN6P, the precursor of the reaction
promoted by the glmM enzyme; GlcN1P, the isomer of GlcN6P
and the product of the reaction; glucose (Glc); Glc6P and glu-
cosamine (GlcN) – the last two were chosen due to their chemi-
cal similarity to the metabolites, because each carries a different
chemical group present in both metabolites of the enzymatic
reaction (the amino and the phosphate groups). The kinetic rate
constants were normalized to no–metabolite cleavage kinetics at
the same conditions, and showed that GlcN1P, the product of the
reaction catalyzed by glmM, activated only the Fpra–2 ribozyme
– albeit with low statistical significance, whereas Glc slightly in-
creased the activity of both ribozymes (Figure S1). GlcN and
Glc6P increased the cleavage rate of both drz–Fpra–1 and drz–
Fpra–2 ribozymes to a similar extent (Figure S1). This is an unex-
pected result for drz–Fpra–2, because it was not inhibited by
GlcN and Glc6P, as it was in the presence of GlcN6P. These re-
sults suggest that both the amino and the phosphate groups are
important in modulating the self–scission rate of the ribozymes,
and their relative positioning around the sugar ring leads to the
differential effect on the two ribozymes. Finally, we decided to
probe the ribozymes in the presence of UDP–GlcNAc, the final
product of the hexosamine biosynthesis pathway, but no signifi-
cant effect was observed (Figure S2).
(
open diamonds), normalized to a control band at G69. The data
were fit to a model based on Equation 2. The average dissociation
constant is estimated in 4.7 ± 0.2 mM. The positions where the band
intensities respond to metabolite concentration are also indicated
left of the gel in (A).
2+
same concentrations of the metabolite, while varying the Mg
concentration. At 20 mM metabolite concentration, the
kGlcN6P/kGlcN1P ratios for drz–Fpra–1 were 1.4 ± 0.1, 2.2 ± 0.4, and
2
+
2
.5 ±0.5 at 1, 5, and 10 mM Mg . Interestingly, when both
GlcN1P and GlcN6P were included in the drz–Fpra–1 reaction,
the effect of GlcN6P was enhanced (Figure S3).
The structure of the ribozyme drz–Fpra–1 is stabilized by
GlcN6P. In order to investigate the effect of the metabolite on
the ribozyme structure, we performed an in–line probing exper-
iment in the presence of different concentrations of GlcN6P. This
technique profiles the natural degradation of an RNA molecule at
different conditions, providing information on the relative stabil-
46
ity of individual phosphodiesters . More flexible, solvent–
exposed regions (such as single–strand regions) and specific con-
formations are more likely to promote a 2′–OH attack on the
scissile phosphate, cleaving the molecule. The pattern of the
Another way to compare the effects of GlcN6P and GlcN1P is
to calculate the ratio of the self–scission rate constants at the
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Biochemistry
6, 7
cleavage at different conditions can indicate structural changes,
from the hepatitis delta virus
. No significant change in the
1
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3
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5
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9
1
1
1
1
1
1
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6
suggesting differences in the flexibility and protection promoted
by a ligand. Our results showed that GlcN6P promotes an appar-
ent stabilization of the ribozyme at specific sites, suggesting that
the metabolite directly interacts with the RNA (Figure 4A). The
regions showing the most prominent change in degradation at
higher GlcN6P concentration are P3/L3, J1.1/4, L4 and A60 (J4/2).
Three of those regions (P3/L3, J1.1/4 and A60) surround the ac-
tive site of the ribozyme, with A60 only 2 nucleotides away from
the catalytic cytosine residue (C58). No region of increased deg-
radation was observed in this experiment.
cleavage rate was observed for these ribozymes (Figure 5), sug-
gesting that the kinetic modulation by the metabolite is specific
to the drz–Fpra ribozymes.
To narrow down the cause of the opposite effect promoted by
GlcN6P on drz–Fpra–1 and drz–Fpra–2, we decided to study the
discrepant nucleotides near the active sites of the two otherwise
similar ribozymes. The catalytic cores of the two ribozymes differ
by only three nucleotides. We tested three hybrids C23A and
U26C in the L3, and A60G in the J4/2 regions of the drz–Fpra–1
sequence. We found that U26C retains, and even slightly in-
creases the effect of GlcN6P on the cleavage rate constant; C23A
seems to abrogate the metabolite effect; and A60G reverts the
metabolite effect, decreasing the cleavage rate of the ribozyme
when compared to the kWT (Figure 5), to a level similar to the
drz–Fpra–2. These results indicate that the nucleotides C23 and
A60 are involved in the allosteric modulation of drz–Fpra–1.
