THE,JOURNAL OF BIOLOGICAL
CHEMISTRY
1992 by T h e American Society for Biochemistry and Molecular Biology, Inc
Vol. 267, No. 4 , Issue of February 5, pp. 2429-2436, 1992
Prmted rn U S A
IC
Influence of Metal Ions on the Ribonuclease P Reaction
DISTINGUISHINGSUBSTRATEBINDINGFROM
CATALYSIS*
(Received for publication, April 24, 1991)
Drew Smith$, AlexB. Burgin, Elizabeth S. Haas, and Norman R.Pace$
From the DeDartment of Biolom and Institute for Molecular and Cellular Biology, Indiana University,
Bloomington; Indiana 47405
”
A high yield, photoactivated cross-linking reaction
between a modified tRNA and RNase P RNA was used
as a quantitative assay of substrate binding affinity.
The cross-linking assay allowsthe effects of metal ions
on substrate binding to bemeasured independently and
in the absence of the pre-tRNA cleavage reaction. The
results of this assay, in conjunction with the conventional cleavage assay, support the following conclusions about the nature of the RNase P RNA-tRNA
binding interaction. (i) Monovalent cations actprimarily to enhance enzyme-substrate binding, presumably
by functioning as counterions. This enhancement can
be attributed to a reduction in the tRNA off-rate. (ii)
Although divalent cation is required for cleavage, the
enzyme-substrate complex can form in the absence of
divalent cation; the essential role of divalent cation in
the reaction is thus catalytic. (iii)Caz+is as efficient as
Mg2+in promoting binding but supports catalysis only
at a low rate.
of cleavage. Since quantitative changes in elementary rate
constants can lead to qualitative changes in substratespecificity (4, 26), reports of altered cleavage site specificity (5-9)
also cannoteasily be interpreted.
To circumvent these problems, we studied the RNase P
reaction using an assay for binding of substrate that is independent of the cleavage reaction. This assay is based on a
high efficiency cross-linking reaction (10) in which an azidophenacyl-derivatized tRNA is mixed with RNase P RNA and
is then covalently conjugated to RNase P RNA by illumination with 302 nm light. In conditions of enzyme excess up to
30% of the azidophenacyl-tRNA is conjugated to RNase P
RNA. The azidophenacyl group is located uniquely at the 5’
phosphate of mature tRNA. This phosphateis the productof
theRNase P phosphodiester cleavage reaction and so is
expected to occupy the enzyme active site. The sitesof crosslinking to RNase P RNA are within the conserved core (11)
of the enzyme and map toequivalent regions of the secondary
structures of RNase P RNAs from three different eubacteria
(10).
We provide evidence that pre-tRNA, mature tRNA, and
RNase P removes 5’ leadersequences
fromprecursor
P RNA
azidophenacyl-tRNA occupy the same sites on RNase
tRNAs by endonucleolytic cleavage to form the mature5’ end and show that comparable binding constants are obtainedin
of tRNA (1,2). In eubacteria thisenzyme is a ribonucleopro- both the cleavage and cross-linking reactions. These results
tein composed of a small (about 14 kDa) protein and a large supportthe use of cross-linkingextentas
a quantitative
(about 130 kDa) RNA. The RNA is the catalytic subunit and measure of substrate binding. We then compare theeffects of
performsthe cleavage reactionaccurately in vitro inthe
metal ion type and concentration on substrate binding (as
absence of the protein. The nature of substrate recognition measured by cross-linking extent) andcleavage rate andevalby this RNA catalyst is of particular interest because (i) it uate ionic contributions to the RNase P RNA-tRNA interbinds andcleaves free substratewhereas other RNA catalysts action.
undergo self-cleavage in vivo, and (ii) thesequence heterogeneity of precursor tRNAs largely precludes substrate recogEXPERIMENTALPROCEDURES
nition by simple base pairingrules.
RNAs-RNAs were produced by in uitro run-off transcription using
Attempts at understanding substrate binding by RNase P
SP6 (New England Biolabs) or T7 (B. Pace, Indiana
RNA have been limited by the nature of the assayemployed, bacteriophage
University) RNA polymerase under the conditions described (12).
namely, the rate or extent of cleavage of precursor tRNA.
RNAs were separated by electrophoresis in polyacrylamide, 7M urea,
Because product release can be the rate-limiting step of this TBE (90 mM Tris borate, pH 8.0, 9 mM EDTA) gels, visualized by
UV shadowing, cut out from the gel, and then eluted from crushed
reaction (3), the effects of environmental factors (temperature, pH,ionic strength) or structural alterations
of either the gel fragments. Uniform labeling was accomplished by the inclusion
enzyme or substrate often cannot
be unambiguously inter- of 50-100 pCi of [a-”PIGTP (3,000 Ci/mmol, from Amersham Corp.)
in the transcriptionreactions. RNase P RNAs were transcribed from
preted. For example, a reduction in cleavage rate may, with plasmids
DW66 (Bacillus subtilis), AB12 (Chromatium uinosum), and
equal plausibility,be attributed to weaker binding of the DW98 (Escherichia coli); substrates were transcribed from plasmids
substrate, tighter binding of the product, or slower catalysis DW152 (B. subtilispre-tRNAASP),67CFO ( E .coli tRNAPhe), and
67YFO
* The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
$ Recipient of postdoctoral fellowships from the Indiana Institute
for Molecular and Cellular Biology and National Institutes of Health
Grant GM13712.
5 Recipient of National Institutes of Health Research Grant
GM34527. To whom correspondence should be addressed. Tel.: 812855-6152: Fax: 812-855-6705.
(Saccharomyces cereuiseae tRNAPhe),which have been described (10).
RNA concentrations were determined by specific activities of labeled
RNAs or by absorbance at 260 nm.
Preparation of Azidophenucyl-tRNA-As detailed previously (lo),
transcription of the tRNA template was carried out in the presence
of a-thio GMP,which is incorporated only at the5’ nucleotide of the
transcript. This tRNA transcript was reacted with 4-azidophenacylbromide (Fluka), thereby attaching 4-azidobenzoate uniquely at the
5’ thiophosphate. Unless otherwise noted, the E. coli tRNAPhe transcript was used.
