Gutell 072.jmb.2000.301.0265

Function of Tyrosyl-tRNA Synthetase in Splicing
Group I Introns: An Induced-fit Model for Binding to
the P4-P6 Domain Based on Analysis of Mutations at
the Junction of the P4-P6 Stacked Helices
Xin Chen, Robin R. Gutell and Alan M. Lambowitz*
Institute for Cellular and
Molecular Biology, Department
of Chemistry and Biochemistry
and Sections of Molecular
Genetics and Microbiology and
Integrative Biology, School of
Biological Sciences, University
of Texas at Austin, Austin
TX 78712, USA
We used an Escherichia coli genetic assay based on the phage T4 td intron
to test the ability of the Neurospora crassa mitochondrial tyrosyl-tRNA
synthetase (CYT-18 protein) to suppress mutations that cause structural
defects around its binding site in the P4-P6 domain of the group I intron
catalytic core. We analyzed all possible combinations of nucleotides at
either P4 bp-1 or P6 bp-1, which together form the junction of the P4-P6
stacked helices, and looked for synergistic effects in double mutants.
Most mutations at either position inhibit self-splicing, but can be sup-
pressed by CYT-18. CYT-18 can compensate ef®ciently for mutations that
disrupt base-pairing at either P4 bp-1 or P6 bp-1, for mutations at P6 bp-
1 that disrupt the base-triple interaction with J3/4-3, and for nucleotide
substitutions at either position that are predicted to be suboptimal for
base stacking, based on the analysis of DNA four-way junctions. How-
ever, CYT-18 has dif®culty suppressing combinations of mutations at
P4 bp-1 and P6 bp-1 that simultaneously disrupt base-pairing and base
stacking. Thermal denaturation and Fe(II)-EDTA analysis showed that
mutations at the junction of the P4-P6 stacked helices lead to grossly
impaired tertiary-structure formation centered in the P4-P6 domain. CYT-
18-suppressible mutants bind the protein with Kd values up to 79-fold
higher than that for the wild-type intron, but in all cases tested, the koff
value for the complex remains within twofold of the wild-type value,
suggesting that the binding site can be formed properly and that the
increased Kd value re¯ects primarily an increased kon value for the bind-
ing of CYT-18 to the misfolded intron. Our results indicate that the P4/
P6 junction is a linchpin region, where even small nucleotide substi-
tutions grossly disrupt the catalytically-active group I intron tertiary
structure, and that CYT-18 binding induces the formation of the correct
structure in this region, leading to folding of the group I intron catalytic
core.
# 2000 Academic Press
Keywords: aminoacyl-tRNA synthetase; group I intron; ribozyme;
RNA splicing; RNA structure*Corresponding author
Introduction
Group I introns fold into a conserved tertiary
structure that is required for catalytic activity
(Michel & Westhof, 1990; Cech & Golden, 1999).
Although some group I introns are self-splicing
in vitro, most, if not all, require proteins to facilitate
RNA folding in vivo (reviewed by Lambowitz &
Perlman, 1990; Lambowitz et al., 1999). These pro-
teins either bind speci®cally to the intron RNAs
(Weeks & Cech, 1995; Shaw & Lewin, 1995;
E-mail address of the corresponding author:
lambowitz@mail.utexas.edu
Abbreviations used: DMS, dimethyl sulfate; Kan,
kanamycin; LSU, large subunit ribosomal RNA; MM,
minimal medium; MMT, minimal medium plus
thymine; mt, mitochondria; ORF, open reading frame;
phenol-CIA, phenol/chloroform/isoamyl alcohol
(25:24:1); TS, thymidylate synthase; TTM, minimal
medium plus thymine and trimethoprim; TyrRS,
tyrosyl-tRNA synthetase.
