From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
Received for publication, October 20, 2000
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ABSTRACT |
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Ribonuclease H (RNase H) selectively degrades the
RNA strand of RNA·DNA hybrids in a divalent
cation-dependent manner. Previous structural studies
revealed a single Mg2+ ion-binding site in
Escherichia coli RNase HI. In the crystal structure of the
related RNase H domain of human immunodeficiency virus reverse
transcriptase, however, two Mn2+ ions were observed
suggesting a different mode of metal binding. E. coli RNase
HI shows catalytic activity in the presence of Mg2+ or
Mn2+ ions, but these two metals show strikingly different
optimal concentrations. Mg2+ ions are required in
millimolar concentrations, but Mn2+ ions are only required
in micromolar quantities. Based upon the metal dependence of E. coli RNase HI activity, we proposed an activation/attenuation
model in which one metal is required for catalysis, and binding of a
second metal is inhibitory. We have now solved the co-crystal structure
of E. coli RNase HI with Mn2+ ions at 1.9-Å
resolution. Two octahedrally coordinated Mn2+ ions are seen
to bind to the enzyme-active site. Residues Asp-10, Glu-48, and Asp-70
make direct (inner sphere) coordination contacts to the first
(activating) metal, whereas residues Asp-10 and Asp-134 make direct
contacts to the second (attenuating) metal. This structure is
consistent with biochemical evidence suggesting that two metal ions may
bind RNase H but liganding a second ion inhibits RNase H activity.
The RNase H class of enzymes cleaves the RNA moiety of RNA·DNA
hybrids in a divalent cation-dependent manner leaving
5'-phosphate and 3'-hydroxyl products. RNase H proteins are found in a
wide variety of organisms ranging from bacteria to vertebrates (for review see Refs. 1 and 2). From a medical perspective, the most
significant function of RNase H is its critical action in the life
cycle of retroviruses such as the human immunodeficiency virus
(HIV).1 This activity arises
from the C-terminal region of reverse transcriptase. The RNase H domain
from HIV is homologous to other members of this class including RNase
HI from Escherichia coli. Because inhibiting RNase H
activity prohibits production of infectious virions (3), understanding
the function and mechanism of RNase H is an important avenue toward the
development of anti-retroviral compounds.
RNase H requires divalent cations (either Mg2+ or
Mn2+) for catalysis. Five conserved residues form the
metal-binding active site of RNase H (Fig. 1a). Two RNases H
with metal ions bound have been studied by x-ray crystallography.
E. coli RNase HI binds a single Mg2+ ion via
three carboxylates (Asp-10, Glu-48, and Asp-70) that form Site 1 (4,
5), whereas the HIV RNase H domain shows two Mn2+ ions, one
in a position similar to Site 1 and another (Site 2) liganded by the
equivalents of Asp-10 and Asp-134 (6). Mutations in RNases H, which
eliminate Mg2+-dependent activity, often allow
retention of Mn2+-dependent activity (7-11).
This, together with the different stoichiometry observed via
crystallography, raises the possibility of alternate binding modes
and/or catalytic requirements for these metals. In E. coli
RNase HI, conservative mutation of the residues comprising Site 1 (Asp-10, Glu-48, and Asp-70) eliminates activity (12), whereas
mutations of the conserved histidine (His-124) (13) and aspartate
(Asp-134) of Site 2 (14) have smaller effects on catalysis. Despite
structural efforts to identify additional weaker binding sites (5), no
metal binding to Site 2 in E. coli RNase HI has been reported.
Our laboratory has recently proposed a model for the metal dependence
of E. coli RNase HI termed an "activation/attenuation mechanism" (15). In this model, a single metal bound at Site 1 is
required for catalysis, and the binding of a second metal at Site 2 reduces catalysis ~100-fold. In this report, we present a high
resolution crystal structure of E. coli RNase HI in complex with Mn2+ ions. These data demonstrate that the active site
of E. coli RNase HI can bind two Mn2+ ions in
the sites predicted by the activation/attenuation hypothesis. Furthermore, we show that the surprising dependence of RNase H activity
in the presence of Mn2+ upon ionic strength can also be
explained by this model.
Production of RNase H*--
Kunkel mutagenesis (16) of pSM101
(carrying a synthetic gene directing the expression of RNase H*, a
cysteine-free variant of E. coli RNase HI) was used to
create pEG120 which encodes for K87A RNase H*. DNA sequencing confirmed
the incorporation of the desired mutation. RNase H* and K87A proteins
were overexpressed in E. coli and purified by
cation-exchange chromatography as described previously (15). Mass
spectrometry confirmed the expected molecular mass of purified
proteins within 1 Da (data not shown).
