Tài liệu Báo cáo khoa học: Unusual metal specificity and structure of the group I ribozyme fromChlamydomonas reinhardtii23S rRNA pptx

Unusual metal specificity and structure of the group I
ribozyme from Chlamydomonas reinhardtii 23S rRNA
Tai-Chih Kuo
1
, Obed W. Odom
2
and David L. Herrin
2
1 Department of Biochemistry, Tapei Medical University, Taiwan
2 Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas, Austin, TX, USA
Group I introns are cis-acting ribozymes whose sub-
strates (5¢ and 3¢ splice sites) are attached intramolecu-
larly. These introns have conserved uridine and
guanosine nucleotides at the ends of the 5¢ exon and
intron segments, respectively. Although sequence con-
servation of group I introns is poor, their folded forms
share a common core structure composed of two
stacked-helix domains (P5–P4–P6 and P7–P3–P8) [1,2].
Group I introns can be differentiated into five major
subgroups (IA, IB, IC, ID, and IE) with further subdi-
visions that depend on the presence of peripheral
domains that stabilize the core [3,4]. Studies of several
group I ribozymes, but especially the intron from
the large rRNA gene of Tetrahymena thermophila
(Tt.LSU), indicate that some domains are modular,
and that the catalytic site is buried inside the folded
ribozyme [5–7]. The tertiary structure is stabilized by
domain–domain interactions, such as hydrogen bond-
ing of loop–receptor pairs, base triples, and pseudo-
knots [1,2].
The group I self-splicing pathway consists of two

consecutive transesterification reactions with the acti-
vated phosphodiesters at the splice sites. First, the
3¢-OH of an exogenous guanosine nucleotide (GTP)
Keywords
Fe
2+
–EDTA; group I intron; Mn
2+
; RNA
structure; RNA–metal interactions
Correspondence
D. L. Herrin, Section of Molecular Cell and
Developmental Biology, 1 University Station
A6700, University of Texas at Austin,
Austin, TX 78712, USA
Fax: +1 512 4713843
Tel: +1 512 4713843
E-mail:
Website: />MCDB/
(Received 9 February 2006, revised 3 April
2006, accepted 12 April 2006)
doi:10.1111/j.1742-4658.2006.05280.x
Group I intron ribozymes require cations for folding and catalysis, and the
current literature indicates that a number of cations can promote folding,
but only Mg
2+
and Mn
2+
support both processes. However, some group I
introns are active only with Mg

2+
, e.g. three of the five group I introns in
Chlamydomonas reinhardtii. We have investigated one of these ribozymes,
an intron from the 23S LSU rRNA gene of Chlamydomonas reinhardtii
(Cr.LSU), by determining if the inhibition by Mn
2+
involves catalysis,
folding, or both. Kinetic analysis of guanosine-dependent cleavage by a
Cr.LSU ribozyme, 23S.5DG
b
, that lacks the 3¢ exon and intron-terminal G
shows that Mn
2+
does not affect guanosine binding or catalysis, but
instead promotes misfolding of the ribozyme. Surprisingly, ribozyme mis-
folding induced by Mn
2+
is highly cooperative, with a Hill coefficient
larger than that of native folding induced by Mg
2+
. At lower Mn
2+
concentrations, metal inhibition is largely alleviated by the guanosine
cosubstrate (GMP). The concentration dependence of guanosine cosub-
strate-induced folding suggests that it functions by interacting with the G
binding site, perhaps by displacing an inhibitory Mn
2+
. Because of these
and other properties of Cr.LSU, the tertiary structure of the intron from
23S.5DG

b
was examined using Fe
2+
-EDTA cleavage. The ground-state
structure shows evidence of an unusually open ribozyme core: the catalytic
P3–P7 domain and the nucleotides that connect it to the P4–P5–P6 domain
are exposed to solvent. The implications of this structure for the in vitro
and in vivo properties of this intron ribozyme are discussed.
Abbreviations
Cr.LSU, intron from the 23S LSU rRNA gene of Chlamydomonas reinhardtii; oligo, oligodeoxynucleotide; Tt.LSU, intron from the large rRNA
gene of Tetrahymena thermophila.
FEBS Journal 273 (2006) 2631–2644 ª 2006 The Authors Journal compilation ª 2006 FEBS 2631
attacks the 5¢ splice site (G-dependent cleavage), gener-
ating 5¢ exon and intron-3¢ exon intermediates. Then,
the 3¢-OH of the 5¢ exon attacks the 3¢ splice site,
forming ligated exons and a free intron [9]. The liber-
ated intron can react with itself, forming a circular
RNA [9], or with another RNA [10], via the 3¢-ter-
minal G (XG) of the intron; it can attack a phosphodi-
ester that becomes properly positioned in the catalytic
center [9,10].
Owing to the complexity of the ribozyme reactions
and the polyanionic nature of RNA, the catalytic chem-
istry and folding of group I ribozymes (and other large
ribozymes) require divalent metals [11,12]. Whereas
only Mg
2+
and Mn
2+
are able to support the chemistry

of the Tetrahymena ribozyme [13], several divalent cati-
ons (e.g. Mg
2+
,Mn
2+
,Ca
2+
,Sr
2+
, and Ba
2+
) [14],
and even monovalent cations [15], are able to promote
the formation of a native, or native-like, structure. With
divalent and monovalent salts as the only aids to RNA
folding, however, the formation of alternative, nonpro-
ductive base pairs can trap a fraction of a large ribo-
zyme in inactive conformations [16–18]. The conversion
of these forms into an active ribozyme is sometimes
hampered, ironically, by native domain–domain interac-
tions or high Mg
2+
concentrations [19]. Thus, in vivo,
the folding of most large ribozymes is probably assisted
by proteins. In a few cases, it has been shown that in
the presence of the proper protein, group I ribozymes
that would otherwise be inactive, or become active only
at high temperatures and high Mg
2+
concentrations,

perform catalysis in vitro under mild conditions [20,21].
We have been studying the group I ribozyme,
Cr.LSU, from the chloroplast 23S (LSU) rRNA gene
of the green alga Chlamydomonas reinhardtii [22–24].
Splicing of Cr.LSU in vivo is required for ribosome
formation, and could be a limiting step in ribosome
biogenesis, as it is one of the slowest steps in rRNA
maturation [24]. This subgroup IA3 intron self-splices
efficiently in vitro, but requires higher Mg
2+
concen-
trations than the model intron, Tt.LSU, and it is more
sensitive to nucleotide substitutions in the core [23,25].
Kinetic analysis indicated that a Cr.LSU pre-RNA
containing the full-length intron and relatively long
exon sequences tends to misfold in vitro , although the
active fraction self-spliced rapidly [18]. Self-splicing of
Cr.LSU occurs only with Mg
2+
, and is inhibited by
equivalent concentrations of Ca
2+
or Mn
2+
[26]. The
Mn
2+
inhibition was unexpected, because Mn
2+
is