doi:10.1006/jmbi.2000.3963 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 301, 265±283
0022-2836/00/020265±19 $35.00/0 # 2000 Academic Press

Saldanha et al., 1996; Ho et al., 1997) or interact
non-speci®cally as RNA chaperones (Coetzee et al.,
1994; Zhang et al., 1995). The Neurospora crassa
mitochondrial tyrosyl-tRNA synthetase (mt
TyrRS), or CYT-18 protein, functions in splicing by
binding speci®cally to the group I intron catalytic
core (Guo & Lambowitz, 1992; Caprara et al.,
1996a,b). The CYT-18 protein normally functions in
splicing the mt large rRNA (mt LSU) intron and
other group I introns in Neurospora mitochondria
(Mannella et al., 1979; Akins & Lambowitz, 1987;
Wallweber et al., 1997). However, it can also bind
to and splice diverse group I introns from other
organisms, including introns belonging to different
structural subgroups, so long as its binding site in
the catalytic core is not obscured by peripheral
RNA structures (Guo & Lambowitz, 1992; Mohr
et al., 1992, 1994). As discussed below, the splicing
function of CYT-18 may re¯ect its ability to recog-
nize conserved tRNA-like structural features of the
group I intron catalytic core (Caprara et al., 1996b).
The group I intron catalytic core consists of two
extended helical domains: the P4-P6 domain
formed by the coaxial stacking of the secondary
structure elements P5, P4, P6 and P6a, and the P3-
P9 domain formed by P8, P3, P7 and P9 (Figure 1;
Michel & Westhof, 1990; Golden et al., 1998). The
juxtaposition of the two domains forms the intron's
active site, which contains the binding sites for the
5H
and 3H
splice sites (helices P1 and P10, respect-
ively) and the guanosine cofactor (P7). The orien-
tation of the two helical domains is maintained by
the base-triple interactions between the P4-P6
domain and the adjoining single-stranded junction
regions J3/4 and J6/7, as well as long-range ter-
tiary interactions, such as the base triple between
P4 bp-5 and J8/7-5 and the tetraloop/minor
groove interaction between L9 and P5 (Michel &
Westhof, 1990; Tanner & Cech, 1997). RNA folding
studies with the Tetrahymena LSU intron indicate
that the P4-P6 domain folds ®rst to form a scaffold
for the assembly of the P3-P9 domain (Murphy &
Cech, 1993; Laggerbauer et al., 1994; Zarrinkar &
Williamson, 1994; Scalvi et al., 1998).
The N. crassa mt LSU and ND1 introns, which
are dependent on CYT-18 for splicing both in vitro
and in vivo, are not detectably self-splicing
(Mannella et al., 1979; Garriga & Lambowitz, 1986;
Wallweber et al., 1997). RNA structure mapping
showed that these introns could form most of the
short-range helices of the conserved group I intron
secondary structure, but otherwise remain largely
unfolded in the absence of CYT-18. CYT-18
restores splicing by binding to the unfolded intron
RNAs and promoting formation of the catalyti-
cally-active group I intron tertiary structure
(Caprara et al., 1996a,b). RNA footprinting exper-
iments with the N. crassa mt LSU and ND1 introns
showed that CYT-18 protects the phosphodiester
backbone on the surface of the catalytic core oppo-
site the active-site cleft. Most of the putative CYT-
18 protection sites were in the P4-P6 domain, clus-
tered around the junction of the P4-P6 stacked
helix, but additional sites were found in P3, P8 and
P9 in both introns and in P7.1/P7.1a and L9 in the
mt LSU intron (Caprara et al., 1996a). Consistent
with these results, a small RNA containing only
the P4-P6 domain of the mt LSU intron could bind
CYT-18 independently (Kd ˆ 130 pM at 22
C), but
additional sequences from the P3-P9 domain were
required for maximal binding (Kd ˆ 22 pM; Guo
Lambowitz, 1992; Saldanha et al., 1996). By con-
trast, the isolated P3-P9 domain could not bind
CYT-18 independently, presumably because it does
not fold correctly in the absence of the P4-P6
domain (Saldanha et al., 1996). Together, these stu-
dies suggested a model in which CYT-18 binds
®rst to the P4-P6 domain to form a scaffold for the
assembly of P3-P9 and then makes additional con-
tacts with the P3-P9 domain to stabilize the two
helical domains in the correct relative orientation
to form the intron's active site (Caprara et al.,
1996a,b).