Reactions-Cross-linking and cleavage reactions were performed
2429
Ions and Substrate Bindingby RNase P
2430
Cross-linking Cleavage
PRNA 0 +
pre-tRNAmat-tRNA
-
P
RNA
C. vin.. E.
None
-. mfi
-..
. .. .
NH4CI O + + 0 + + 0 + + 0
M g C l z O O + O O + O O + O
PRNAtRNA
=&
"_
"."_.
t R N A : f R N A & & - G e
..
%
.
5' leader
FIG. 1. Cleavage and cross-linking assays. The left panel
shows an autoradiogram of a polyacrylamide gel used to separate the
products (mature tRNA,5' leader) from the substrate(pre-tRNA) of
the RNase P cleavage reaction (see "Experimental Procedures"). The
right panel is an autoradiogram of a polyacrylamide gel used to
separate the products of the cross-linking reaction (see "Experimental
Procedures") and demonstrates the cation dependentof cross-linking.
C.vinosum or E. coli RNase P RNA, when present (+),was at40 nM;
azidophenacyl-tRNA was a t 40 nM. NH4CI,when present (+), was at
1.0 M; MgCI2, when present (+), was a t 25 mM. The sample in the
right-most lane was not irradiated with UV light. The upper group of
bands are RNase P RNA.tRNA conjugates. The formation of these
conjugates depends on the presence of RNase P RNA and cation.
The middle set of bands are uncharacterized tRNA.RNA conjugates
whose formation does not depend on RNase P RNA or cation. At the
bottom is unconjugated azidophenacyl-tRNA. Just below this band is
a band of intramolecularly cross-linked tRNA.
typically less than 0.002 of the total incross-linking experiments and
less than 0.01 of the total in cleavage experiments. These control
values were subtracted from the experimental values to determine
the extent of conjugation or cleavage. Errors reported are the standard
error of the mean.
For studies employing Ca2+extra care was taken to avoid contamination with Mg" or other metals. The CaC12(Aldrich) used contains
< 1 ppm of M e . Adventitious metals were removed from buffers
and stock solutions by extraction with diphenylthiocarbazone (13).
To ensure that Mg2' was not carried over from transcription reactions, RNAs were purified on denaturing polyacrylamide gels in TBE
buffer and subsequently maintained in M$+-free buffers in the presenceofEDTA.
Oxidation of manganese was avoidedby storage of MnC12solutions
in pH 6 buffer a t -20 "C and thawing just prior to use. The hydrolytic
activity of Mn2+was monitored by degradation of substrate. Incubation times were minimized to avoid spontaneous RNA hydrolysis.
This hydrolysis was monitored by the degradation of tRNA, which is
well known to have tight divalent cation binding sites and so should
serve as an adequate internal control.
Cross-linking sites on RNase P RNA were determined by primer
extension analysis of purified tRNA.RNase P RNA conjugates, as
described (10).
RESULTS
Cross-linking as an Assayof Binding
Mature tRNA, Pre-tRNA, and Azidophenacyl-tRNA Compete for the Same Site in the Cleavage Reaction-Our assay
for the binding of tRNA to RNase P RNA is the extent of
in a standard reaction buffer: 1.0 M NH4CI, 25 mMMgC;CL, 50 mM cross-linking of azidophenacyl-tRNA to RNase P RNA. We
Tris-HCl or HEPES'/NaOH, pH 8.0 (37T ) , 0.1% sodium dodecyl first show that mature tRNA and itsderivative azidophenasulfate, 0.05% Nonidet P-40,with additions or alterations as noted. cyl-tRNA bind to the same site as pre-tRNA and that preThe titration of HEPES buffer contributed less than 10 mM Na+ to tRNA and mature tRNAhave comparable apparent binding
the final reaction mixture. Cross-linking reactions typically contained constants as assayed in both the cross-linking and cleavage
40-100 nM derivatized tRNA and a 0.5-10-fold molar excess of RNase reactions.
P RNA. All components of the reactions were mixed in a microcenFig. 2a shows that mature tRNAis a competitive inhibitor
trifuge tube and allowed to equilibrate in the dark for 2-5 min. Open
tubes were exposed to 302 nm light at room temperature for 15 min. of the RNaseP RNA cleavage reaction, withK, = 0.4-0.8 K,,,
A polystyrene Petri dish lid was usedas afilter to screen wavelengths of pre-tRNA (Table I, compare line 5 with lines 3 and 4). As
< 300 nm. Because the half-life of the cross-linking reaction is about expected for a purely competitiveinhibitor, the correlationof
1 min (data not shown), the data reported from these experiments
K,,,' (the apparent Michaelis constant) with [I] is excellent,
are extents rather thanrates. Cross-linking extent as well as the K, whereas the correlation
between Vmax'(the apparentmaximal
of the cleavage reaction (data not shown) change little over a temreaction
velocity)
and
[I]
is not significant. These data indiperature range of 24-37 "C, so it is valid to compare the cross-linking
site
pre-tRNA.
extent a t room temperature with binding parameters obtained in cate that mature tRNA binds the same as
Fig. 2b shows that thecross-linking substrate, azidophena37 "C cleavage reactions. Cross-linking extent was not reduced after
longer preincubations in the presence of excess enzyme, indicating cyl-tRNA, is also a competitive inhibitor of the cleavage
that RNase P RNA does not degrade the cross-linking substrate.
reaction, albeit a weaker onethan underivatized mature
Kinetic analyses of the cleavage reaction were performed in the t R N A Kiis 12-fold greater than the Kiof mature tRNA
reaction buffer described above, a t 37 "C, with "P-labeled pre-tRNA
substrate present at 5-250 nM and C. vinosum or E. coli RNase P (Table I, compare line 6 with line 5 ) . We presume that the
RNA a t 0.4-25 nM. The substrate used was an in vitro transcript of addition of the azidophenacyl group or the substitution of
B. subtilis pre-tRNAA"p,and the mature tRNA used as inhibitor was sulfurfor oxygen onthe 5' phosphate of tRNAhinders
E. coli tRNAPhe(Boehringer Mannheim). Enzymes and substrates binding, causing a loss of binding energy roughly equivalent
were equilibrated separately in the reaction buffer at the reaction to one hydrogen bond (15). Azidophenacyl-tRNA has a weak
temperature for 2-5 min, and reactions were started by mixing effect on V,,,', perhaps because of adventitious cross-linking
enzyme with substrate. Unless otherwise noted, velocities represent
initial rates a t steady-state conditions, with substrate in excess of in ambient light, which inactivates theenzyme (10).