0
1
2
3
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9
0
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0
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2
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8
9
0
The data obtained for each concentration of metabolite, in-
cluding the no–metabolite control, were normalized to G69 near
the 3′ terminus of the ribozyme, used as a control position with
ligand–independent degradation. The data were modeled by a
dissociation constant equation (Equation 2) for a single ligand
and a K was calculated independently for each affected region
d
(
Figure 4B). The K estimations for all 4 regions were consistent,
d
resulting in an average K of 4.7 ± 0.2 mM. Performing the same
d
DISCUSSION
experiment with the metabolite GlcN1P, the isomer of GlcN6P
and product of the enzymatic reaction that does not affect the
cleavage rate of the ribozyme, we did not observe any region
with significant degradation pattern changes (Figure S4A and B).
We measured the degradation for the regions P3/L3, L4 and A60
One of the nine natural families of self–cleaving ribozymes,
the HDV–like ribozymes are found in several bacteria, many eu-
karyotes (including humans), Chilo iredescent virus, and in mi-
1
4, 22-27
crobial metagenomic datasets
. Little is known about their
biological functions and regulation, particularly any roles that
cofactors and metabolites have in altering their activity. Drz–
(
J4/2), and used 3 different control bands for normalization. No
apparent K was revealed for any of the regions, demonstrating
d
22
Fpra–1 and drz–Fpra–2 HDV–like ribozymes were found in the
that GlcN1P does not affect the degradation pattern of the ribo-
zyme.
human gut bacterium F. prausnitzii genome, surrounding the
phosphoglucosamine mutase (glmM) open reading frame (ORF)
Metabolite effect is ribozyme– and position–specific. To veri-
fy that the effects promoted by GlcN6P in both drz–Fpra–1 and
drz–Fpra–2 were specific, we decided to test the ligand influence
on different ribozymes of the same family, starting with two
inactive drz–Fpra–1 mutants, C58A and C58U. Based on previous
49
.
The enzyme glmM catalyzes the transformation of glucosa-
mine 6–phosphate (GlcN6P) into glucosamine 1–phosphate
31
(
GlcN1P) in the hexosamine biosynthesis pathway . We have
studied the effect of the metabolites from this pathway on the
self–cleavage rate of the drz–Fpra ribozymes. Whereas GlcN6P
increases the cleavage rate of the drz–Fpra–1 ribozyme and ap-
pears to stabilize its structure, GlcN1P, the product of the glmM
enzymatic reaction, does not significantly affect the ribozyme.
1
6, 17, 19, 22, 23, 51, 53
mutagenesis of HDV ribozymes
, we expected a
very slow self–cleavage of C58A and an abolishment of catalysis
for C58U. In two–hour–long experiments, neither mutant of drz–
Fpra–1 showed self–scission in the presence of GlcN6P (data not
shown). These results demonstrate that GlcN6P does not rescue
the activity of the ribozyme, further supporting a role as an allo-
steric modulator and not a catalytic co–factor for the ligand. To
further investigate the specificity of GlcN6P, we probed two con-
trol ribozymes with the metabolite, the genomic HDV (gHDV) and
the antigenomic HDV (aHDV), the two distinct HDV ribozymes
The GlcN6P effect on drz–Fpra–1 showed that the ligand mod-
estly increases the self–cleavage rate of the ribozyme when
2+
compared to the no–metabolite control. Titration of Mg at
2+
constant GlcN6P or titration of ligand at constant Mg showed
similar results, increasing the self–cleavage rate of the ribozyme.
2+
When we examined the influence of GlcN6P on the Mg de-
pendence of the ribozymes, we observed that the ligand effect is
not seen at low concentrations of the divalent cation, behaving
2+
similarly to the no–metabolite control. Mg coordinated with a
hydroxide anion likely acts as the general base in the self–
cleavage reaction of HDV ribozymes, deprotonating the 2′–OH of
17
the base that promotes the attack in the scissile phosphate ,
while the essential cytosine in J4/2 (C58 in drz–Fpra–1) acts as a
general acid, mediating a proton transfer to the 5′–oxygen of G1,
17
the leaving group of the self–scission reaction . If the effect
2+
promoted by the metabolite directly influenced the role of Mg
in the mechanism of the reaction, the kGlcN6P would be expected
to increase the cleavage rate in all instances throughout the lig-
Figure 5. Effect of GlcN6P on self–scission of drz–Fpra–1/2 hybrids
and HDV ribozymes. Comparison of modulation of the ribozyme
activity by 20 mM GlcN6P normalized to the no–metabolite control
2+
and titration, including at low concentrations of Mg . This result
suggests that the effect caused by the ligand does not directly
influence the catalytic role of the metal ion in the self–cleavage
reaction.