This model for CYT-18 function is supported
further by studies with the Tetrahymena LSU
intron. The Tetrahymena LSU intron contains a
large peripheral RNA structure, P5abc, which ordi-
narily blocks the CYT-18 binding site in the P4-P6
domain. The deletion of the P5abc structure results
in loss of self-splicing at low Mg2‡
concentrations,
but splicing can be restored by adding either the
CYT-18 protein or P5abc RNA in trans (van der
Horst et al., 1991; Mohr et al., 1994). CYT-18 and
P5abc bind to overlapping sites around the junc-
tion of the P4-P6 stacked helices and likely play
similar roles in stabilizing the structure of the P4-
P6 domain, exemplifying how a protein might
assume a role played by an RNA in the course of
evolution (Mohr et al., 1994).
Remarkably, comparison of the CYT-18 binding
sites in the N. crassa mt LSU and ND1 introns with
that in N. crassa mt tRNATyr
by RNA footprinting
in conjunction with graphic modeling revealed an
extended three-dimensional overlap between the
tRNA and the group I intron catalytic core. In this
overlap, the P4-P6 domain corresponds to the
anticodon/D-arm stacked helices of the tRNA, P7
to the long variable arm, and P9 to the acceptor
stem (Caprara et al., 1996b). These ®ndings support
the hypothesis that CYT-18 adapted to function in
splicing by recognizing conserved tRNA-like struc-
tural features of group I introns, and raise the
possibility of an evolutionary relationship between
group I introns and tRNAs.
The interaction of CYT-18 with the P4-P6
domain, the cognate of the tRNA's anticodon/D-
arm stacked helices, is critical to its role in splicing.
To investigate this interaction in detail, we took
advantage of the ability of CYT-18 to bind speci®-
cally to the isolated P4-P6 domain of the N. crassa
mt LSU intron (Guo Lambowitz, 1992; Saldanha
et al., 1996). In vitro selection experiments to ident-
ify features required for CYT-18 binding identi®ed
ten invariant residues around the junction of the
P4-P6 stacked helices, including the four residues
comprising P4 bp-1 and P6 bp-1 at the stacked
266 Tyrosyl-tRNA Synthetase in Group I Intron Splicing

helical junction, as well as P4 bp-3, P6 bp-2, J3/4-1
and J3/4-2 (Saldanha et al., 1996). RNA footprint-
ing and modi®cation-interference experiments
showed that the interaction of CYT-18 with the iso-
lated domain is similar to that in the intact intron,
with phosphate backbone protections on both sides
of the junction of the P4-P6 stacked helix, but few
if any base-speci®c contacts in this region (M.G.
Caprara A.M.L., unpublished results). Finally, it
was shown that CYT-18 could not bind to a per-
muted version of the P4-P6 domain, in which P4-
P6 is a continuous rather than a stacked helix,
suggesting that the con®guration recognized by
CYT-18 is not perfectly coaxial (Saldanha et al.,
1996).
Previously, we developed an E. coli genetic assay
that permits us to identify rapidly structural
defects that can or cannot be suppressed by CYT-
18. This assay is based on the ability of CYT-18 to
restore the splicing of structurally-defective
mutants of a plasmid-borne phage T4 td intron
(Mohr et al., 1992). Splicing of the intron leads to
the synthesis of thymidylate synthase, which
enables thyAÀ
cells to grow on minimal medium
and confers sensitivity to trimethoprim on medium
containing thymine. By using this assay, we
showed that CYT-18 could suppress splicing-defec-
tive td introns containing mutations in different
regions of the catalytic core, including J3/4, P4, P6,
J6/7, P7, P8 and P9 (Mohr et al., 1992; Myers et al.,
1996). Here, we further tested our model of CYT-
18 function by using this bacterial genetic assay to
investigate systematically the effect of nucleotide
substitutions at the junction of the P4-P6 stacked
helices. Our results indicate that the P4/P6 junction
is a linchpin region, where even small nucleotide
substitutions grossly disrupt formation of the cata-
lytically-active group I intron tertiary structure,
and that CYT-18 binding induces the formation of
the correct structure in this region, leading to fold-
ing of the group I intron catalytic core.