Mature tRNA, Pre-tRNA, and Azidophemcyl-tRNA Comenzyme and thefraction of substrate cleaved < 0.2.Multiple turnover
reactions were quenched in 2.5 volumes of ice-cold ethanol. This pete for the Same Site in the Cross-linking Reaction-If the
method of quenching proved too slow for time points < 45 s; accord- cross-linking reaction reflects the bindingof ligands to RNase
ingly, single turnover reactions were quenched in 5 volumes of 10 M P RNA accurately then it should be competitively inhibited
urea, 10 mM EDTA a t 60 "C, whichprovides linear time courses down by pre-tRNA andby underivatized mature tRNA. Inhibition
to 10 s.
Reaction mixes were electrophoresed in a 5% (for cross-linking of cross-linking by mature tRNA and pre-tRNA isshown in
reactions) or 8% (for cleavage reactions) polyacrylamide, 7 M urea, Fig. 3. The data are presented as a Dixon plot (that is, the
TBE gel to resolve tRNA-RNase P RNA conjugates from unconju- fraction conjugated is taken as velocity and its reciprocal
gated tRNA or pre-tRNA from maturetRNA and the 5' leader plotted asa function of inhibitor concentration) and fitted to
sequence. Autoradiograms of the fixed and dried gels (for example, a least squares lineby linear regression. The fit of the line is
appropriate RNA bands, excellent ( r > 0.995 for allplots), indicating that
Fig. 1)were used as templates to cut out the
cross-linking
and the relative amount of RNA in each band was determined by
extent
correlates
well
with
the
steady-state
concentration
of
Cerenkov scintillation. Background levels in negative controls were
free enzyme binding sites. The substrate of the cross-linking
The abbreviation used is: HEPES, 4-(2-hydroxyethyl)-l-pipera- reaction (azidophenacyl-tRNA) is a t subsaturating concentrazineethanesulfonic acid.
tions, and so Kivalues for pre-tRNA and mature tRNA are
Ions and Substrate Binding byRNase P
LUU
eK”
TABLEI
Binding and kinetic constants
APA-tRNA,azidophenacyl-tRNA.Where the substrate is pretRNA, data are the results of cleavage assays; wherethe substrate is
azidophenacyl-tRNA, data refer to the results of cross-linking assays.
In line 1, data are from (3). Lines 2, 7, and 8 are from Fig. 3. Lines 3
and 9 were determined froma series of cleavage reactionsat standard
conditions (see “Experimental Procedures”) with 25 mM MgCl,, 0.5
nM RNase P RNA, and 5-150 nM pre-tRNAPhe
(not shown). In line
4, data were averaged from cleavageinhibition plots for tRNAPh‘ (Fig.
2, line 5 ) azidophenacyl-tRNA (Fig. 2, line 6 ) , and tRNAPhewith an
azidophenacyl group attached to s4U8 (not shown). Line 10 is from
(25). Line 11 was determined from a series of cleavage reactions at
standard conditions with 25 mM MgCl,, 1.0 M NaC1, 0.5 nM RNase
P RNA,10-600 nM pre-tRNAASP
(data notshown).Line 12 was
determined from a series of cleavage reactions at standard conditions
with 0 mM MgC12,50 mM CaC12,1 nM RNase P RNA, 5-150 nM pretRNAAnp
(data not shown).
RNase P RNA
Substrate
Inhibitor
Km Kt
kcat
a
0 1Nmar
2431
c 150.
nM
0
50
100
150
200
I
250
[azldophenacyl-tRNA], nM
FIG. 2. Product inhibition of the RNase P RNA cleavage
reaction. The apparent values for K, (K,,,’) or 1/Vmax(1/Vmax’)
are
plotted against inhibitor concentration. Each value is from a determination of these steady-state parameters at the indicated inhibitor
concentration (not shown).Reactionconditionswere:0.4
nM C.
vinosum RNase P RNA, 10-120 nM pre-tRNA in six increments, in
the standard reaction buffer, at 37 “C,for 30-75 min time, increasing
as the inhibitor concentrationwas increased. Panel a,the correlation
coefficient of the least squares line is 0.99, p < 0.001. Panel b, the
correlation coefficient for the least squares line for the plot of K,’
versus [azidophenacyl-tRNA]is 0.92, 0.1 > p > 0.01. The correlation
coefficient for Vmax’versus [azidophenacyl-tRNA]is0.62, p > 0.2.
Reaction conditions were: 0.4 nM C. vinosum RNase P RNA, 10-120
nM pre-tRNA in six increments, inthe standard reaction buffer (see
“ExperimentalProcedures”),at 37 “C, for 30-75 min time, increasing
as the inhibitor concentration was increased. Cleavage extents were
from 0.03 to 0.15.
approximated by the negative of the x intercept (K:app = K,
(1 [SI/&), where azidophenacyl-tRNA is the substrate[SI
and Ks is its equilibrium binding constant).Because cleavage
of pre-tRNA is expected under the cross-linking conditions,
K, pre.tRNA is equivalent to K,,, in the cleavage assay. We find
that the constants from both types of assays are in good
quantitative agreement for all combinations of tRNAs and
RNase P RNAs (Table I, compare line 1 with line 2; lines 3
and 4 with line 7; line 5 with line 8). We conclude from these
experiments that the cross-linking reaction
a suitable
is
quantitative assay for the binding of tRNA ligands to RNase P
RNA.