similar to Mg
2+
[27,28], and has been shown to sup-
port the activity of group I and other large and small
ribozymes [27,29,30]. Thus, this group I intron exhibits
several properties that distinguish it from the more
well-studied Tt.LSU and phage introns.
We wished to know whether Mn
2+
inhibits the for-
mation of active Cr.LSU or whether it interferes with
catalysis, and have addressed this question using ribo-
zyme kinetics. We also wanted to probe the folding
and tertiary structure of the ribozyme using Fe
2+
-
EDTA, which promotes cleavage of the sugar–phos-
phate backbone, and can determine, for example, if
the active site of Cr.LSU is internalized like those of
other group I ribozymes [7,31]. The wild-type Cr.LSU
intron was unsuitable for this, because of its large size
(888 nucleotides). Moreover, high concentrations of
NH
4
+
and Mg
2+
were required for efficient self-spli-
cing of the large 23S.1 precursor [23]. Hence, a smaller
RNA, 23S.5 (448 nucleotides), in which the intron was

shortened by replacing the long P6 extension (which
encodes the I-CreI endonuclease [32]) with a short
stem–loop, and the exons were reduced, was generated.
This pre-RNA self-splices efficiently without monova-
lent salt, and approximately 85% of the RNA is of the
same kinetic competence (k
cat
¼ 1 min
)1
and K
1/2
G
¼
26 lm) [18]. For this study, we have taken the 23S.5
pre-RNA and generated a ribozyme, 23S.5DG
b
, that
performs GMP cleavage at the 5¢ splice site, but not
exon ligation or intron circularization, as it lacks the
3¢ exon and the XG. This RNA was used to assay
ribozyme activity, and to generate end-labeled RNA
for structural probing.
Results
Inhibition of self-splicing and G-dependent
cleavage by Mn
2+
In splicing reactions with 23S.5 pre-RNA, substituting
part (> 1 ⁄ 3) of the Mg
2+
with Mn

2+
reduced the
amount of products, which were undetectable when
Mn
2+
was the only divalent cation (not shown [26]).
Varying the Mn
2+
concentration (0.1–50 mm), pH
(5.5–7.5), monovalent salt, temperature (37 or 47 °C),
and reaction time (0.25–60 min) also did not yield any
splicing products (data not shown). Mn
2+
inhibits self-
splicing of the 23S.3 and 23S.4 pre-RNAs, which have
different lengths of 5¢ exon [23], and it inhibits a trans-
reaction [10] that involves the free intron reacting with
5.8S rRNA (not shown). Together, these data sugges-
ted that inhibition by Mn
2+
probably involved the
core ribozyme, and not the intron open reading frame
(ORF) or exon sequences.
23S.5DG
b
pre-RNA is a truncated version of 23S.5
that terminates 3 nucleotides before the end of the
intron (Fig. 1A; see Fig. 5D for the intron sequence
and structure). Core catalytic activity is preserved,
however, in the form of G-dependent cleavage at the

Mn
2+
inhibition and structure of Cr.LSU ribozyme T C. Kuo et al.
2632 FEBS Journal 273 (2006) 2631–2644 ª 2006 The Authors Journal compilation ª 2006 FEBS
5¢ splice site. Incubation of 23S.5DG
b
with saturating
Mg
2+
(25 mm) and GMP (150 lm) produces the
InDG
b
and 5¢ exon (not shown) molecules as expected
(Fig. 1B). In the presence of 10 mm Mn
2+
, however,
less of the pre-RNA reacts (compare Fig. 1B,C). Thus,
the inhibition of 23S.5 self-splicing by Mn
2+
is recapit-
ulated by the G-dependent cleavage of 23S.5DG
b
pre-
RNA. Mixed-metal titrations over a range of total
metal concentrations showed that the ratio of the two
metals is somewhat more important than the absolute
concentrations; a ratio of Mn
2+
⁄ Mg
2+

of approxi-
mately 1 : 2 or higher inhibited G-dependent cleavage
of 23S.5DG
b
RNA (and self-splicing of 23S.5, not
shown).
Quantification of time-course reactions similar to
those in Fig. 1B,C, except at two different GMP con-
centrations (Fig. 1D), show that approximately 85%
of the 23S.5DG
b
RNA is kinetically homogeneous and
highly active (k
obs
approximately 0.9 min
)1
at 150 lm
GMP). The remaining fraction (approximately 15%) is
relatively inactive, reacting 20–30 times more slowly
(k
obs
¼ 0.032 min
)1
at 150 lm GMP, and 0.017 min
)1
at 20 lm GMP). It can also be inferred from Fig. 1D
that the inactive RNA fraction increases substantially
when 10 mm Mn
2+
is added, from 15% to 32% at

150 lm GMP, or 50% at 20 lm GMP. The inverse is
true for the active fraction, which decreased from 85%
to 68% and 50%, respectively. The observed rate of
G-dependent cleavage by the active fraction is not sub-
stantially affected by Mn
2+
: the k
obs
at 150 lm GMP
is 0.96 min
)1
with Mn
2+
and 0.87 min
)1
without it,
and at 20 lm GMP, the k
obs
is 0.49 min
)1
with Mn
2+
and 0.55 min
)1
without it. We conclude that Mn
2+
increases the proportion of 23S.5DG
b
pre-RNA that is
inactive, whereas GMP increases the proportion that is

active.
An extensive kinetic analysis was performed at
5–300 lm GMP and 10 or 15 mm Mn
2+
in the pres-
ence of 25 mm Mg
2+
. Figure 2A shows that Mn
2+
has little or no effect on the K
G
1/2
or k
cat
for the active
fraction of the ribozyme. However, as Fig. 2B shows
quite dramatically, the metal decreases the size of this
fraction. It should be noted that the proportion of act-
ive ribozyme without Mn
2+
is approximately 88% at
all GMP concentrations tested. In the presence of
10 mm Mn
2+
, the maximum size of this fraction is
66% (> 100 lm GMP), and it decreases dramatically
at GMP concentrations < 100 lm (Fig. 2B). At 15 mm
Mn
2+
, the percentage of active ribozyme is even lower