Results
Analysis of mutations at P4 bp-1
The experimental strategy was to introduce
mutations at P4 bp-1 and/or P6 bp-1 of the td
Figure 1. Secondary-structure
model of the phage T4 td intron.
The td intron (Genebank accession
number M12742) belongs to struc-
tural subgroup IA. The
Figure shows the predicted second-
ary structure of the 265 nt ÁORF
derivative of the td intron
(pTZtd1304) used in this study. The
structure is drawn in the format of
Cech et al. (1994) and numbered
according to Belfort et al. (1987).
Nucleotide residues in the intron
and exons are indicated in upper
and lowercase letters, respectively.
The 5H
and 3H
splice sites (5H
SS and
3H
SS) are indicated by arrows. Ter-
tiary interactions are indicated by
thin connecting lines. ÁORF indi-
cates the deleted region containing
the intron ORF. Nucleotide resi-
dues changed to create restriction
sites are circled. The box indicates
nucleotide residues in P4 bp-1 and
P6 bp-1 that were mutagenized
here.
Tyrosyl-tRNA Synthetase in Group I Intron Splicing 267

intron, and then use the E. coli genetic assay to
rapidly identify those with splicing defects that
could or could not be suppressed by CYT-18.
pTZtd plasmids carrying a phage T4 td gene with
mutations at the P4-P6 junction region were trans-
formed into an E. coli thyAÀ
strain in the presence
or absence of pA550, which expresses wild-type
CYT-18 protein. The transformants were then pla-
ted on minimal medium (MM), minimal medium
plus thymine (MMT), and minimal medium plus
thymine and trimethoprim (TTM) (Belfort et al.,
1987; Mohr et al., 1992). Cells harboring splicing-
defective td introns lack functional TS and are
unable to grow on MM, but grow on MMT and
are insensitive to trimethoprim. Restoration of spli-
cing by CYT-18 results in the synthesis of TS,
which enables the cells to grow on MM and con-
fers sensitivity to trimethoprim. Previous studies
showed a good correlation between the growth
phenotype in the plating assay and the amount of
spliced td mRNA, with 1 % splicing giving a tdÀ
phenotype and 38 % splicing giving a fully wild-
type phenotype (Mohr et al., 1992; Brion et al.,
1999b).
Results of td plating assays for all 16 possible
nucleotide combinations at P4 bp-1 are summar-
ized in Table 1A, and representative plating assays
are shown in Figure 2. The wild-type intron, which
contains an AU base-pair at P4 bp-1, gave a
td‡
phenotype in the presence or absence of
CYT-18, indicating that it could splice ef®ciently
under both conditions. Most of the P4 bp-1 mutant
introns gave tdÀ
phenotypes in the absence of
CYT-18, indicating that they are unable to self-
splice ef®ciently, but gave td‡
phenotypes indica-
tive of restored splicing in the presence of CYT-18.
Two mutants, Aa and ua, showed weak splicing in
the absence of CYT-18, and two other mutants, Ag
and gg, showed no detectable splicing in the pre-
sence or absence of CYT-18. (Note: wild-type
nucleotide residues are indicated in uppercase
letters and mutant nucleotide residues in lowercase
letters.) The ®nding that self-splicing was inhibited
by nearly all nucleotide substitutions, including
those that retain the ability to form a Watson-Crick
or a wobble base-pair (cg, gc, gU, ug), indicates a
requirement for speci®c nucleotide residues at
P4 bp-1. Indeed, this base-pair is highly con-
strained, corresponding to AU or UA in 98 % of
all naturally occurring group I introns (see Discus-
sion). Since nearly all of the P4 bp-1 mutants can
be ef®ciently suppressed by CYT-18, we infer that
the structural defect resulting from the nucleotide
substitutions can be compensated for by the pro-
tein, and that CYT-18 does not make an indispen-
sable base-speci®c contact with P4 bp-1.