Soluble RNase H Activity Assays--
Radiolabeled RNA·DNA
hybrid was synthesized from M13K07 single-stranded DNA, and RNase H
assays were conducted as described previously (8). RNase H reaction
assays were carried out in standard conditions of 50 mM
Tris, pH 8.0, 1 µM (base pairs) RNA·DNA hybrid, 2.5%
glycerol, 0.1 mg/ml linear polyacrylamide, 1.5 µM bovine
serum albumin with the indicated concentrations of divalent cation
(MnCl2 or MgCl2) and NaCl at 37 °C. The
acid-soluble radioactivity (cleaved product) present in the supernatant
was determined by liquid scintillation counting. Specific activity
(units/mg) was determined from the slope of multiple assay time points
in the linear range of enzymatic activity. One unit of RNase H activity is defined as the amount of enzyme needed to generate 1 µmol of acid-soluble product in 15 min under our reaction conditions.
Crystal Growth--
Crystals of K87A RNase H* were grown by the
hanging-drop vapor diffusion method in 1 mM
MnCl2, 20 mM Hepes, pH 8, 16% PEG-3350 with 6 mg/ml protein. Except for the metal ions, these conditions are similar
to those used previously for the crystallization of wild-type E. coli RNase HI (17) and for RNase H* (Goedken et al.
(38)). Small needle-like crystals unsuitable for x-ray analysis frequently grew under these conditions. Occasionally, larger rod-shaped crystals suitable for x-ray diffraction appeared, growing to a maximum
size of ~600 × ~15 × ~15 µm within 4 weeks.
Crystals were flash-frozen in liquid nitrogen directly from the drop
conditions without the need for additional cryo-protectants.
X-ray Diffraction and Refinement of Model--
X-ray diffraction
data were collected at the Advanced Light Source (Beamline 5.0.2) at
Lawrence Berkeley National Laboratory. Diffraction data were integrated
and scaled using MOSFLM and SCALA (18) under the control of Wedger
Elves (kindly provided by J. Holton).
The K87A RNase H*/Mn2+ co-crystal structure was solved by
molecular replacement via AMoRe (19) using the RNase H* coordinates (Protein Data Bank code 1F21) (38). Models were initially refined using
REFMAC (20) and later by the crystallography and NMR system
(21). Between refinement cycles, manual rebuilding into
2Fo
The coordinates of the RNase H*/Mn2+ structure have been
deposited in the Protein Data Bank (1G15).
Metal-dependent Activation/Attenuation of RNase
H--
Although E. coli RNase HI can use either
Mn2+ or Mg2+ for activity, these divalent
metals lead to very different activity profiles (9, 15, 23). The metal
dependence of RNase H*, the cysteine-free version of E. coli
RNase HI, is shown in Fig. 1b.
(This variant shows metal dependence that is identical to that of the
wild-type enzyme.) In the presence of Mg2+ ions and
moderate ionic strength (50 mM NaCl), the enzyme is activated in the low mM range (0.5-10 mM) and
is repressed by very large concentrations of divalent metal (>10
mM). This activation profile is in contrast to that in the
presence of Mn2+. This ion activates the enzyme at low
metal concentrations over a narrow range (to ~2 µM),
and additional metal inhibits the protein. The activity at 1 mM Mn2+ is ~100-fold decreased from its
maximum at 2 µM Mn2+ (15). Mutations in
residue Asp-134 produce an enzyme that shares the activation profiles
of the wild-type protein but shows a large increase in the
Mn2+ concentrations required to inhibit the enzyme (15).
These observations led to the proposal of the activation/attenuation
model for RNase H (15) in which a metal ion in Site 1 activates
catalysis and the binding of another ion in Site 2 attenuates
activity.
Crystal Structure of RNase H and Two Mn2+ Ions--
To
bolster the activation/attenuation model, we sought structural
identification of the location and liganding geometry of Mn2+ ions at the activating and attenuating sites of
E. coli RNase HI. Cysteine-free E. coli RNase H*
crystallizes in an orthorhombic lattice (38), but our attempts to
identify bound metal ions in these crystal forms either by
co-crystallization or by soaking into existing crystals were
unsuccessful. That is, diffraction data to greater than 1.8-Å
resolution were obtained for a crystal grown in the presence of 5 mM MgCl2 that was isomorphous to the apo-crystals. After molecular replacement with the apo-structure, however, no electron density attributable to metal ions was observed in
the active site (data not shown). Similar results were found for 24-h
soaks of RNase H* crystals in 20 mM MgCl2 or 8 mM MnCl2 (data not shown).