(approximately 30% at > 25 lm GMP) and it decrea-
ses further at GMP < 20 lm. These data extend the
above result, and support the conclusion that Mn
2+
affects mainly the correct folding of the ribozyme. The
Ct 0 .3 .7 1 1.5 3 5 10 25 60
0 .3 .6 1 1.5 3 5 10 20 40 60
Fig. 1. Mn
2+
inhibits the activity of the 23S.5DG
b
ribozyme.
(A) Schematic diagram of 23S.5DG
b
pre-RNA and the reaction being
assayed. 23S.5DG
b
pre-RNA contains a partial 5¢ exon (rectangle)
and a shortened Cr.LSU intron (line), which lacks the large P6
extension and the last three nucleotides (AU XG) of the intron. The
sizes of the intron (InDG
b
) and 5¢ exon (5E) are indicated. The arrow
indicates cleavage of the pre-RNA at the 5¢ splice site by GMPG*.
(B, C) G-dependent cleavage reactions with 0 m
M (B) or 10 mM (C)
MnCl
2
. The reactions with
32

P-labeled 23S.5DG
b
pre-RNA (Pre)
included 25 m
M MgCl
2
and 150 lM GMP. The large product, InDG
b
,
was separated on a denaturing gel and phosphorimaged. (D) Quan-
tification of 23S.5DG
b
pre-RNA decay in the presence (10 mM)or
absence (0 m
M)ofMn
2+
, and either 20 or 150 lM GMP. The GMP
cleavage reactions were performed as in (B) and (C), except at two
different concentrations of GMP. The %23S.5DG
b
pre-RNA remain-
ing was measured, and the data fitted to an equation for two-
phase, exponential decay kinetics (see Experimental procedures).
T C. Kuo et al. Mn
2+
inhibition and structure of Cr.LSU ribozyme
FEBS Journal 273 (2006) 2631–2644 ª 2006 The Authors Journal compilation ª 2006 FEBS 2633
results also show that most of the inhibition by 10 mm
Mn
2+

is reversed by saturating GMP (Fig. 2B). More-
over, the fact that this GMP activation curve is similar
to the GMP cleavage plot (Fig. 2A) indicates that
GMP is promoting ribozyme folding via the G binding
site in P7.
The rate of G-dependent cleavage by the inactive
fraction that forms in Mg
2+
increases slowly with
GMP concentration (K
1/2
G
¼ 60 lm and k
cat
¼
0.035 min
)1
, not shown). However, the inactive fraction
in Mn
2+
(10 mm in Fig. 2B) reacts much more slowly
(approximately 0.007 min
)1
) and independently of the
GMP concentration (not shown). This result suggests
that Mn
2+
induces a distinctive slow-reacting fraction
that must go through a rate-limiting conformational
change before it can bind GMP and catalyze cleavage.

Model for the effect of Mn
2+
on the 23S.5DG
b
ribozyme
Since Mn
2+
does not substantially affect the kinetic
parameters for the active ribozyme, but instead reduces
the size of this fraction, the following scheme
(Scheme 1) is proposed to describe the inhibition of
G-dependent cleavage by the metal ion.
U þ nMg
2þ
+
(
pre-RNAÁnMg
2þ
active
U þ mMn
2þ
+
(
pre-RNAÁmMn
2þ
inactive
Scheme 1
U is unfolded 23S.5DG
b
pre-RNA, and the binding of

a minimum of n Mg
2+
ions leads to formation of the
active complex, whereas binding of a minimum of m
Mn
2+
ions forms the inactive complex. The sizes of
the active and inactive fractions are the result of com-
petitive metal binding to RNA. The values of n and m
are estimated from Hill analysis of G-dependent clea-
vage of 23S.5DG
b
. It should be noted that Scheme 1
indicates only the initial and final states of the pre-
RNA; it does not invoke or rule out any misfolded
intermediates that might form.
To determine n, G-dependent cleavage of 23S.5DG
b
was analyzed at varying MgCl
2
concentrations
(0–50 mm) and either 0 or 12 mm MnCl
2
(plus satur-
ating GMP). Figure 3A shows that in the presence
of Mn
2+
, a much higher concentration of Mg
2+
is

required to form the same amount of cleavage product
(InDG
b
). A quantitative analysis of similar experiments
(Fig. 3B), but using 0–100 mm Mg
2+
and several fixed
Mn
2+
concentrations (0, 7, 12 and 17 mm), reveals
that cleavage increases cooperatively with increasing
Mg
2+
, in the absence or presence of Mn
2+
. The mid-
point of the Mg
2+
titration curve in the absence of
Mn
2+
is approximately 4.5 mm, and nearly full
activity is reached by 10 mm Mg
2+
. Hill analysis of
the data gives n-values of 2.6, 2.2, 2.7 and 2.7, for
reactions in 0, 7, 12 and 17 mm Mn
2+
, respectively.
These results indicate that formation of the active

RNA–Mg
2+
complex involves the binding of at least
three Mg
2+
ions by the ribozyme. The data also show
that Mg
2+
can completely block the inhibition caused
by Mn
2+
.
Fig. 2. Ribozyme activity at varying GMP and fixed Mn
2+
concentra-
tions. The G-dependent cleavage reactions were performed at dif-
ferent concentrations of GMP (0–300 l
M) and Mn
2+
(0, 10 or
15 m
M) in the presence of 25 mM MgCl
2
. The reactions were ana-
lyzed as described in Experimental procedures, and the observed
rate constants (A) and percentages (B) of active ribozyme were
plotted versus GMP concentration. In (A), the line was fitted using
the 0 m
M Mn
2+

data. In (B), the k
cat
values are 1.1, 1.0 and
1.0 min
)1
, respectively, for the reactions at 0, 10 and 15 mM Mn
2+
,
and the corresponding K
G
1/2
values are 22, 24 and 21 lM, respect-
ively.
Mn
2+
inhibition and structure of Cr.LSU ribozyme T C. Kuo et al.
2634 FEBS Journal 273 (2006) 2631–2644 ª 2006 The Authors Journal compilation ª 2006 FEBS
To determine the minimal number of Mn
2+
involved in forming the inactive RNA–metal com-
plex, G-dependent cleavage reactions were performed
at varying Mn
2+
and fixed Mg
2+
concentrations.
Figure 4A shows representative gels of reactions that
were performed at varying (0–30 mm) MnCl
2
con-