2+
at 5mM Mg . U26C, C23A and A60G are drz–Fpra–1/drz–Fpra–2
hybrids referenced to the drz–Fpra–1 positions. gHDV and aHDV are
the genomic and antigenomic HDV ribozymes, respectively. All p-
values were calculated with respect to the wild–type Fpra–1 ribo-
zyme and show statistical significance of the results.
In the case of titration of GlcN6P, the ribozyme responds to
the ligand, cleaving faster than the no–metabolite control. Again,
this effect is not seen at low concentrations of the metabolite,
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Biochemistry
Page 6 of 15
indicating that the effect is not directly related to the catalysis in
ribozymes. C23 is part of L3 and is important to maintain the
active site structure, stacking on C24 and possibly hydrogen
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5
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7
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9
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6
the active site, otherwise we would expect to see an appreciably
higher kGlcN6P even at lower concentrations of GlcN6P. Moreover,
testing the effect of the metabolite in the two inactive mutants
of drz–Fpra–1, C58A and C58U, we found that the ligand does
not rescue their activity, indicating that GlcN6P does not act as
the general acid. This finding contrasts with the role of GlcN6P in
the glmS ribozyme, where the cofactor actively participates in
the active site, mediating the protonation of the 5′–oxyanion
16, 18, 19
bonding with neighboring residues
portant for the stacking with U–1, similarly to the role of U23 in
. C23 may also be im-
1
6, 18, 19
the HDV ribozyme
. A mutation at C23 may promote a
conformational change that prevents the interaction of GlcN6P
with the ribozyme. A60 is part of the trefoil turn of the ribozyme
structure and makes an A–minor interaction with P3, participat-
ing in a network of hydrogen bonding between P3/L3 and J4/2
3
6-39, 42-44, 54
16, 18, 19
leaving group
. The glmS cofactor has been implicated
regions
. Thus, the A60G mutation would sterically hinder
in other roles in catalysis, including helping to align the active
site, stabilizing the developing charge during the self–cleavage
the interaction of the nucleobase, causing the J4/2 to move away
from P3 and potentially disrupting the interactions of the A60
ribose as well. This change may be sufficient to abrogate the
already weak interaction between the ribozyme and the metabo-
lite.
0
1
2
3
4
5
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7
8
9
0
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2
3
4
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9
0
reaction, and participating in a set of competing hydrogen bonds
54, 55
to ensure potent activation and regulation of the catalysis
.
In contrast, our results suggest that the effect produced by
GlcN6P on drz–Fpra–1 is not directly in the catalytic step of the
self–cleavage reaction, suggesting an allosteric modulation of the
ribozyme activity.
Testing ribozyme kinetics in the presence of other metabolites
elucidated the functional moieties critical in GlcN6P sensitivity in
drz–Fpra–1. The aforementioned GlcN1P, the product of the
enzymatic reaction of glmM, showed no significant effect on drz–
Fpra–1 and a small effect on drz–Fpra–2. Similarly, Glc and UDP–
GlcNAc, had little to no effect on the ribozymes. On the other
hand, GlcN and Glc6P increased the cleavage rate for both ribo-
zymes, leading us to speculate that both the amino and phos-
phate groups are relevant for the effect of the ligand in the ribo-
zyme, as long as they are not at adjacent carbons of the sugar
ring, as seen in GlcN1P. The concentration of Glc6P in the cytosol
is likely very low and a previous report on the concentration of
metabolites in E. coli with a detection limit of 130 nM did detect
The structural probing of the drz–Fpra–1 in the presence of
GlcN6P and GlcN1P showed decreased conformational flexibility
of the RNA solely in the presence of GlcN6P (Figures 4 and S4).
The active site of HDV ribozymes is formed by the P3/L3, P1.1
1
6, 18, 19
and J4/2 elements
, and our probing data show that the
ligand decreases the degradation of P3/L3, J1.1/4, L4 and A60 in
J4/2. Even though GlcN6P clearly affects the active site stability
and promotes faster self–scission, the in–line probing experiment
does not pinpoint the site of interaction between the metabolite
and the ribozyme, because the changes in degradation pattern
may result from a conformational change promoted by the in-
termolecular interaction, and additional studies would be need-
ed to map the exact position of interaction.