Examination of lattice contacts in these orthorhombic crystals showed
that the positively charged side chain of residue Lys-87 from a
symmetry mate in the lattice protruded into the negatively charged
enzyme-active site. We hypothesized that this crystal contact altered
the electrostatic environment and accessibility of the metal-binding
pocket precluding the entry of divalent cations. In a single enzyme
monomer, Lys-87 is distant from the active site and is unlikely to
influence metal binding. We therefore created a mutant of E. coli RNase H* lacking this residue (K87A) expecting that this
variant would crystallize in the same lattice but allow liganding of
divalent metals. K87A did not form the expected orthorhombic crystals
in the presence or absence of divalent metal even when microseeded with
RNase H* crystals; however, high quality hexagonal crystals did appear
in conditions containing 1 mM MnCl2 at pH 8.
The K87A hexagonal crystals diffract to 1.9 Å at a synchotron source
and belong to the space group P63, having one protein molecule per asymmetric unit. We solved the structure of K87A RNase H*
using the molecular replacement method with the metal-free RNase H*
structure as the probe molecule. Data collection
and refinement statistics are summarized
in Table I. Overall the structure of
K87A (Fig. 2) is highly similar to the
RNase H* structure; comparison of the C-
After molecular replacement, two difference density peaks lying 4.0 Å apart were easily identified in the electron density. Because they
displayed canonical octahedral liganding geometry and no other divalent
metals were present in the crystallization conditions, these peaks can
be clearly identified as Mn2+ ions. Representative
2Fo
Both metal ions have octahedral coordination with positions occupied
either by protein atoms or solvent molecules. The first Mn2+ ion (Mn2+ 1) makes inner sphere
(nonwater-mediated) contacts to the carboxylate oxygens of Asp-10,
Glu-48, and Asp-70. The three remaining coordination sites on the ion
are occupied by water molecules, one of which (Water 2) appears to
interact with the other carboxylate oxygen of Asp-70. The second
Mn2+ ion (Mn2+ 2) is coordinated by inner
sphere contacts to Asp-10 and Asp-134 of the same molecule; an inner
sphere contact to Glu-131 from an adjacent symmetry mate is also
observed. Water molecules occupy the remaining coordination sites of
the Mn2+ 2, one of which, Water 2, is shared between the
two metal ions. Both metals are therefore "bridged" by having
contacts both to Asp-10 and Water 2 suggesting that both metals can
bind simultaneously. The water molecules making coordination contacts
to the divalent metal ions may be replaced by contacts from the nucleic
acid substrate during the RNase H reaction.
Comparison to Other Metals in RNase H Active Pocket--
How do
these metal-binding sites compare with those previously observed? Fig.
3 shows a comparison of crystallographic
metal binding studies in RNases H. The position of the Mn2+
ions and the conformation of liganding homologous residues in the HIV
RNase H domain from reverse transcriptase (observed at 2.4 Å resolution) (6) are similar to that observed for K87A E. coli RNase HI in the presence of Mn2+. In contrast,
the conformation of the active site residues Asp-10 and Glu-48 in the
Mg2+ co-crystal with E. coli RNase HI (observed
at 2.8 Å resolution (5)) is different from what we observed in our
1.9-Å structure with Mn2+ ions. In particular, the single
Mg2+ ion is displaced away from Asp-70 such that it can no
longer make direct contact with the metal ion as observed for
Mn2+ 1 in our structure. The difference in precise
positioning of the ion in Site 1 is interesting because solution
studies on both E. coli RNase HI (25) and Moloney murine
leukemia virus RNase H (26) suggest that Asp-70 (or its homologue) is
more important for metal affinity than is Glu-48. Given the variable
position of metal ions observed crystallographically, it is conceivable that in solution metals can adopt several positions in the active site.
This may prove relevant for the enzymatic mechanism as first suggested
by Kanaya et al. (25). Alternatively, the presence of a
metal at Site 2 may induce conformational changes in the Site 1 causing
movement of the ion toward Asp-70.