centrations and either 15 or 25 mm MgCl
2
. The
GMP-dependent cleavage decreases sharply above 5
and 7 mm MnCl
2
, respectively. Quantitative analysis
(Fig. 4B) gives a Hill value (m) of 5.7 for the experi-
ments with 15 and 25 mm MgCl
2
. Thus, formation
of the inactive ribozyme is highly cooperative. The
data also suggest that binding of a minimum of six
Mn
2+
ions is involved in the misfolding that forms
the inactive ribozyme.
Structure of the Cr.LSU intron in the 23S.5DG
b
pre-RNA
The unusual metal specificity, as well as other atypical
features of Cr.LSU (see Discussion), led us to
study the global tertiary structure of the intron using
Fig. 3. Mg
2+
dependence of ribozyme activity at fixed Mn
2+
con-
centrations. (A) G-dependent cleavage of 23S.5DG
b

pre-RNA at
varying Mg
2+
concentration, and 0 mM (top) or 12 mM (bottom)
MnCl
2
; the reactions also contained 150 lM GMP, and were incuba-
ted for 40 s. They were separated on a denaturing polyacrylamide
gel, which was phosphorimaged. (B) Mg
2+
concentration curves at
fixed Mn
2+
concentrations. G-dependent cleavage of 23S.5DG
b
was performed as in (A), except for using the indicated Mn
2+
con-
centrations. The cleavage product (InDG
b
) was quantified, and
expressed as a percentage of total RNA [Relative InDG
b
(%)]. The
data were curve-fitted to obtain Hill coefficients as described in
Experimental procedures.
Fig. 4. Mn
2+
dependence of ribozyme inhibition at fixed Mg
2+

con-
centrations. G-dependent cleavage of 23S.5DG
b
pre-RNA was per-
formed with the indicated concentrations of MnCl
2
(0–30 mM), 10
or 25 m
M MgCl
2
, and 150 lM GMP for 40 s. The reactions were
analyzed as in Fig. 3.
T C. Kuo et al. Mn
2+
inhibition and structure of Cr.LSU ribozyme
FEBS Journal 273 (2006) 2631–2644 ª 2006 The Authors Journal compilation ª 2006 FEBS 2635
hydroxyl radical cleavage. This analysis was carried
out by incubating end-labeled, Mg
2+
-folded intron
(InDG
b
) with Fe
2+
-EDTA [7,8]. It should be empha-
sized that the RNA structure revealed by Fe
2+
-EDTA
is an averaged image of the RNA molecules in solu-
tion. However, since the kinetic data indicate that 85–

90% of 23S.5DG
b
pre-RNA is functionally similar, we
assume that the predominant signal in the protection
pattern is from active ribozyme.
Figure 5A shows an Fe
2+
-EDTA cleavage analysis
performed at 0–25 mm Mg
2+
. In the absence of Mg
2+
(lanes 1 and 12), cleavage occurs throughout the mole-
cule; this profile was defined as background. As the
Mg
2+
concentration increased, different regions of the
RNA became either more sensitive to, or more protec-
ted from, cleavage or cleavage was relatively
unchanged. Figure 5A shows that the overall cleavage
profile changes gradually with Mg
2+
, a trend that
appears to hold for most nucleotide positions (solid
and open bars in Fig. 5A, lanes 2–11). It should be
noted, however, that the degree of change at some
nucleotides, such as the protection of P3¢ (Fig. 5A),
appears to be greater at the higher concentrations of
Mg
2+

(> 5 or 6 mm), suggesting that this region may
have a higher Mg
2+
requirement for folding. There is
also a general increase in the amplitudes of the clea-
vage ⁄ protection peaks at the highest (25 mm)Mg
2+
concentration tested. A similar result is apparent in
some of the Tt.LSU L-21 ScaI protection data [33],
and may reflect a greater overall stability of the RNA
at high Mg
2+
.
Since 23S.5DG
b
pre-RNA is fully active at 25 mm
Mg
2+
, we inferred the native structure of the ribozyme
by comparing the cleavage profiles at 0 and 25 mm
Mg
2+
(Fig. 5B). For those nucleotides that showed
differential cleavage, the extent of the difference was
2–4-fold at most positions; Fig. 5C is the difference
profile (for 0 and 25 mm Mg
2+
) plotted by nucleotide
position. It should be noted that similar results were
obtained when cleavage time, or concentration of

Fe
2+
-EDTA, was varied over a four-fold range, or
when dithiothreitol was replaced by ascorbate and
hydrogen peroxide (not shown).
To better visualize the locations of exposed and pro-
tected regions of the InDG
b
intron, data from the
difference plot were converted to a color palette and
mapped onto the proposed secondary structure
(Fig. 5D). Focusing on the critical P7–P3–P8 domain,
the 5¢ strand of P7, which includes the G-binding site,
is cleaved, whereas the 3¢ strand is protected. The
3¢ strand of P3 is also protected, but the 5¢ strand is
neither protected nor particularly exposed. The part of
J8 ⁄ 7 proximal to P8 is protected, whereas the residues
close to P7 are strongly cleaved; also, P8 itself is pro-
tected, but L8 is not. For the P9 subdomains, most of
P9 is protected, but most of P9.1 is neutral. The
5¢ strand of P9.0 is protected despite the absence of
the 3¢ strand. For the idiosyncratic P7.1 and P7.2
domains, the helices are weakly protected, except for
the 3¢ strand of P7.2, which is strongly protected, and
both loops are cleaved.
For the other major stacked-helix domain, P5–P4–P6,
as well as P2, the extent of protection or exposure was
mostly neutral or relatively weak compared with the
P7–P3–P8 domain. However, P5, P6 and minor parts of
P5a and P6a are protected. Also, J4 ⁄ 5 is weakly protec-

ted, but J5 ⁄ 4 is weakly cleaved. Of particular signifi-
cance is the observed cleavage of J6 ⁄ 7 and the neutrality
of J3 ⁄ 4; these joining segments link the two major
domains and form base triples with P4 and P6.
The overall Fe
2+
-EDTA cleavage ⁄ protection pat-
tern for this group IA3 intron (InDG
b
) has many
Fig. 5. Hydroxyl radical cleavage with Fe
2+
-EDTA of the intron ribozyme. (A) Fe
2+
-EDTA cleavage and protection pattern of 5¢ end-labeled
InDG
b
RNA as a function of Mg
2+
concentration. The InDG
b
RNA, labeled at its 5¢ end through G-dependent cleavage of 23S.5DG
b
pre-RNA,
was incubated with Mg
2+
(final concentration indicated above lanes 2–12) and then cleaved with Fe
2+
-EDTA. The reactions were resolved
on gels of 5–12% polyacrylamide with ladders and other markers. The locations of structural elements (P5, P6, etc.) are marked to the left