+
Monovalent Cations in Binding andCleavage
nM
rnin“
Pre-tRNAAsp None0.6
200
1 B. subtilis
APA-tRNA
2 B. subtilis
Pre-tRNAAnp
170
Pre-tRNAPhe None
42
0.9
3 C. vinosum
Pre-tRNAASPNone0.39
20
4 C. uinosum
16
Pre-tRNAAaP tRNAPhe
5 C. uinosum
190
Pre-tRNAASPAPA-tRNA
6 C. uinosum
34
Pre-tRNAAsp
APA-tRNA
7 C. vinosum
50
APA-tRNA tRNAPh“
8 C. vinosum
Pre-tRNAPhe None
9 E. coli
48
0.4
Pre-tRNAAsp None
40
0.4
10 E. coli
130
1.7
11 E. coli (Na’) Pre-tRNAAsp None
1 2 E. coli (Ca2+) Pre-tRNAAsp None
24
0.05
0
0.1
0.3
0.2
04
0.5
[pre- or mature-tRNA], pM
FIG. 3. Inhibition of cross-linking. Cross-linkingreactions
were performed in the presence of increasing concentrations of underivatizedpre- ormaturetRNA
at a constant concentration of
RNase P RNA and azidophenacyl-tRNA. Reaction conditions were:
100 nM C. vinosum (Cv)RNase P RNA, 40 nM azidophenacyl-tRNA,
or 100 nM B. subtilis (Bs)RNase P RNA, 100 nM azidophenacyltRNA, with pre-tRNAAsp or mature
tRNAPheat the indicated concentrations, under standard cross-linking conditions (see “Experimental
Procedures”). Apparent K, values (K,’) were determined as the negative of the x intercept of the least squares lines.
rate. For the minimalreaction scheme
Effects of Cation Concentration-Fig. 4 shows the effects of
monovalent cation type and concentration on substrate binding (a) andonpre-tRNAsteady-state
cleavage rates ( b ) .
Several patterns are apparent
by inspection. First, both bind- the rateof cleavage a t subsaturating substrate concentrations
ing and cleavage generally increase with ionic strength. This is proportionalto k l k 2 / ( k - 1 k,) (we assume,here
and
is the behavior expected if monovalent cations act as coun- throughout, that the reaction is essentially irreversible; that
terions toreduce electrostatic repulsion between enzyme and is, k-2 is negligible). In contrast, cross-linking extent should
substrate RNAs(3). Second, an increase in monovalent cationreflect KD= k - , / k l . Consequently, although both binding and
concentration stimulates binding much more than it stimu- cleavage will respond proportionately to changes in k l , bindlates cleavage rate: cross-linking extent generally increases
ing, but not cleavage (unless k-l >> k2), will respond propor10-15-fold as monovalent cation concentration
increases from tionately to k-l. The response of binding and cleavage to
0 to 1.0 M whereas the increase in cleavage rate is only 2-4- monovalent salt concentration can thereforebe attributed to
fold over the same range. This result can be explained if the tRNA off-rate. Third, a significant amount of binding and
of addedmonovalent
primary effect of salt concentration is on the substrate off- cleavage is detectedintheabsence
+
Ions and Substrate Binding by RNase P
2432
0.04
a
0
0.1M
RbCl CsCl
TMACl
LiCl
NaCl
KC1
NH4CI
I
-
LiCl
NaCl
KC1
NH4CI
RbCl
CsCl
TMACl
Increasing radius
FIG.4. Effects of monovalent cation on binding and cleavage. Cross-linkingandcleavagereactions wereperformed in the
presence of increasing concentrations of different monovalent cations. Cations are arranged in order of increasing nonhydrated ionic
radius from left to right. Cross-linking conditionswere 50 nM E. coli
RNase P RNA, 50 nM azidophenacyl-tRNA, standard cross-linking
buffer (see "Experimental Procedures"), and monovalent cation as
indicated, at 24 "C.Cleavagereaction conditions were: 10 nM pre0.5 nM E. coli RNase P RNA, standard cleavage buffer
(see
tRNAABp,
"Experimental Procedures") and monovalent cation as indicated, at
37 "C,.
for 55 min. These conditions are subsaturating with respectto
substratefor 1.0 M NH&l (Table I); weassume that K,,, does not
decrease significantly under the other ionic conditions.
This assumption is consistent with the effect of the other ionic conditions on
cross-link extent. TMA, tetramethyl ammonium; nd, no data.
actions. These ions have similar ionicradii' (1.3-1.7 A),
suggesting that this property, or other properties dependent
upon it, is an important factor in ion selectivity (16). Ions of
a radius outside this range do not show so simple a correlation
between binding and cleavage. Li', the smallest ion tested,
promotes binding reasonably well but actually reduces the
cleavage rate below that found for no monovalent cation. The
ionic radius of Li+ is comparable to that ofMg2+ (0.60 and
0.65 A,respectively), suggestingthe possibility that Li' is able
to compete with Mg2f for occupation of a catalytically important site and so reduce the cleavage rate. Na' (radius = 0.95
A) is 3-fold less effective than NH: in promoting binding at
1.0 M concentration but supports a cleavage rate about onethird faster. These properties are also observed when the
steady-state parameters for cleavage in Na' and NH: are
compared (Table I, lines 10 and 11):K , in Na' is 3-fold higher
than in NH:, but kc.&, is one-third greater in Na' than in
NH: (1.3 X lo7 M" min" versus 1.0 X lo7 M" min", respectively). Tetramethyl ammonium, which is much larger than
the other ions tested(radius E 2.5 A), shows little or no
enhancement of binding and reduces the cleavage rate. We
speculate that its large size reduces the charge density (and
hence the electrostatic attraction to phosphate) below the
level required for an effective counterion. The reduction in
cleavage rate may also be a denaturation effect: tetramethyl
ammonium is known to bind specifically to U-A pairs (17)
and may thus interfere with RNA tertiaryorquaternary
interactions.
Divalent Cations and Polyamines in Binding and Cleavage
Divalent cation is absolutely required for cleavage3by
RNase P (Mg2' is optimal) and has been proposed to act asa
catalytic cofactor in thereaction mechanism (18).Because of
the limitations of the cleavage assay, however, it has never
been clear whether the requirement for Mg2' is catalytic,
structural, or both.
Binding Competence in the Absence of Divalent CationFig. 1 shows an assay of cross-linking for two RNase P RNA
species in theabsence of all cations, monovalent cation alone,
cation, at 25 mM MgC12.This result indicates that unusually and monovalent plus divalent cation. Although the cleavage
high concentrations of monovalent cations are not required reaction takes place only in the presence of divalent cation,
cross-linking, and hence binding, does not require divalent
to fold RNase P RNA properly.
An alternative interpretation of the correlation of binding cation. A comparison of the cross-linking extents for several
with ionic strength is that less enzyme is properly folded at combinations of RNase P RNAs and tRNAs in the presence
low salt concentration. Two observations argue against this and absence of Mg2+ is shown in Table 11. Theseresults
interpretation. First, enzyme was equimolar to substrate in suggest that Mg2+is notgenerally required for proper formathe cross-linking experiments but was limiting in the cleavage tion of the RNase P RNA' tRNA complex.