56
Glc6P . Thus, despite an in vitro effect of Glc6P on the ribo-
zymes, it is unlikely that it affects their activity in vivo.
The cleavage rate constants obtained for gHDV and aHDV ri-
bozymes in the presence of GlcN6P were similar to those of the
no–metabolite control. These results suggest that the interaction
between the metabolite and drz–Fpra–1 is specific. Concerning
For all regions of intensity change of the in–line probing bands,
the obtained estimated K was similar, with an average of 4.7 ±
d
0
.2 mM. The intracellular concentration of GlcN6P in F.
prausnitzii is not known, but the reported steady–state concen-
the structure of the ribozymes, to our knowledge, the first P1.1
5
6
22, 24, 25, 27, 49
tration in E. coli cells is 1.2 mM . Although it is around 4 times
base pair is found to be A–U only in bacteria
, and the
lower than the estimated K for drz–Fpra–1 ribozyme, subcellular
drz–Fpra–1 was the only case of a bacterial HDV–like ribozyme
d
localization and spikes in metabolic activity may result in a higher
concentration of the metabolite than the cell culture average.
Thus, the metabolite concentration may reach intracellular levels
that significantly alter the activity of the ribozyme.
upstream of a neighboring gene. However, a recent discovery of
14
several ribozymes using comparative genomic analysis
also
revealed HDV–like ribozymes in environmental samples, of which
4 showed the glmM gene 34–to–42 nts downstream of the ribo-
zymes, similarly to drz–Fpra–1 in F. prausnitzii (37 nucleotides).
Seven other HDV–like ribozymes showed the glmM gene up-
stream of the ribozymes. All of these ribozymes have an A–U
base pair in P1.1, suggesting that they originate from closely
related bacteria. The alignment of the drz–Fpra–1, drz–Fpra–2
and other microbial HDV–like ribozymes to the secondary struc-
ture is shown in Figure S5. The alignment, combined with the
results presented here, suggests that env–26 HDV–like ribozyme
would respond to GlcN6P, because it is the only sequence with a
cytosine in the same position of L3 as in drz–Fpra–1 (C23),
whereas all other ribozymes bear an adenosine at this position,
like in drz–Fpra–2, where we found no effect of the metabolite.
Regarding the J4/2 strand, only drz–Fpra–2 contains a guanosine
residue at the position equivalent to A60 in drz–Fpra–1, here
reported to be responsible for the inhibitory effect of GlcN6P on
the activity of the ribozyme.
The drz–Fpra–2 ribozyme, located 558 nucleotides down-
stream of the glmM gene, was found by sequence similarity
when searching for drz–Fpra–1. Interestingly, GlcN6P promoted
an opposite effect on drz–Fpra–2 than on drz–Fpra–1, decreasing
the self–cleavage rate when compared to the kcontrol. This effect
was unexpected and intriguing, bringing to our attention those
nucleotides of the active site, where the ribozymes differ. We
tested the effect of GlcN6P on the activity of the hybrids of drz–
Fpra–1 and drz–Fpra–2, with the following mutations made to
drz–Fpra–1: C23A and U26C in the L3, and A60G in the J4/2 re-
gions. The results demonstrate that U26C retains the effect of
GlcN6P on the cleavage rate, maintaining the elevated kGlcN6P and
suggesting that the metabolite does not interact with the ribo-
zyme at this nucleotide. On the other hand, the C23A mutation
abrogates the metabolite effect, bringing the self–cleavage rate
to the same level as the no–metabolite control. The third muta-
tion, A60G, reverts the metabolite effect, decreasing the cleav-
age rate of the ribozyme below the kcontrol, similarly to what is
observed in drz–Fpra–2. Thus, our data suggest that C23A and
A60G are the discrepant nucleotides from the catalytic core that
play a role in the opposite effect caused by GlcN6P in the two
We show that the metabolite GlcN6P interacts with the drz–
Fpra–1 ribozyme and increases its self–cleavage activity; howev-
er, we do not know the biological significance of these findings,
and future studies will include biological assays addressing this
question. In contrast to the glmS riboswitch–ribozyme, which is
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Biochemistry
part of a feedback regulatory loop utilizing the product of the
ACKNOWLEDGMENT
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
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4
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5
5
5
5
6
downstream metabolic step to induce the ribozyme self–scission
and mRNA degradation, the activity of drz–Fpra–1 increases with
the concentration of the substrate, providing an example of a
putative feed–forward mechanism. The downstream RNA prod-
uct, which starts with the cleaved drz–Fpra–1 ribozyme and is
followed by the GlmM open reading frame, is terminated with a
We thank all members of the Lupták laboratory for helpful dis-
cussions.