The two non-splicing mutants, which could not
be rescued by CYT-18, are the purine-purine mis-
matches Ag and gg. However, the other two pur-
ine-purine mismatches, Aa and ga, could be
rescued. The difference could re¯ect that depend-
ing on sequence context purine-purine mismatches
can potentially form either an imino hydrogen-
bonded pair or a sheared base-pair (Wu Turner,
1993; Chou et al., 1997). The latter is not readily
accommodated in a standard A-form helix and can
cause structural disruption by changing the widths
of the major and minor grooves (Wu Turner,
1993; Gautheret et al., 1994). In addition, the non-
splicing mutants gg and Ag can potentially form
an alternative, presumably non-productive second-
ary structure, which is not predicted for the CYT-
18 suppressible mutants Aa and ga (Figure 3).
Chemical structure mapping with DMS indicated
that this alternative secondary structure does in
fact form in the mutants (see Figure 3).
Analysis of mutations at P6 bp-1
An analogous set of mutants was constructed for
P6 bp-1. Unlike P4 bp-1, this base-pair is involved
in a known tertiary interaction, a minor-groove
nucleotide triple with J3/4-3, which imposes
additional sequence constraints (Figures 1 and
Table 1. In vivo splicing phenotypes of td intron
mutants
Splicing phenotype
Mutants ÀCYT-18 ‡CYT-18
A. P4 bp-1
AU (WT) ‡ ‡ ‡ ‡ ‡ ‡
Aa ‡ ‡‡
Ac À ‡ ‡ ‡
Ag À À
ca À ‡ ‡ ‡
cc À ‡ ‡ ‡
cg À ‡ ‡ ‡
cU À ‡ ‡ ‡
ga À ‡‡
gc À ‡ ‡ ‡
gg À À
gU À ‡ ‡ ‡
ua ‡ ‡ ‡ ‡
uc À ‡ ‡ ‡
ug À ‡‡
uU À ‡ ‡ ‡
B. P6 bp-1
GC (WT) ‡ ‡ ‡ ‡ ‡ ‡
aa À À
aC À ‡‡
ag À ‡
au ‡ ‡‡
ca À ‡ ‡ ‡
cC À ‡
cg ‡ ‡ ‡ ‡
cu À ‡ ‡ ‡
Ga À ‡‡
Gg À ‡‡
Gu À ‡‡
ua À ‡‡
uC À ‡‡
ug À ‡‡
uu À ‡
This summarizes in vivo splicing phenotypes of the wild-type
(WT) and mutant td introns having nucleotide substitutions at
either (A) P4 bp-1 or (b) P6 bp-1, in the presence (‡) or absence
(À) of the CYT-18 protein, based on plating assays. Wild-type
nucleotide residues are indicated in uppercase letters, and
mutant nucleotide residues are indicated in lowercase letters.
Splicing phenotypes are as de®ned in Materials and Methods.

4(a)). In a previous mutational analysis of P6 bp-1
using the full-length td intron, Ehrenman et al.
(1989) found that the mutants cC and Gg could not
self-splice in vivo and that the compensatory
mutant cg only weakly restored splicing, implying
either additional structural constraints or a dual
function of this base-pair.
The results of the td plating assay for the more
complete set of P6 bp-1 mutants analyzed here are
summarized in Table 1B. The wild-type intron,
which has a GC base-pair at this position, spliced
ef®ciently in the presence or absence of CYT-18.
Again, most of the mutants failed to splice in the
absence of CYT-18, but could be suppressed by the
protein. The only two mutants that showed weak
splicing in the absence of CYT-18 were cg, as
reported by Ehrenman et al. (1989), and au. Both of
these mutants retain a canonical base-pair at
P6 bp-1, as well as nucleotide triple combinations
that are found in naturally occurring group I
introns (A-U:A and C-G:A; Figure 4(b,c), Table 2).
The mutant ua, which also retains a Watson-Crick
base-pair at P6 bp-1, could not self-splice and was
not fully suppressed by CYT-18. Examination of
potential base triples using the ISOPAIR program
(Gautheret Gutell, 1997) showed that U-A:A
could form an alternative, more stable base triple
that would disrupt the normal phosphodiester
backbone geometry, possibly accounting for the
impaired self-splicing of the mutant (Figure 4(d)).