RNase H Metal Dependence as a Function of Ionic
Strength--
Although x-ray crystallography demonstrates that a
Mn2+ ion can bind at Site 2, proving that this metal is
inhibitory as predicted by the activation/attenuation model is more
difficult. Examination of the metal-dependent activity of
RNase H* in the presence of Mn2+ or Mg2+ ions
as a function of ionic strength, however, has proved informative (see
Fig. 4a). In particular, we
noted that as NaCl concentrations were increased, the catalytic
behavior of RNase H in the presence of 1 mM
MnCl2 was very different from that in 1 mM
MgCl2. The enzyme was strongly inhibited by increasing
ionic strength in the presence of fixed amounts of Mg2+
(where based upon crystallographic data we expect the involvement of
one ion). In contrast, in the presence of Mn2+ (where we
expect two ions to be bound by RNase H), activity levels increased as
NaCl concentrations rose.
This was a surprising result; a priori we had expected that
NaCl would inhibit activity in the presence of either metal due to
shielding of electrostatic effects that contribute to the affinity of
the enzyme for its substrate and/or for its cofactor. Nonetheless, under conditions where the enzyme is attenuated by high
Mn2+ concentrations, this attenuation appears to be partly
relieved by increased ionic strength. Furthermore, it seems unlikely
that the conformation of the RNA·DNA hybrid was altered by additional NaCl making it less susceptible to RNase H degradation because NaCl has
the opposite effect (inhibition) in the presence of Mg2+.
We subsequently found that addition of NaCl did indeed repress activity
at other lower Mn2+ (0.5 and 2 µM)
concentrations (Fig. 4b). Hence, under metal concentrations where we expect at most one ion to be bound (the activation regime in
Fig. 1a), we detected strict inhibition by increasing NaCl concentrations. At 20 µM Mn2+ where the
attenuating site becomes populated at low ionic strength (10-50
mM NaCl), we observed a maximum activity at intermediate (200 mM) concentrations of NaCl, followed by a decrease of
activity at higher concentrations of salt.
Fig. 4c shows the Mn2+ dependence of RNase H
activity at a series of different NaCl concentrations. This shows a
displacement to higher divalent metal concentration on both the
activation and attenuation regimes as the NaCl concentration increases.
Furthermore, the maximum activity of the enzyme was also decreased at
high ionic strengths, probably because of electrostatic shielding of RNase H from nucleic acid. We believe these data indicate that NaCl
nonspecifically competes for the same sites as the Mn2+
ions and thereby reduces the apparent binding affinity of each divalent
metal. The unexpected increases in activity as the NaCl concentrations
increase in 1 mM Mn2+ samples likely stem from
the reduction in affinity for the second (attenuating) Mn2+
ion. In this case, then, the overall effect of increased ionic strength
was a loss of a Mn2+ ion in Site 2. The removal of the
metal ion in Site 2 is expected to relieve the attenuation and overcome
the decreased affinity for the substrate to give rise to a net increase
in activity. Consistent with this idea, in the presence of
Mg2+ concentrations from 0.1 to 50 mM (where we
expect a single metal in Site 1), we found only inhibition as NaCl
concentrations are increased (data not shown).
RNase H catalyzes the disruption of the phosphodiester backbone of
the RNA portion of RNA·DNA heteroduplexes in a divalent cation-dependent manner. This reaction requires a concerted
deprotonation of water to form a nucleophilic hydroxide to attack the
scissile phosphate of the RNA. Several different conserved active-site residues have been proposed to be the nucleophile-producing general base for this reaction including Glu-48, Asp-70, and His-124 (5, 13,
25-28), but it remains an open question which (if any) serve in this role.
RNase H is believed to require metal cofactors because divalent cations
stabilize the negatively charged pentacovalent phosphorane intermediate
of the nuclease reaction. The stoichiometry of metal ions to the RNase
H active site has been evaluated by a number of methods. X-ray
crystallography (5, 29) as well as a large battery of solution
techniques (30-32) have detected only a single Mg2+
ion-binding site in E. coli RNase HI. In contrast, two
Mn2+ ions were observed bound to the isolated HIV RNase H
domain, and stoichiometries of one Mg2+ ion and two
Mn2+ ions binding at the RNase H active site in full-length
HIV-reverse transcriptase have been determined by calorimetric studies
(33). Based upon these data and the metal dependence of RNase H
activity (Fig. 1b), our laboratory proposed an
"activation/attenuation model." This model posits that the first
metal to bind RNase H is required for activity and occupies Site 1 (formed by the side chains of Asp-10, Glu-48, and Asp-70). A second
metal with weaker affinity can occupy Site 2 (composed of the side
chains of Asp-134 and possibly Asp-10 and His-124). This inhibitory
metal accounts for the reduction of activity to ~1% of maximal value
at higher Mn2+ concentrations.