of the gel image, and RNA sizes to the right. Rectangular bars indicate areas where cleavage is enhanced (filled rectangles), or reduced
(open rectangles), with increasing Mg
2+
. Other lanes are: S, starting RNA; Mn, S RNA cleaved with Mn
2+
at GAAA sequences (nucleotides
56, 257 and 323); A, G, A ⁄ U, and C, enzymatic sequence ladder of S RNA; OH, partial alkaline hydrolysis of S RNA; and M, 5¢ end-labeled
DNA size markers. The composite figure is from 8% (upper) and 12% (lower) polyacrylamide gels. (B) Phosphorimager scans of Fe
2+
-EDTA
cleavage of InDG
b
RNA with 0 and 25 mM Mg
2+
. The RNA was probed as in (A) and the samples resolved on a 12% polyacrylamide gel,
which was phosphorimaged. (C) Difference plot of Fe
2+
-EDTA cleavage at 0 and 25 mM Mg
2+
. Regions of cleavage are positive (> 0), and
regions of protection are negative (< 0). The plot was compiled from cleavage profiles obtained on a series of 5–12% polyacrylamide gels.
(D) Protection and cleavage patterns mapped onto the predicted secondary structure; the data are from (C). Protected nucleotides are
shades of red and orange, whereas cleaved nucleotides are blue shades; a color bar is given on the bottom right. The analysis under these
conditions was repeated twice with similar results. The accuracy of the protection data at the nucleotide level is ± 0 for residues £ 180, ± 1
for residues 181–270, and ± 2 for residues 271–342. The cleavage ⁄ protection patterns of nucleotides 1–14 and 343–378 were insufficient
relative to background, and are not indicated. The red arrowheads indicate sites of cleavage by Mn
2+
-GAAA ribozymes [38].
Mn
2+

inhibition and structure of Cr.LSU ribozyme T C. Kuo et al.
2636 FEBS Journal 273 (2006) 2631–2644 ª 2006 The Authors Journal compilation ª 2006 FEBS
similarities to the introns from other subgroups [7,8]
(see also below). However, it is atypical because of the
relative lack of protection of the catalytic domain and
the junction nucleotides that link it to the other major
domain. We attempted to probe the InDG
b
RNA with
Fe
2+
-EDTA in the presence of Mn
2+
, but cleavage was
T C. Kuo et al. Mn
2+
inhibition and structure of Cr.LSU ribozyme
FEBS Journal 273 (2006) 2631–2644 ª 2006 The Authors Journal compilation ª 2006 FEBS 2637
too poor to obtain a clear result. This is most likely due
to the similar affinities of EDTA for Mn
2+
and Fe
2+
,
and the fact that Mn
2+
is in large excess in these
reactions [34].
Discussion
Kinetic analysis of Mn

2+
inhibition
The group I ribozyme literature indicates that divalent
metals are required for tertiary folding and catalysis,
and that Mg
2+
or Mn
2+
can satisfy these functions.
However, the activity of some group I introns, inclu-
ding three of the five introns in Chlamydomonas rein-
hardtii (Cr.psbA2, Cr.psbA3, and Cr.LSU) is inhibited
by Mn
2+
[26]. We wanted to determine for the
Cr.LSU intron if this metal specificity is due to a more
stringent requirement for catalysis, or folding, or both.
The data show that Mn
2+
does not have a significant
effect on the kinetic parameters (K
1/2
G
and k
cat
) of the
23S.5DG
b
ribozyme. Thus, Mn
2+

does not inhibit
binding of the guanosine nucleotide, and nor does it
suppress cleavage chemistry, suggesting that, as in the
case of Tt.LSU and some other group I ribozymes,
Mn
2+
can support catalysis by Cr.LSU. The data do
indicate, however, that Mn
2+
inhibits formation of the
active ribozyme, presumably by causing misfolding.
This result was unexpected, because of the extensive
work with the Tt.LSU ribozyme indicating that the
metal requirement for ribozyme folding is less restrict-
ive than that for catalysis [14,15].
It is also surprising that Mn
2+
-induced misfolding of
this ribozyme is highly cooperative, with a Hill coeffi-
cient of approximately 6. In fact, it is more cooperative
than Mg
2+
induction of active ribozyme (Hill coeffi-
cient of approximately 3). High cooperativity is consis-
tent with the great stability of the RNA formed in
Mn
2+
, which converts very slowly to a form capable of
binding GMP and reacting. The higher Hill coefficient
could also indicate that Mn

2+
binds to additional
(three) sites on the ribozyme that do not bind Mg
2+
.
However, the fact that Mg
2+
can completely block
Mn
2+
inhibition would suggest that they bind to the
same sites. Hence, it may be that they bind to the same
basic locations, but that Mn
2+
binds with slightly dif-
ferent configurations at some key sites, resulting in
inhibition. In this respect, it should be noted that,
based on mixed-metal titrations of 23S.5 self-splicing,
Mn
2+
can functionally replace Mg
2+
at some sites [26].
Mn
2+
inhibition of 23S.5DG
b
is partially alleviated
by GMP, especially at lower metal concentrations.
The GMP concentration dependence of this effect

indicates that the nucleotide is acting via the G
binding site in P7. An obvious explanation for this
result is that binding of GMP prevents an inhibi-
tory Mn
2+
from binding to this site. Interestingly,
2¢-dGTP inhibits Pb
2+
cleavage of the T4-td intron
at the bulge nucleotide in P7 [30], which is very close
to the bound XG in recent crystal structures [35–37].
With Cr.LSU, we did not see a similar specific clea-
vage with Pb
2+
or other metals [38], so the same
experiment could not be performed. It should be
noted, however, that metal-dependent cleavage reveals
only a small fraction of metal-binding sites in RNA
[29]. An alternative explanation for GMP-induced
folding of the ribozyme in Mn
2+
is that binding of
the cosubstrate to its site induces a conformational
change in the RNA that inhibits Mn
2+
binding at
another inhibitory site(s). It may be relevant that
in vitro-evolved Tt.LSU ribozymes capable of using
Ca
2+

as sole divalent cation had several nucleotide
substitutions clustered about the G binding site and
the triple base pairs at the P4–P6 junction [39]. We
tried unsuccessfully to select variants of Cr.LSU act-
ive with Mn
2+
from pools of mutants generated by
error-prone PCR (T C. Kuo & D. L. Herrin, unpub-
lished results). Based on the high Hill coefficient
reported here, however, the failure of that experiment
may be attributed to the difficulty in overcoming the
relatively high number of inhibitory Mn
2+
-binding
sites in Cr.LSU.
We previously identified six Mn
2+
-binding sites in
Cr.LSU based on site-specific cleavages at pH > 7
[38]. These sites all contain the sequence GAAA, and
cleavage occurs between G and A. The cleavage effi-
ciency, however, varied between sites, and correlated
with the predicted secondary structure. Also, the addi-
tion of sufficient Mg
2+
to induce self-splicing did not
affect the Mn
2+
cleavage rates at the various sites, sug-
gesting that most of the intron’s secondary structure