This alternative base-triple con®guration is found
naturally in yeast tRNAPhe
(U12:A23:A9) (Quigley
Rich, 1976). If this interpretation is correct, the
U-A:A combination may be deleterious in group I
introns, and indeed it has been reported in only
two subgroup IB mutants of unknown self-splicing
capability (Table 2). Mutants Gu and ug, which
introduce a wobble base-pair at Pb bp-1, did not
self-splice and were only moderately suppressed
by CYT-18. Since CYT-18 can suppress nearly all
substitutions at P6 bp-1, we infer that it can com-
pensate generally for impaired base-pairing and
base-stacking interactions, as well as for the loss of
the normal base-triple interaction with J3/4-3, con-
sistent with previous analysis of J3/4 mutations
(Myers et al., 1996). In addition, as for P4 bp-1, we
can infer that CYT-18 makes no indispensable
base-speci®c contacts with P6 bp-1.
CYT-18 had particular dif®culty suppressing
P6 bp-1 mutants aa, ag, cC, and uu. The dif®culty
in suppressing purine-purine mismatches is remi-
niscent of the situation at P4 bp-1. However, the
most deleterious mutation at P6 bp-1 was aa,
rather than ag or Gg, which were the most deleter-
ious at P4 bp-1. Again, a possible explanation is
that aa in this sequence context forms a sheared
Figure 2. Plating assay for spli-
cing of P4 bp-1 and P6 bp-1
mutants in the presence or absence
of the CYT-18 protein. E. coli 1904
(C600 thyA::Kanr
) containing wild-
type or mutant pTZtd plasmids
plus pA550 (‡CYT18) or the vector
pACYC184 (ÀCYT18) were plated
on MMT, MM and TTM containing
kanamycin, chloramphenicol and
ampicillin and grown overnight at
37
C. The grids at the top show
the pattern of P4 bp-1 (left) and
P6 bp-1 (right) mutants inoculated
on each plate. The negative control,
pTZtdÁP4-7, contains an intron
with an internal deletion, which
does not splice in the presence or
absence of CYT-18 (Mohr et al.,
1992).

purine-purine pair, thereby causing greater distor-
tion of the major and minor grooves. Unfavorable
base-stacking interactions may also contribute,
since aa, along with the pyrimidine-pyrimidine
mismatches uu, cu, and uc, have been found to
cause the greatest disruption of base stacking in
studies with DNA four-way junctions (Duckett
Lilley, 1991). In extrapolating these results to RNA,
the tacit assumption is that stacking preferences
are determined largely by base interactions (see
Duckett et al., 1995). However, cu and uC, which
are also expected to be unfavorable for base stack-
ing, were more readily suppressed than cC and uu
at P6 bp-1. The difference here could re¯ect that cu
and uC have greater propensity for base-pairing
than uu or cC in certain sequence contexts (cf.
Aboul-ela et al., 1985; Werntges et al., 1986). This
explanation is compatible with the conclusion
below that both base-pairing and base stacking
contribute to the formation of the correct geometry
at the junction of the P4-P6 stacked helix.
Analysis of double mutants at P4 bp-1 and
P6 bp-1
In order to assess the possible interaction
between P4 bp-1 and P6 bp-1, we analyzed 26
mutants selected randomly from a pool having
mutations at all four positions. The results of td
plating assays are summarized in Table 3. None of
the mutants was self-splicing. A total of 13 mutants
were CYT-18 dependent, and the remaining 13 did
not splice in the presence or absence of CYT-18. All
13 CYT-18-dependent mutants retain at least one
Watson-Crick or wobble base-pair at either P4 bp-1
or P6 bp-1, and indeed six of these retain the wild-
type base-pair at one of these positions. Only two
of the CYT-18-suppressible mutants, gc/ua and
ua/ug, have substitutions at all four positions, and
both retain the ability to form Watson-Crick or
wobble base-pairs at both P4 bp-1 and P6 bp-1.