By using x-ray crystallography, we have demonstrated that a
Mn2+ ion can bind to Site 2. Mutagenesis of Asp-134 to less
polar residues His and Asn results in an active enzyme with a decreased attenuation at high Mn2+ concentrations (15). We observed
that Asp-134 (along with Asp-10) makes direct inner sphere contacts to
the second Mn2+ ion. Those results together with the
observation that increasing ionic strength unexpectedly relieves the
inhibition of RNase H at high Mn2+ concentration (Fig. 4)
strongly suggest that the ion observed in Site 2 is inhibitory.
E. coli RNase HI requires very different concentrations of
Mg2+ ions than Mn2+ ions for optimal activity
(Fig. 1b). The enzyme shows a greater maximum activity in
the presence of Mg2+ than in the presence of
Mn2+ ions. Whether the inhibition observed at very high
Mg2+ concentration (Fig. 1b) results from weak
binding by Mg2+ at Site 2 is unclear. However, given the
large ionic strength of the reaction conditions of those samples, it
seems equally likely that a reduction in the electrostatic affinity for
the nucleic acid substrate gives rise to this inhibition. Although it
is difficult to evaluate the free concentrations of divalent metal ions
in vivo, it should be noted that the concentration at which
Mg2+ activates the enzyme (0.5-5 mM)
approximates physiological values (34, 35). Mn2+ ions,
which are found at much smaller concentrations in vivo, bind
the enzyme much more tightly (apparent Kd ~1
µM), and Mn2+ can inhibit RNase H activity in
the presence of Mg2+ (15). Therefore, we speculate that
both ions may be relevant for the physiological regulation of RNase H activity.
Notably, the attenuation of E. coli RNase HI via second
metal binding does not eliminate all activity. Mutation in the
conserved active-site histidine (His-124) eliminates attenuation at
high Mn2+ concentrations but allows maximal activity that
reaches only the attenuated rates of the wild-type protein (15). This
led to the suggestion that attenuation at Site 2 occurs by removing the
action of His-124. Two alternative ways metal binding at Site 2 could
neutralize the histidine (presumably by eliminating a role in acid/base
catalysis) were proposed: 1) that His-124 was directly involved in
liganding the second metal-binding site or 2) that binding of a metal
ion at Site 2 hinders activity because the His-containing loop is
flipped out away from the active site. This loop has been seen
in separate conformations in the apo-form (4, 36), one with the
histidine side chain near the active site as in Fig. 1a and
the other where it is displaced away from the catalytic pocket. As the
entire His-containing loop is disordered in the Mn2+
co-crystal, it is unlikely that His-124 is directly involved in the
liganding of the Site 2 metal. Thus, in light of the x-ray structure,
the second possibility that His-124 is neutralized by second metal
binding via increased flexibility of the His-containing loop seems more reasonable.
The RNase H/Mn2+ co-crystal structure presented here
provides direct structural evidence that E. coli RNase HI
can bind two metal ions in the absence of substrate. Numerous
conflicting proposals have been made regarding the number of metal ions
necessary for activity and the precise catalytic mechanism of
ribonuclease H (5, 6, 13, 15, 25, 28, 37), but a consensus is developing that a single metal is required for activity. It is unclear
which co-crystal (Mg2+ or Mn2+) most closely
approximates the position of the metal in Site 1 during catalysis on a
nucleic acid. Our x-ray data indicate that all of the conserved
carboxylates in the active site of RNase H can make direct contacts to
divalent metals. Whether RNase H uses any of these side chains as a
general base to deprotonate solvent molecule remains an open question.