Two further experiments support this view. Primer extenexperiments, in which substrate was in 20-fold excess. Cleavsion
analysis of the C.vinosum RNase P RNA-tRNA conjuage rate should therefore be moresensitive than cross-linking
extent to a hypothetical reduction in enzyme concentration gate determined that thesites of cross-linking in thepresence
at low ionic strength. Instead, cross-linking extent is more and absence of Mg2+are identical with regard to location and
sensitive to ionic strength. Second, the amount of active relative intensity (not shown). By this measure, the conforenzyme in the cross-linking experiment was titrated using mation of the enzyme in the vicinity of the 5"terminal
tRNAPheas inhibitor, with [ E ] >> Ki,>> [azidophenacyl- phosphate of tRNA (the location of the cross-linking reagent,
tRNA]. Under these conditions the fractional inhibitor of and the site of action by RNase P) is not highly dependent
cross-linking extent is proportional to [ E .11, and extrapola- on Mg+.
The result is complemented by the results of a cleavage
tion to 100%inhibition gives the active enzyme concentration.
At NH,C1 concentrations of 0.1 and 1.0 M we found the active assay designed to detect enzyme-substrate complex formation
concentrations of enzyme to be within the error of the nominal in the absence of Mg2' (Fig. 5). Radiolabeled pre-tRNA that
concentrations: 2.5 f 0.6 FM active versus 2.0 p M nominal at is bound to RNase P RNA in the absence of Mg2+ should be
0.1 M NH4C1,and 0.55 .C 0.1p~ active versus 0.50 PM nominal cleaved upon addition of Mg2' before it can be diluted by a
at 1.0 M NH4C1 (not shown). Note that this assay measures nonradioactive chase of unlabeled pre-tRNA, which will reonly binding competence and not catalytic competence (see
' Values given are for nonhydrated radii, which vary inversely
with
Fig. 5 legend).
hydrated radii.
Effect of Cation Type-Cs+, K', NH4+, and Rb+have comkcat/& in the absence of addeddivalent cationis < 200 M" min"
parable effects on both the cross-linking and cleavage re- (not shown).
Ions and Substrate
Binding
TABLEI1
Effects of Mg2+and spermidine on binding
APA-tRNA,azidophenacyl-tRNA.Reactionconditions
were: 28
nM azidophenacyl-tRNA, 270 nM RNase P RNA, 1.0 M NH4C1, 20
mM Tris, pH7.5, and cross-linking was performed as described under
"Experimental Procedures." MgCl,, when added, was at 16 nM; spermidine, when added, was at 10 mM. Spermine and putrescine were
also tested and showed no enhancement of cross-linking extent (not
shown).
by RNase P
2433
release is rate limiting), a control is shown in which radiolabeled pre-tRNA, unlabeled pre-tRNA and MgC1, are allowed
to equilibrate and then are added to the enzyme simultaneously. The burst resulting from prebinding of pre-tRNA is
significantly larger than the control; subsequently, the rates
of the experimental and control reactions areidentical. This
result indicates that RNaseP RNA does not require Mg2+to
bind pre-tRNA in a productive conformation or a t least in a
conformation that is in rapid
equilibrium with theproductive
Additives"
RNAs
one. We conclude from these experiments that the requireNone
MgCI,
Spermidine
RNase
P RNA
APA-tRNA
ment for Mg2+in the RNase P reaction is catalytic and that
B. subtilis
E. coli Phe
0.03
0.22
0.03
the enhancementof substrate bindingby Mg2+is secondary.
0.14
0.15
0.15
C. vinosum
E. coli Phe
Mg" Increases Binding Affinity 5-Fold-We repeated the
0.06
0.07
0.17
E. coli
E. coli Phe
cross-linking inhibition experimentsof the typeshown in Fig.
0.02
0.23
0.02
B. subtilis
Yeast Phe
3 in the absence of divalent cation to determine the contri0.18
0.11
0.10
Yeast Phe
C. uinosum
bution of Mg" to the binding interaction (data not shown).
E. coli
Yeast Phe
0.05
0.19
0.05
derived from this analysis is 200 (*
The K, for pre-tRNAASP
Fraction conjugated to RNase P RNA.
6) nM whereas that of an invitro transcript4 of mature E. coli
tRNAPhe is260 (f70) nM. Because there is no cleavage reac0.20tion under these conditions, the K, values in the absence of
0 pre-incubated no Mg 2+
0 no pre-incubation
Mg'+ correspond to equilibrium dissociation constants. Comparingthese valueswith thoseobtained in 25 mM MgC12
(Table I, lines 7 and 8) indicates that bindingaffinity is
increased only about 5-fold by Mg2+.
High Concentrations of Mg2+Reduce the Product Dissociation Rate-Fig. 6a compares theeffects of Mg2+concentration
on the ratesof single and multiple turnover reactions. Under
both conditions, a sharp rise in velocity is seen up to 5 mM
0
50
100
150
200
MgCl,. Velocity continues to increaseslightlywith
MgC12
the, s
concentration
for
the
single
turnover
reaction
but
steadily
FIG. 5. Binding of pre-tRNA toRNase P RNA in the absence
of Mg2+.This experiment was designed to detect formation of RNase decreases under conditionsof multiple turnover. These results
P RNA.pre-tRNA complexes in the absence ofM$+ by diluting are consistent withincreased nonspecific affinity of tRNA for
unbound pre-tRNA with a nonradioactive chasewhen the reaction is enzyme at higher Mg2+ concentrations: as binding becomes
started by the addition ofMgC12. Pre-incubated without M$+:3.75 tighter, product release is slowed, lowering the rate of the
pmol of C. vinosum RNase P RNA and 3.75 pmol of radiolabeled pre- multiple turnover reaction. The rate of the single turnover
tRNA (initial concentrations
of both RNAs = 150 nM) were incubated
in 25 pl of 1.0 M NHaC1,50 mM HEPES, pH8.0,0.1%sodium dodecyl reaction, which is not affected by product release (assuming
sulfate, 0.05% Nonidet P-40, 1 mM EDTA. An equal volume of the lz-, is negligible), increases slightly as the rate of substrate
same buffer containing 50 mM MgCL, 37.5 pmol of unlabeled pre- association is increased and/or the rateof substrate dissociatRNA, and no EDTA was separately incubated for 5 min at 37 "C, tion is decreased.
and the reaction was started by mixture of the two components. No
Ca" Promotes Binding as Effectively as Mg"-Fig.