Interestingly, two of the CYT-18-dependent
mutants, gc/ua and cU/au, have combinations of
nucleotides that spliced more ef®ciently than the
Figure 3. Alternative secondary structure of P4 bp-1
Ag and gg mutants. The predicted secondary structure
of the P4-P6 domain of the wild-type td intron is shown
to the left, and the alternate structure predicted for the
P4 bp-1 Ag and gg mutants is shown to the right.
Nucleotides at positions 48 and 77, which comprise
P4 bp-1 in the wild-type intron are boxed. Open circles
indicate nucleotide positions modi®ed by DMS in
P4 bp-1 Ag and gg mutants, but protected in wild-type
and other P4 bp-1 purine-purine mutants (5®vefold
difference in band intensity). Filled circles indicate pos-
itions protected in the P4 bp-1 Ag and gg mutants, but
strongly modi®ed in wild-type and other P4 bp-1 pur-
ine-purine mutants (5tenfold difference in band inten-
sity). The ®lled triangles indicate positions modi®ed in
both the wild-type and mutant introns.
Figure 4. Diagram of potential base-triple interactions
between P6 bp-1 and J3/4-3 in wild-type and mutant td
introns. (a) Wild-type, G-C:A, (b) mutant c-g:A, (c)
mutant a-u:A, (d) mutant u-a:A. Base-triple con®gur-
ations were predicted by the ISOPAIR program
(Gautheret Gutell, 1997). The Figure shows that the
U-A:A combination could form an alternate triple,
which has a greater number of hydrogen bonds, but
would distort the phosphodiester backbone geometry in
this region. The equilibrium arrows with a question
mark represent the hypothesis that this alternate con-
®guration might be favored in the td intron, accounting
for the impaired self-splicing of the P6 bp-1 ua mutant.
Numbers indicate nucleotide positions in the td intron.
Broken lines indicate hydrogen bonds. Filled pentagons
indicate the position and orientation of ribose sugars.

corresponding P6 bp-1 single mutants ua and au
(cf. Table 1).
All 13 non-splicing mutants had P4 bp-1 and
P6 bp-1 substitutions that were individually rescu-
able by CYT-18. Thus, their non-splicing pheno-
type must result from cumulative effects or
disfavored nucleotide combinations at the two pos-
itions. Notably, all of the non-splicing mutants con-
tain at least one of the combinations UU, CU, UC,
or AA that are expected to interfere most strongly
with base-stacking interactions, based on analysis
of DNA four-way junctions (Duckett Lilley,
1991). Further, except for Aa/Gg, which contains
purine-purine mismatches at both positions, all of
these non-splicing double mutants contain at least
one pyrimidine-pyrimidine mismatch at either
P4 bp-1 or P6 bp-1.
In addition to the possible effects on base stack-
ing, seven of the double mutants that could not be
rescued by CYT-18 lack a Watson-Crick or wobble
base-pair at either position. Of the remaining six
mutants, four have a Watson-Crick or wobble pair
at P4 bp-1 coupled with a pyrimidine-pyrimidine
mismatch at P6 bp-1, and two have a wobble pair
at P6 bp-1 coupled with a pyrimidine-pyrimidine
mismatch at P4 bp-1. Together, these ®ndings
suggest that, in addition to the nucleotide triple
with J3/4, both base-pairing and base-stacking
contribute to establishing the functional geometry
at the junction of the P4 and P6 helices, and that
disruption of base-pairing at P4 bp-1 and/or
P6 bp-1 makes base-stacking defects more dif®cult
to rescue by CYT-18. The intron appears to be par-
ticularly sensitive to disruption of the base-pair at
P6 bp-1, since 11 of the 13 non-splicing mutants
lack a Watson-Crick base-pair at this position, and
the remaining two have only a wobble pair. The
greater sensitivity to the disruption of P6 bp-1
likely re¯ects that P6 in the td intron is only two
base-pairs long.