forms correctly in Mn
2+
. The experiments herein were
performed at pH 6 (self-splicing of Cr.LSU is efficient
at pH 6–9 [23]), and for shorter times to limit the
Mn
2+
-induced cleavages. Thus, these data would sug-
gest that Mn
2+
is probably inhibiting tertiary folding
of Cr.LSU . In this respect, we note the published evi-
dence [40] that correct tertiary folding of the Azoarcus
intron is specific for Mg
2+
. However, it is possible that
subtle but important changes in secondary structure
could also be involved. For example, the crystal struc-
tures of yeast tRNA
Phe
in Mg
2+
, or in a mixture of
Mg
2+
and Mn
2+
, are quite similar but not identical;
residue D16 (D loop) forms a base pair with U59
(TWC loop) only in the latter condition [41].

It is unlikely that the three remaining GAAA-Mn
2+
cleavage sites in the 23S.5DG
b
ribozyme (three are
Mn
2+
inhibition and structure of Cr.LSU ribozyme T C. Kuo et al.
2638 FEBS Journal 273 (2006) 2631–2644 ª 2006 The Authors Journal compilation ª 2006 FEBS
deleted) are principal targets of Mn
2+
inhibition, since
Mn
2+
cleavage is not cooperative [38]; however, one
of them could be a site of Mn
2+
inhibition. The best
candidate would probably be J4 ⁄ 5, which is strongly
cleaved with Mn
2+
[38], and is involved in substrate
recognition during splicing [35,36]. Changing the
GAAA in J4 ⁄ 5 to GACA blocked Mn
2+
cleavage and
doubled the Mg
2+
requirement for Cr.LSU self-spli-
cing (T C. Kuo, S. P. Holloway & D. L. Herrin,

unpublished results), suggesting that it might be an
important metal-binding site. Evidence for a functional
metal interaction with the J4 ⁄ 5-GAAA region of an
Anabaena intron was reported recently [42].
Structural and functional idiosyncrasies of the
23S.5DG
b
intron ribozyme
To help us understand the noncanonical properties of
Cr.LSU, the intron’s (InDG
b
) structure was analyzed
using Fe
2+
-EDTA, which has been used extensively to
view tertiary-folded group I ribozymes. The chelated
iron generates hydroxyl radicals that cleave riboses,
unless they are protected by RNA–RNA interactions
[7]. Figure 6 compares the Cr.LSU protection pattern,
which is the first for a subgroup IA3 intron, with five
ribozymes from other subgroups: L-21 ScaI of Tt.LSU
[7], T4.nrdD [8], T4.td [8], Sc.bI5 [43], and Azoarcus
Fig. 6. Comparison of the Fe
2+
-EDTA protection patterns of group I ribozymes. Residues protected from Fe
2+
-EDTA cleavage are indicated
by filled squares with white letters. The data for Cr.LSU (A) are from Fig. 5; for Tt.LSU (B) from [7]; for T
4
.sunY (C) and T

4
.td (D) from [8];
for bI5 (E) from [43]; and for Azoarcus pre-tRNA
Ile
(F) from [44]. Domain–domain interactions in introns B–F are indicated by dashed lines. In
(A), the lightly shaded nucleotides in the L9.1 and P7.1 loops may form a novel base-pairing interaction; the gray dashed line between L2
and P8 also indicates a possible interaction. Solid arrows indicate sites of Mn
2+
cleavage in Cr.LSU and T4-td; open arrows are sites of Pb
2+
cleavage in Tt.LSU, T4.td, and T4.nrdD; and arrowheads in Azoarcus indicate the 5¢ and 3¢ splice sites.
T C. Kuo et al. Mn
2+
inhibition and structure of Cr.LSU ribozyme
FEBS Journal 273 (2006) 2631–2644 ª 2006 The Authors Journal compilation ª 2006 FEBS 2639
pre-tRNA
Ile
[44]. All of these introns can self-splice,
although the Mg
2+
requirement for bI5 is higher than
that for the others. Focusing on the catalytic domain
(P8–P3–P7), the P3 3¢-strand is the only element that is
uniformly protected in all ribozymes, although they all
have parts of P7, J8 ⁄ 7 and P9.0 protected. The
Cr.LSU pattern is distinct in that the first three nucle-
otides of P7, and the 3¢-half of J8 ⁄ 7, are not protected.
The first three nucleotides of P7 are part of the G
binding site, as are the terminal nucleotides of J6 ⁄ 7
and J8 ⁄ 7 [35–37]; the latter nucleotides are also not

protected in the InDG
b
RNA. It should be noted that
the three terminal nucleotides of Cr.LSU, including
the XG, are not present in the InDG
b
RNA, which
presumably also lacks P9.0 (Fig. 6A). It is possible this
has an effect on the protection pattern. However, the
kinetic parameters (k
cat
and K
1/2
G
) for G-dependent
cleavage by 23S.5DG
b
are very similar to those
obtained for the first step of self-splicing by 23S.5 [18],
indicating that P9.0 is not important for core ribozyme
activity (it is probably important for the second step
of splicing [45]). It is also noteworthy that the 3¢-ter-
minal nucleotides were also absent from the Tt.LSU
ribozyme mapped with Fe
2+
-EDTA [7]. To conclude,
the G binding site and flanking nucleotides of Cr.LSU
are more accessible to solvent (i.e. less internalized)
than are those of other group I ribozymes studied to
date. It is also intriguing that the kinetic data implicate

the G binding site as playing an important role in
Mn
2+
inhibition.
Why is the active site less internalized in the
InDG
b
RNA? The lack of protection of J6 ⁄ 7 and
J3 ⁄ 4, which are involved in triple base pairs with P4
and P6 [3,35–37], and the relatively weak overall
protection of the P4–P5–P6 domain (Fig. 5C), indi-
cate that this domain is not tightly packed against
the catalytic domain. Analysis of the predicted sec-
ondary structure suggests that Cr.LSU may be some-
what deficient in interactions between these domains.
For example, it seems to lack the L9 · P5 interac-
tion found in the other ribozymes (Fig. 6). This
interaction is primarily between the L9 tetraloop and
the second and third nucleotides of P5 [3]; however,
in Cr.LSU, P5 is only two base pairs. Although
deletion of the P4–P5–P6 domain from the T4.td
intron suggests that it is not essential [46], disruption
of P6 in Cr.LSU obliterated self-splicing, and point
mutations in P4 strongly decreased splicing in vitro
and in vivo, indicating that the P5–P4–P6 domain is
important for Cr.LSU [25]. The Li et al. study [25]
also indicates that Cr.LSU is more sensitive to single
nucleotide substitutions in the core than Tt.LSU
or T4.td, which is consistent with fewer tertiary
interactions. Li et al. [25] also isolated nuclear gene