To evaluate which residues (if any) are the general base and acid of
the nuclease reaction and how the RNA strand of RNA·DNA hybrids is
recognized, a co-crystal of RNase H bound to substrate and/or substrate
analogs will likely be required.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Fc and
Fo
Fc maps was performed using
O (22). Immediately after molecular replacement, difference
density attributable to two octahedrally coordinated Mn2+
ions was observed in the enzyme-active site. Refinement statistics are
summarized in Table I.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Activation/attenuation model.
a, conserved active site residues and position of divalent
metal ions are drawn schematically based upon the E. coli
and HIV RNase H crystal structures (4, 6, 36). The location of the
lower metal ion (Site 1) is based upon the Mg2+
in the E. coli structure and the Mn2+ in the HIV
structure, and that of the upper metal ion (Site 2) is based
upon the second Mn2+ observed in HIV RNase H. b,
activity of E. coli RNase H* as a function of varying
concentrations of divalent metal in the presence of MnCl2
(closed circles) or MgCl2 (open
squares). Assays were performed in 50 mM Tris, pH 8.0, 50 mM NaCl, 1.5 µM bovine serum albumin, 1 µM (base pairs) RNA·DNA hybrid at 37 °C with the
indicated divalent cation concentrations.
coordinates (24) of K87A to
structures determined previously gave a root mean square deviation of
0.8 Å to the cysteine-free RNase H* and 1.0 Å to the wild-type
protein. In addition to disordered N and C termini (residues
1-2 and 153-155), two loops, those between helices C and D (residues
92-96) and the loop between strand V and helix E containing a
conserved histidine (residues 122-125, the "His-containing loop"),
were disordered in the crystal.
RNase H/Mn2+ crystallographic statistics
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Fig. 2.
Co-crystal structure of
Mn2+ and K87A RNase H*. Mn2+ ions are
shown in purple with oxygens and metal-coordinated waters in
red. Left, ribbon depiction of crystal structure
highlighting residues liganding metal ions. The dashed lines
indicate two loops (residues 92-96 and residues 122-125) that show no
electron density. Image prepared using INSIGHTII (Biosym Inc.).
Right, electron density around the active site of RNase H. A
2Fo Fc map contoured at
1.3
is shown. Image prepared using O. Water 2 which
makes contacts with Asp-70 and both Mn2+ ions as noted in
the text is noted with an asterisk.
Fc maps calculated using
the phases from the final model are shown in Fig. 2. At full occupancy
both ions refined to low temperature factors (~15 Å2)
similar to the surrounding residues, suggesting these binding sites are
fully occupied and that the data are well accounted for by the
Mn2+ ions in the model. In comparison to the metal-free
structure (Protein Data Bank accession code 1F21) (38), there are
notable changes in the geometry of the active site. The side chain of Glu-48 flips dramatically toward the carboxylate pocket, and His-124 is
no longer visible in the structure. The positions of the side chains
Asp-10, Asp-70, and Asp-134, however, change very little in the
co-crystal structure.
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Fig. 3.
Comparison of metal ion coordination in
RNases H. E. coli RNase HI was crystallized in the
presence of greater than 100 mM Mg2+ (5)
(Protein Data Bank code 1RDD); K87A E. coli RNase H* was
crystallized in the presence of 1 mM Mn2+ (this
study, Protein Data Bank code 1G15), and the isolated RNase H domain of
HIV-reverse transcriptase was soaked with 45 mM
Mn2+ (6) (Protein Data Bank code 1HRH). Image prepared
using INSIGHT (Biosym Inc.).
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Fig. 4.
Salt dependence of the activity of E. coli RNase H*. Buffer conditions are 50 mM
Tris, pH 8.0, 50 mM NaCl, 1.5 µM bovine serum
albumin, 1 µM (base pairs) RNA·DNA hybrid at 37 °C.
a, dependence of NaCl upon activity in 1 mM
divalent cation. MgCl2 (closed circles) or
MnCl2 (open squares). b, dependence
of NaCl activity in various concentration of MnCl2: 1 mM (closed circles), 20 µM
(open squares), 2 µM (closed
diamonds), 0.5 µM (open triangles).
c, dependence of MnCl2 of activity in various
concentration of NaCl: 50 mM (closed circles),
150 mM (open squares), 350 mM
(open diamonds), 500 mM (closed
triangles).
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank James Keck for assistance with x-ray crystallography and helpful discussions and the critical reading of this manuscript. We thank James Holton and Prof. James Berger for assistance with x-ray crystallography and the critical reading of this manuscript and David King for mass spectrometry.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant 50945.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1G15) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence should be addressed: Dept. of Molecular and
Cell Biology, University of California, 229 Stanley Hall, Berkeley, CA
94720. Tel.: 510-642-7678; Fax: 510-643-9290; E-mail: marqusee@zebra.berkeley.edu.
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M009626200
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ABBREVIATIONS |
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The abbreviation used is: HIV, human immunodeficiency virus.
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REFERENCES |
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