ABBREVIATIONS
HDV, hepatitis delta virus; glmS, glucosamine–6–phosphate syn-
thase; glmM, phosphoglucosamine mutase; ORF, open reading
frame; GlcN6P, glucosamine–6–phosphate; GlcN1P, glucosa-
mine–1–phosphate; UDP–GlcNAc, uridine diphosphate N–acetyl–
glucosamine; Glc, glucose; Glc6P, glucose–6–phosphate; GlcN,
glucosamine; PAGE, polyacrylamide gel electrophoresis; nt, nu-
cleotide.
5
′–OH that is sequestered by the structure of the cleaved ribo-
16, 18, 19
zyme
. This structure may protect the 5′ terminus against
endonucleases, thus increasing the mRNA stability. Moreover,
RNAs with 5′–OH termini have an extended half–life when com-
pared with 5′–phosphorylated mRNAs, because 5′-hydroxyls are
inferior substrates for the endonucleolytic degradation by RNase
E
phosphorylated 5′ termini has been used in bacterial metabolic
engineering to design a number of aptazymes that increased
stability of the downstream transcripts upon ligand–dependent
self–scission , and similar mechanism may be acting in the case
of the F. prausnitzii ribozyme–terminated glmM mRNA. To the
best of our knowledge, the drz–Fpra–1 activation by GlcN6P is
the first example of an allosteric modulation of a natural self–
cleaving ribozyme by a metabolite. We believe that this is not a
unique case and that other examples of natural allostericly–
regulated self–cleaving ribozymes exist, providing another exam-
ple of gene expression regulation at the RNA level.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
57, 58
. The extended half–life of mRNAs with 5′–OH termini over
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Effect of multiple ligands in drz–Fpra–1 and drz–Fpra2 (Figure
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AUTHOR INFORMATION
Corresponding Author
*Phone: (949) 824–9132; E–mail: aluptak.uci.edu.
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Author Contributions
L.F.M.P., R.M.J. and A.L. designed the study; L.F.M.P., R.M.J. and
J.Y.F performed the experiments; all authors analyzed the data;
and L.F.M.P., R.M.J. and A.L. wrote the manuscript.
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a novel RNA in Neurospora
‡
These authors contributed equally.
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Funding Sources
This work was supported by Science Without Boarders Program –
CAPES Foundation, Ministry of Education of Brazil – Process
[14] Weinberg, Z., Kim, P. B., Chen, T. H., Li, S., Harris, K. A., Lunse, C. E.,
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9
9999.013571/2013–03 (L.F.M.P.), NIH 5F31GM103241–02
(
R.M.J.), NSF MCB 1330606 and a grant from the John Templeton
Foundation (to A.L.). This project/publication was made possible
through the support of a grant from the John Templeton Founda-
tion. The opinions expressed in this publication are those of the
author(s) and do not necessarily reflect the views of the John
Templeton Foundation.
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Characterization of self-cleaving RNA sequences on the
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17] Das, S. R., and Piccirilli, J. A. (2005) General acid catalysis by the
The authors declare no competing financial interests.
hepatitis delta virus ribozyme. Nat Chem Biol 1, 45-52.
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[
18] Chen, J. H., Yajima, R., Chadalavada, D. M., Chase, E., Bevilacqua, P.
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Biochemistry
[
58] Deana, A., Celesnik, H., and Belasco, J. G. (2008) The bacterial
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enzyme RppH triggers messenger RNA degradation by 5'
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program gene expression. Science 334, 1716-1719.
0
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B
Biochemistry
Page 10 of 15
glmM
drz-Fpra-1
phosphoglucosamine mutase
drz-Fpra-2
glmM
1
2
3
6
75962
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1385 nt
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Glucosamine 6-P
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drz-Fpra-1
drz-Fpra-2
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Biochemistry
A
drz-Fpra-1
drz-Fpra-2
1
2
3
sec
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sec
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Time
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4
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6
Precursor
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3' Product
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0
1
2
3
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2
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Biochemistry
Page 12 of 15
2
.5
.0
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47
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52
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[
Mg ] (mM)
2+
2+
[
GlcN6P] (mM)
[Mg ] (mM)
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Biochemistry
A C G
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[GlcN6P] (mM)
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ACS Paragon Plus Environment

Biochemistry
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ACS Paragon Plus Environment