6 also
pre-incubation: 3.75 pmol of C. uinosum RNase P RNA was incubated shows a comparison of Mg", Ca2+, and Mn2+ in enhancing
in 25 pl of the same no-Mg2i- buffer as above, in parallel with 3.75
the binding of tRNA to RNase P RNA. Guerrier-Takada et
pmol of radiolabeled pre-tRNA and37.5 pmol of unlabeled pre-tRNA
in 25 pl of the same Mg2+-containingbuffer as above, then started by al. (18, but see below) have suggested that ea2+ canperform
mixture of the two components. Final concentrations of reactants in the structural role ofMg'+ but not the catalytic role. The
both experiments were 75 nM RNase P RNA, 75 nM radiolabeled pre- cross-linkingassayshows
that Ca2+ is indeed effective in
tRNA, 750 nM unlabeled pre-tRNA, and 25 mM MgCL. 6-pl aliquots promoting the binding
of tRNA to RNaseP RNA; at concenwere removed at theindicated times and mixed with 15 pl of ice-cold trations greater than 10mM, it is more effective than Mg2'.
ethanol toquench the reaction. Least squares linesare drawn through
Ca2+ Supports the Cleavage Reaction-In contrast to prethe data points, and the y intercepts are given with the standard
cleavage
error. The y intercepts represent an extrapolation from the steady vious reports that Caz+ is inert in promoting the
state back to the amountof radiolabeled pre-tRNA initially bound to reaction (16, 18) we found that it does support cleavage (Fig.
the enzyme, less any pre-tRNA that dissociates before cleavage oc- 6b), albeit at a reduced rate: k,,,/K, is reduced 5-fold comcurs. Assuming that such dissociation is minimal, the y intercept of pared with Mg'+ (from Table I, kCat/Km
Ca2+= 2.1 X lo6 M"
the "no pre-incubation" experiment indicates that only = 30% of the min", kcat/K,, Mg2+= 1.0 X lo7 M" min"). Several precauenzyme is catalytically active.
tions against adventitious contamination
of the reaction with
Mg2+and other metals were taken (see "Experimental Produce the apparent cleavage rate. This behavior will be char- cedures"), and the deliberate addition
of trace amounts(lo+acterized by an initial "burst"of product formation, inwhich lo-" M) ofMgC1, did not increase therate of theCa2+cleavage of the bound, labeled pre-tRNA is followed a phase promoted reaction (data not shown). We conclude that the
of apparently slower cleavage, as the pools of labeled and Ca2+ reaction isgenuine.
unlabeled pre-tRNA equilibrate. If pre-tRNA is unable to
The slower rate of the Ca2+ reaction can be attributed to
of Mg" it should be the catalytic step. In conditions of excess E. coli RNase P
bind RNase P RNA in the absence
diluted by the chase before binding and cleavage can occur, RNA (up to 1.0 p ~ ) the
, limiting rate of the single turnover
and no burstof cleavage will be detected.
reaction is found to be the same as kc,, in the steady-state
The data presented in Fig. 5 show the burst of product
formation expected if pre-tRNA is able to bind RNase
P RNA
Surprisingly, we found that mature tRNA purified from E. coli
in the absence ofMg". To distinguish this burst from that was unable to act as an inhibitor of the cross-linking reaction in the
caused by a fast first round of cleavage (expected if product absence of Mg'+.
2434
Ions and Substrate Binding by RNase
P
[Maz+l,m M
[Ca2+l,m M
[Mn2’], m M
FIG. 6. Effects of divalent cation concentration on
cleavage and binding. Cross-linking conditionswere:
50 nM E. coliRNase P RNA, 50 nM azidophenacyl-tRNA, standard cross-linkingbuffer(see“Experimental
Procedures”), and divalent cationas indicated, at 24 “C. Reaction conditions for the single turnover experiment
( a ) were: 10 nM E. coliRNase P RNA, 10 nM pre-tRNAAaP,
standard cross-linking buffer (see “Experimental
Procedures”),MgC1, as indicated,at 37 “C for 2 min. Reaction conditions forCa2+cleavage ( b ) were: 0.5 nM E. coli
RNase P RNA, 5.0 nM pre-tRNAAsp,standard cleavage buffer, CaC1, as indicated, at 37 “C for 227 min. The extent
of cleavage in the Caz+reactions was up to 0.12. Reaction conditions for M e ( a ) and Mn2+(c) cleavage were: 0.5
nM E. coli RNase P RNA, 10 nM pre-tRNAAsp,
standard cleavage buffer, MgCl, or MnC12as indicated, at 37 “C for
30 min. kcat/Km‘is calculated as u/[Et,,.,[
[StotaI].
of the true kCat/Km
proportional
.
..
. This is a systematic underestimate
to [Efreel/[Etota~I.
reaction, 0.05 min” (not shown). The catalytic step in the
Ca2+ reaction is thus at least 10-fold less than that of the
Mg2+ reaction(taking kcat,Table I, lines 9 and 10,as the lower
limit) andpossibly much slower.
Mn2+-Fig. 6c shows that Mn2+ is capable of supporting
the cleavage reaction, aspreviously reported (16, 18).Mn2+ is
more effective at lower concentrations than Ca2+ or Mg2+,
peaking in cleavage activity at 2 mM and in bindinga t 5 mM.
This behavior is consistent with the
generally greater affinity
of Mn2+ for nucleotides (19). The cleavage reaction in the
presence of Mn2+ is almost as fast as that of Mg2+at their
respective optima. Unlike Ca2+or Mg’+, Mn2+ causesa sharp
at relatively low
dropinboth
cleavage rateandbinding
concentrations. We attribute this behavior to the ability of
Mn2+ to coordinate to nucleotide bases in addition to phosphates; the sharpdecrease in binding andcleavage may therefore be a denaturation effect.