suppressors of the P4 mutations; thus, based on these
data, it is reasonable to speculate that one or more
of these suppressors promote interaction between the
two major domains.
There are other functional differences between
Cr.LSU and the Tt.LSU and phage T4 introns besides
Mn
2+
inhibition that may reflect the distinctive struc-
tures. It was shown that Pb
2+
promotes specific cleav-
ages in the P7 and J8 ⁄ 7 regions of Tt.LSU and the
phage introns (Fig. 6B–D) in the presence of Mg
2+
[30]. However, we did not observe similar specific
cleavages in Cr.LSU with Pb
2+
(or other cations) [38].
Cr.LSU is also more resistant to inhibition by polycat-
ionic aminoglycoside antibiotics, such as neomycin B
[47], requiring 25–50-fold higher concentrations to
inhibit self-splicing in vitro by 50% (T C. Kuo,
Y. Bao & D. L. Herrin, unpublished results). It is
noteworthy that neomycin inhibits the T4.td intron by
binding at the G binding site and displacing one or
two critical Mg
2+
ions [48]. We propose that the lack
of Pb

2+
cleavage, and the apparent absence of a high-
affinity, inhibitory site for neomycin, could be conse-
quences of the more open structure of Cr.LSU, which
should present a less electronegative environment at
the active site. It would be interesting to know if other
group I introns that are inactive with Mn
2+
[24] have
properties similar to those of Cr.LSU, including the
more stringent metal requirement for ribozyme forma-
tion.
The tertiary-folded structure of the InDG
b
RNA in
Mn
2+
would probably have been instructive, but
unfortunately, the Fe
2+
-EDTA cleavage pattern was
strongly inhibited by Mn
2+
under these conditions,
presumably due to the similar affinities of EDTA for
Mn
2+
and Fe
2+
(K

d
approximately 10
)14.1
) [34]. It
may be possible to accomplish this with synchrotron
X-ray [49] or peroxynitrous [50] cleavage, although the
former requires special equipment, and the latter rea-
gent is not as easy to use as Fe
2+
-EDTA.
An intriguing, though speculative, implication of the
relatively open ground-state structure of InDG
b
is that
the ribozyme might undergo a transient internalization
of P7, J8 ⁄ 7 and J6 ⁄ 7 after binding GMP to start the
reaction. Binding of the guanosine nucleotide by the
Tt.LSU ribozyme is very slow, and is believed to
induce a local rearrangement of the G binding site
[51]; these authors also argued that an incompletely
preformed G binding site could promote specificity.
The posited conformational rearrangement of Cr.LSU
would seem to be more extensive, but if it does happen
and is necessary, then that dynamic change could be a
key step that is inhibited by Mn
2+
.
Mn
2+
inhibition and structure of Cr.LSU ribozyme T C. Kuo et al.

2640 FEBS Journal 273 (2006) 2631–2644 ª 2006 The Authors Journal compilation ª 2006 FEBS
Experimental procedures
DNA templates and in vitro RNA synthesis
The DNA template for in vitro synthesis of 23S.5DG
b
pre-
RNA, PCR23S.5DG
b
, was generated by PCR using Taq
polymerase and the manufacturer’s conditions (Perkin
Elmer-Cetus, Norwalk, CT, USA). The template was plas-
mid pGEM23S.5 [18], and oligos 104 and 158 served as the
5¢ and 3¢ primers, respectively. Plasmid pGEM23S.5 contains
a shortened intron (380 bp), 26 bp of the 5¢ exon, and 25 bp
of the 3¢ exon. Oligo 104 (45 nucleotides, TAATACGACTC
ACTATAGGGATCGAATTCTGGGTTCAAAACGTAA)
contains, in order, a T7 RNA polymerase promoter (nucleo-
tides 1–17), a six-nucleotide transcription enhancer, an
EcoRI restriction site, 14 nucleotides of authentic 5¢ exon,
and the first two nucleotides of the Cr.LSU intron. Oligo 158
(26 nucleotides, GAAATT TTAAAGCCGAATAAAACTTG)
ends 3 nucleotides before the 3¢-end of the intron. The
temperature program was 30 cycles of 94 °C, 65 °C, and
72 °C (1 min each). Transcription of PCR23S.5DG
b
and
purification of the RNA on denaturing gels was performed
as described [18].
G-dependent cleavage of 23S.5DG
b

pre-RNA, and
quantification
Prior to the reaction, the RNA was denatured by heating
(90 °C, 1 min) and cooled slowly (1 C°⁄5s) to 37°Cin
the absence of divalent metals. The RNA (2–8 nm, 1500–
8000 c.p.m.) was preincubated with MgCl
2
and ⁄ or MnCl
2
in 50 mm Mes-KOH pH 6.0 (the relatively low pH redu-
ces Mn
2+
-promoted cleavage at GAAA) at 37 °C for
20 min, and then G-dependent cleavage was initiated by
raising the temperature to 47 °C and adding GMP
(2–300 lm). The reactions were performed in siliconized
tubes in either 5 or 50 lL (time course). The reactions
were stopped with 1.2 volumes of 80% formamide, 0.1%
bromophenol blue, 0.1% xylene cyanol, 100 mm EDTA,
pH 8.0, and the RNA was denatured by heating at 65 °C
for 3 min before electrophoresis on 4% polyacryla-
mide ⁄ 8 m urea gels. The dried gels were imaged with a
phosphorimager, and quantified using ImageQuant
(Molecular Dynamics, Sunnyvale, CA, USA) [18].
Estimation of the catalytic constant, k
cat,
and
K
1/2
G

for G-dependent cleavage
For the 23S.5DG
b
pre-RNA, the percentage remaining at
time t (% Pre) is given by:
ð%PreÞ¼Pre=½Pre þ InDG
b
ð1 þ 26=377Þ Â 100% ð1Þ
Pre and InDG
b
are the amounts in c.p.m. of the pre-RNA
and intron, respectively, and 26 ⁄ 377 accounts for the liber-
ated 5¢ exon, which was not measured. The percentages were
plotted on semilogarithmic scales against time. The
observed decrease in pre-RNA was fitted to the two-com-
ponent first-order equation:
ð%PreÞ
t
¼ A expðÀk
a
 tÞþB expðÀk
b
 tÞð2Þ
A and k
a
are the percentage and observed rate constant for
the fast-reacting pre-RNA, and B and k
b
are the same
parameters for the slow fraction; t is reaction time (min).