Polyamines-Spermine,
spermidine,andputrescineare
multivalent cations that enhance the rates of a number of
reactions involving polynucleotide substrates; spermidine is
commonly added to RNase P RNA reactions. We find, however, that in the presence of sufficientmonovalent cation
polyamines have no effect on binding even in the absence of
presence
Mg2+(Table 11), nor do they enhance binding in its
(data not shown). We conclude that the addition of polyamines to RNaseP RNA reactions isprobably superfluous but
harmless.
the inability to determinereadily the effects of experimental
variables on the RNase
P reaction with regardto the substrate
binding, cleavage, and product release steps. Cleavage assays
cannot distinguish requirements for substrate binding from
requirements for cleavage. Similarly, alterations in reaction
velocity, or kc,, and K,,,, cannot readily be assigned to alterations in any particular elementary step.
The Cross-linking Assay
Our approach to theresolution of binding andcleavage has
been to develop a cleavage-independent assayof binding based
on a high yield photoaffinity cross-linking reaction between
RNase P RNA and tRNA. T o increase the generality of our
findings, we employed RNase P RNAs from threeeubacteria.
C. vinosum and E. coli are both members of the y-division of
P RNAs are structurally(11)
proteobacteria, and their RNase
and kinetically (Table I) similar; the RNase P RNA of B.
subtilis, a member of the “gram-positive” phylum, differs in
both structure andreaction kinetics.
Several previously reported (10) features of the cross-linking reaction render it suitable for use as a binding assay. (i)
The wavelength used for excitation (302 nm) does little or no
damage to RNA. (ii) The arylnitrene generated by illumination is highly reactive, allowing insertion into many types of
covalent bonds. (iii)The yield of conjugates is extremely high,
up to 30% inconditions of enzymeexcess, and 2-15% at
equimolar enzymeand substrate at
s K,,, concentrations. This
point is important: cross-linking and
cleavage assays aredone
DISCUSSION
under closely comparableconditions, in contrast to a UV
Substrate recognitionby RNase P RNA is of particular photodimerization reaction (21) which usedRNase P and preconcentrations of the
order
1,000-fold greater
interest because, unlike other RNA catalysts, it isunlikely to tRNA
make much use of helix formation via complementary base than K,,,. (iv) The sites of azidophenacyl-tRNA attachment
pairing. There is littlesequence conservation in theregion of are conserved in the reactionsbetween tRNAs and RNase P
the cleavage site withwhich pairing could take place, and the RNAs from disparate organisms and are located in the phymature domain of pre-tRNA, the substrate component rec- logenetically conserved core of the RNase P RNA secondary
ognized by RNase P, is already highly structured. Some ele- structure. (v) tRNA conjugated to RNase P RNA reversibly
ments important in substrate recognition have been identi- prevents cleavage activity, consistent with its occupation of
fied one is the 3”terminal
CCA (20); another is thesequence the active site.
We have extendedthese
observations. Azidophenacylnear thecleavage site, which can affect cleavage site selection
(5-9). However, the contributions of these structural motifs tRNA is a derivative of mature tRNA, which is the product,
of RNase P; substantial differences
are not simple: the 3‘ CCA is not essential (6), and the
effects rather than the substrate,
of RNA sequence and structure in the5‘ leader and acceptor in the substrate and product binding sites might exist. Although an early report suggested that product inhibition is
stem on cleavage site selection are not predictable (5-9).
One source of confusion in interpreting results has been noncompetitive (22), the kinetic characterization of cleavage
Ions
Substrate
Binding
by and
RNase P
2435
of the
inhibition (Fig. 2a) shows that mature tRNA is
a purely of turnovers)candeterminetherate-limitingstep
competitive inhibitor, as
expected if the product and substratereaction.
Monovalent Cation Requirements-Of the reaction condibinding sites arecoextensive. We therefore conclude that the
binding properties of pre-tRNA and mature tRNA aresuffi- tions reported here, the fastest steady-state rate of cleavage
is achieved at 1.0 M NaCl with 25 mM MgC12 (Fig. 6a). This
ciently similar to justify extrapolation from one to the other.
high [M'] optimum is in keeping with the concept that high
Azidophenacyl-tRNA, thephotoagent-containingderivaionic strength acts to replace the RNase P protein (3). It is
tive of mature tRNA, is qualitatively similar to mature tRNA
in that it is
also a competitive inhibitor of the cleavage noteworthy, however, that this enhancement is only a fewwas low
reaction (Fig. 2b). However, the Kt of azidophenacyl-tRNA is fold (Fig. 4 ) , even though the substrate concentration
(50.25 K,). This conditionwill emphasize the cationrequiresubstantially higher than that of mature tRNA, indicating
that the presenceof the azidophenacyl group at the5' end of ment if cations act toincrease binding affinity; cleavage rates
tRNA weakens binding to RNase P RNA, presumably by at high substrate concentration will be even less dependent
steric hindrance. We do not believe this property poses a on high cation concentration. Since we could not detect any
serious objection: the cross-linking reagent,a t length 8-9 A, reduction in the amountof active enzyme at 0.1 M versus 1.0
is no larger than a single nucleoside, and the 10-fold increase M NH:,we conclude that high concentrations of monovalent
in K, indicates a n increase of only 1 kcal/mol in the free cation are notrequired for folding of RNase P RNA.
Monovalent cationsprobably enhance substratebinding by
energy of binding, equivalent to the
loss of one hydrogen bond
neutralizingelectrostatic repulsionbetweenRNAs
rather
(15).
than
by
forming
salt
bridges.
A
relatively
small
difference
(<
The most compelling evidence of the applicability of the
cross-linking assay is the finding that the binding constants 2-fold) in binding is seenfor a seriesof cations having a large
range of unhydrated ionic radii (0.55-1.7 A), consistent with
obtained for pre-tRNA and mature tRNA in the cross-linking
assay are comparable to those obtained in the conventional charge neutralization but notexpected if specific contacts are
cleavage assay (Figs. 2 and 3 and Table I). These results give being formed though the cations. Similarly, the binding of
monovalent cations must be weak, as cross-linking extent is
us a firm basis for believing that the cross-linking reactions
not saturatedat 0.5-1.0 M M' (K' appears tobe an exception).
accurately reflect the bindingbehavior of RNase P RNA and
This behavior is not expected if the cations fill binding sites
its ligands.
on the RNAs but is consistent
with electrostatic attraction to
equilib- negatively charged phosphates.