These parameters were determined at different GMP con-
centrations (1–300 lm). The catalytic constant (k
cat
) and
half-maximal GMP concentration (K
1/2
G
) were obtained by
fitting k
a
and [GMP] to:
k
a
¼ k
cat
½GMP=ðK
G
1=2
þ½GMPÞ ð3Þ
K
1/2
G
is not a true Michaelis–Menten constant, because the
ribozyme does not perform turnover catalysis.
Estimating the minimum number of required
metal ions: Hill analysis
Rationale
From Scheme 1, the fraction of active pre-RNA, f(pre-
RNA)
active

, is related to Mg
2+
and Mn
2+
by:
½f ðpre-RNAÞ
active
¼½pre-RNA ÀnMg
2þ

active
=½pre-RNA
total
¼½Mg
2þ

n
=f½Mg
2þ

n
þ K
Mg
2þ
ð1 þ½Mn
2þ

m
=K
Mn

2þ
Þg ð4Þ
The [pre-RNA–nMg
2+
]
active
term is the concentration of
active ribozyme, and [pre-RNA]
total
is the RNA concentra-
tion. K
Mg
2+
and K
Mn
2+
are the apparent dissociation con-
stants for the RNA–nMg
2+
and RNA–mMn
2+
complexes.
To determine n, f(pre-RNA)
active
at varying Mg
2+
concen-
tration was fitted to:
f ðpre-RNAÞ
active

¼½Mg
2þ

n
=ð½Mg
2þ

n
þ C
1
Þð5Þ
where
C
1
¼ K
Mg
2þ
ð1 þ½Mn
2þ

m
=K
Mn
2þ
Þ
To determine m, f(pre-RNA)
active
was fitted to
f ðpre-RNAÞ
active

¼ C
2
=ðC
3
þ½Mn
2þ

m
Þð6Þ
where
C
2
¼½Mg
2þ

n
 K
2þ
Mn
=K
2þ
Mg
; and C
3
¼ C
2
þ K
2þ
Mn
In principle, the absolute value of f(pre-RNA)

active
can
only be obtained from the value of A (Eqn 2) in a time-
dependent reaction. However, for determining n and m,
relative values from single initial-time measurements are
sufficient. At the initial time points, the vast majority of
product comes from the highly active fraction. Hence, plots
of the percentage of total RNA that is InDG
b
yield n and
m from Eqns 5 and 6.
T C. Kuo et al. Mn
2+
inhibition and structure of Cr.LSU ribozyme
FEBS Journal 273 (2006) 2631–2644 ª 2006 The Authors Journal compilation ª 2006 FEBS 2641
Procedure
G-dependent cleavage of internally labeled 23S.5DG
b
pre-
RNA (2000–3000 c.p.m.) was performed for 40 s at the
indicated concentrations of Mg
2+
and Mn
2+
, 150 lm (sat-
urating) GMP, 50 mm Mes-KOH pH 6.0 at 47 °C. The
reactions were separated on denaturing gels, and quantified
as described above. The amount (c.p.m.) of InDG
b
formed

at i m m Mg
2+
and j mm Mn
2+
was first expressed as
%InDG
b
using the following definition:
%InDG
bi;j
¼ 100% Â InD G
b
=½pre-RNA þ InDG
b
ð1 þ 26=377Þ
ð7Þ
InDG
b
and pre-RNA are in c.p.m., and the 26 ⁄ 377
accounts for the 5¢ exon. This normalizes the slight
differences (± 10%) in RNA loaded per lane. Relative
InDG
b
percentage at i mm Mg
2+
and j mm Mn
2+
was
defined as:
Relative InDG

b
% ¼½ð%InDG
bi;j
Þ=ð%InDG
b max
Þ Â 100% ð8Þ
%InDG
b max
is the maximum %InDG
b
obtained at varying
Mg
2+
and fixed Mn
2+
concentrations, or vice versa. The
value of Relative InDG
b
% replaced f(pre-RNA)
active
in
Eqns 5 and 6, and the values of n and m were obtained by
curve fitting with KaleidaGraph (Synergy).
Probing the structure of the 23S.5DG
b
ribozyme
with Fe
2+
-EDTA
The InDG

b
RNA was 5¢-labeled by G-dependent cleavage
of nonradioactive 23S.5DG
b
pre-RNA (3 pmol) with 20 lm
[a-
32
P]GTP (3000 CiÆmmol
)1
), 25 mm Mg
2+
for 5 min at
47 °C. The RNA was purified, renatured as described
above, and then preincubated at 47 °C for 15 min in
0–250 mm MgCl
2
,15mm Tris ⁄ HCl, pH 7.5. Cleavage was
effected by adjusting the mixture to 1 mm Fe(NH
4
)
2
(SO
4
)
2
,
2mm EDTA, 5 mm dithiothreitol [7] and incubating at
42 °C for 50 min. The reactions were terminated with
thiourea (10 mm) and gel-loading solution. For gel mark-
ers, end-labeled InDG

b
RNA (2–4 · 10
5
c.p.m.) was diges-
ted with RNases U2, T1, Phy M, or CL3, cleaved with
alkali, and cleaved with Mn
2+
(3 mm MnCl
2
, 0.2 m KCl,
50 mm Tris ⁄ HCl, pH 7.4, at 47 °C for 15 min). DNA size
markers were prepared by restricting a 5¢ end-labeled
EcoRI ⁄ HindIII insert (443 bp) of plasmid pGEM23S.5
[18] with HphI, BstXI, and XhoI. Samples were denatured
and separated on a series of denaturing polyacrylamide
(5–12%) gels, powered at approximately 50 W (50–55 °C);
the gels were imaged and quantified as above. The
data were corrected for background hydrolysis, and
normalized to adjust for loading differences (by integrating
the signal along the entire length of the lanes) [14].
Using the sequence ladders as a guide, positions in the
lanes could be related to corresponding nucleotides in the
sequence.
Acknowledgements
OWO and DLH were supported by a grant from the
Department of Energy (DE-FG03-02ER15352) and the
Robert A. Welch Foundation (F-1164). TCK was sup-
ported by a grant from the Taiwan National Science
Council (NSC 94-2218E038) and Taipei Medical Uni-
versity (TMU94AE1B05).

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