The cross-linking reaction allows us to compare the
rium binding of the RNase P substrate, pre-tRNA, with that The Dual Role of Divalent Cation-The divalent cation
of mature tRNA. It is reasonable to expect that K, = Kr, for requirement for cleavage has been plausibly interpreted to
mature tRNA: since the cleavage reaction is essentially irre- indicate that Mg2' acts as a catalytic cofactor in the RNase
versible, bothconstants will equal k3/k-:i intheminimal
P reaction (18).These data,however, could also be interpreted
reaction scheme. The cross-linking extent appearsalso to be as a requirement for Mg2+for the properfolding of RNase P
a function of Kn,as the K , values determined by the crossRNA (as with the Tetrahymena IVS (25)) or for binding of
linking assay are close to those determined from cleavage substrate to enzyme. Our results support the conclusions of
kinetics. However, the relationship between K, and KT, of Guerrier-Takada et al. (18). Binding of tRNA to RNase P
pre-tRNA is less clear. In the minimal reaction scheme, K , RNA can take place in the absence of Mg'+ (Table 11), and
= ks(k2 + k - 1 ) / k , ( k 2 k3);assuming that k-,, Iz3 << k2, K , = the conformation of the enzyme-substrate complex in the
k:Jkl,and so resembles Kn (k-Jk1) only to the extent that theabsence ofMg2' is similar or identical to the productive
off-rate of mature tRNA equals the off-rateof pre-tRNA ( k , conformation (Fig. 5). Mg2+may enhance, but is notrequired
= kl).Two observations indicate the similarities
of these for, substrate binding orenzyme folding; the requirement for
rates and thus of K,,, and Kr, of pre-tRNA.Firstisthe
cleavage strongly implies participation in the catalyticmechsimilarity of the K, values of mature and pre-tRNAfor inhi- anism.
bition of cross-linkingintheabsence
ofMg".
Sinceno
Contraryto aprevious report (18), Ca2+promotesthe
cleavage occurs under these conditions,Ki = Kr,. Unless Mg" cleavage reaction, albeit somewhatpoorly: k,,,/K, is reduced
5' leader of pre-tRNA, this 5-fold (Table I). The catalytic step is rate limiting
introduces an interaction with the
for the
relationship should hold under cleavage conditions also, and Ca2+ reaction,whereas product release is rate limitingfor the
KI, tRNA
p r e . t ~ ~K~ , p r e . t ~ Second,
~ ~ .
k, is limiting in the Mg2+ reaction. The 8-fold reduction in kc,, istherefore a
Ca2+reaction (k2 << k l , k3) so that K, = k - , / k , = KO.Cross- minimum estimateof the effect of Ca2+ on catalysis step and
linking extents are similar in thepresence of Ca2+ andMg"
is likely to be much larger. This effect on catalysis is also
(Fig. 6), as are K, values in the cleavage reactions (Table I), consistent with a direct role of divalent cation in thereaction
indicating the similarityof substrate binding in thepresence mechanism (18, 24).
Chemical Nature of the RNaseP RNA-tRNA Interactionof thedifferentdivalentcations.Theextrapolationthat
A priori, three types of interactions might contributespecific
Kllc,2+
KDMg2+ therefore seems reasonable.Weconclude
that under our assay conditions, the binding affinities
of pre- RNase P RNA-tRNA contacts: hydrophobic, ionic, and hydrogenbonds. Extensive specific hydrophobic interactions
tRNA and mature tRNA are
very similar.
seemunlikely to be involved because of the highly polar
The Dichotomy of Binding and Cleavage
surfaces of the RNAs (although stacking interactions could
Conditions that enhance substrate binding do not neces- provide localized hydrophobic stabilization). The data presarily increase the steady-statecleavage rate and indeed can sented here, together with previous observations (3, 16, 18),
ionic contactsarenot
involved in the
reduce it. NH: causes tRNA to bind
more tightly to RNaseP indicatealsothat
RNA than does Na' (Fig. 4),but kc,, is severalfold faster in specificity of the RNaseP RNA-tRNA interaction.
Potential ionic contacts would be mediated by divalent
Na+(TableI).Similarly,bindingandthe
single turnover
cation bridges between phosphates.s The present data show
reaction rate increase withMg2+concentration, but the multiple turnover rate decreases at concentrations above 10 mM
Because the RNAs used in these experiments are not modified,
(Fig. 6a). These results demonstrate that thechoice of reac- they have no positively charged residues to interact with negatively
tion conditions(e.g. type and concentrationof cation, number charged phosphates.
+
-
-
-
2436
Substrate
Binding
and Ions
by RNase P
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that RNase P RNA specifically binds tRNAs in the absence
of divalent cations (Table 11 and Fig. 5). In Some cases, this
binding is as good in the absence of M2' as in its presence;
in no
is the difference
than 10-fold.
Although
nonspecific enhancement of binding by divalent cations is
indicated by the reduced rate of product dissociation a t high
Mg2+concentrations, this effect is also modest, a few-fold a t
most. We conclude that divalent cation bridges do not contribute specifically to this ribozyme-substrate complex. The
noninvolvement of ion-dependent contactsin RNase P RNAtRNA binding is also indicated by the general insensitivity of
theinteractiontothe
presence of high concentrations of
monovalent salts. High monovalent salt concentrations
would
be expected to screen interactions with divalent cations and
thereby reduceaffinity; the opposite effect is observed.
Thus, selectivity and binding of substrate by RNase P seem
mediated largely or solely through hydrogen bonds. Hydrogen
bondsmight occur between thebases or otherelements of
enzyme and substrate. Helix formation is unlikely, however,
because of thetight folding totRNAsand variability intheir
sequences.
The basic phenomenology of eubacterial RNase P is now
and the focus Of the
has shifted toward
obtaining a detailed understanding of enzyme structure, substrate recognition, and catalysis. It is clear from the present
work that cation type and concentration can affect binding
and cleavage in ways that are not intuitively
obvious or easily
distinguished by cleavage assays alone. By extension, we
expect that structural alterations of substrate and enzyme, as
in mutational studies, will have similarly confusing effects.
Progress in these
require
more
refined
we
have described one such tool, the cross-linkingassay, which
allows effectsonbindingandcatalysisto
be determined
independently.