In vitro splicing
To reinforce the conclusions from the in vivo spli-
cing assays, several mutants were tested for their
ability to splice in vitro in the presence or absence
of CYT-18 protein in reaction medium containing
3 mM Mg2‡
, which is near the physiological con-
Table 2. Base-pair frequencies at P6 bp-1 in naturally occurring group I introns with an A at J3/4-3
P6 bp-1c
Subgroupa
A at J3/4-3b
GC GU AU UA AC CG UG
A 20/116 12 7 1
B 102/117 87 10 2 2 1
C1-2 0/318
C3 1/321 1
D 14/17 2 11 1
E 11/59 6 5
a
Intron classi®cation based on Michel Westhof (1990) and Suh et al. (1999).
b
Proportion of group I introns in indicated subgroup with an A at J3/4-3.
c
Number of group I introns in subgroup with indicated base-pair at P6 bp-1.
Table 3. In vivo splicing phenotype of P4-P6 mutants
CYT-18 suppressible Non-splicing
Mutants Splicing phenotype Mutants
P4 P6 ÀCYT-18 ‡CYT-18 P4 P6
AU/GC (WT) ‡ ‡ ‡ ‡ ‡ ‡ cg/uC
AU/Gu À ‡‡ ug/uu
AU/aC À ‡‡ gU/uC
AU/uC À ‡‡ gU/uu
AU/Gg À ‡‡ uU/Gu
gU/GC À ‡ ‡ ‡ Aa/Gg
cU/GC À ‡ ‡ ‡ Ac/uC
cU/ua À ‡‡ cU/cC
uc/Gu À ‡‡ uU/ca
us/cC À ‡ uc/cu
cg/Gg À ‡‡ ga/uu
gc/ua À ‡ ‡ ‡ uc/Gg
us/ug À ‡ cU/ug
cU/au À ‡ ‡ ‡
This summarizes in vivo splicing phenotypes of P4 bp-1/P6 bp-1 double mutants that were suppressed by
CYT-18 (left) or were non-splicing in the presence or absence of CYT-18 (right). Uppercase letters indicate wild-
type nucleotide residues, and lowercase letters indicate mutant nucleotide residues. Splicing phenotypes are as
de®ned in Materials and Methods.

centration (Lusk et al., 1968). As expected, the
wild-type td intron spliced ef®ciently under these
conditions in the absence or presence of CYT-18
(kobs ˆ 0.18 and 0.19 minÀ1
, respectively; 90 %
spliced). In agreement with the results of the plat-
ing assays, the CYT-18-suppressible P4 bp-1
mutants cg, gc, gU, ug, and Ac and the P6 bp-1
mutants Gg, cu, and uC showed little, if any,
splicing in the absence of CYT-18, but spliced
relatively rapidly in the presence of CYT-18
(kobs ˆ 0.053-0.085 minÀ1
for the P4 bp-1 mutants,
and 0.041-0.056 minÀ1
for the P6 bp-1 mutants).
These rates of splicing in the presence of CYT-18
are in agreement with those previously reported
for CYT-18-suppressible mutants in other regions
of the td intron (Myers et al., 1996). Notably, a sub-
stantial proportion of mutant RNAs (42-61 % for
the P4 bp-1 mutants and 60-65 % for the P6 bp-1
mutants) failed to splice even in the presence of
high concentrations of GTP (5 mM) and a tenfold
molar excess of CYT-18, indicating a greater pro-
pensity for these mutants to fold into an inactive
RNA conformation. As expected, the non-splicing
double mutants Ac/uC, cU/ug, uU/Gu, cg/uC,
and gU/uC all failed to splice in the presence or
absence of CYT-18. Together, these ®ndings show
a very good correlation between the in vitro and
in vivo splicing phenotypes for the P4 bp-1 and
P6 bp-1 mutants, as found previously for mutants
in other regions of the td intron (Myers et al., 1996).
Thermal denaturation analysis
In general, the P4 and P6 mutants could inhibit
splicing either by affecting the global folding of the
intron RNA or by creating local structural defects
at the intron's active site. To investigate these pos-
sibilities, we analyzed the thermal stability of the
mutant introns by UV spectroscopy. In this
approach, RNA denaturation is followed by the
increase in absorbance at 260 nm as the tempera-
ture is increased slowly from 25
C to 80
C. Group
I introns show an initial structural transition due to
disruption of tertiary structure and a later tran-
sition due to disruption of secondary structure
(Banerjee et al., 1993; Jaeger et al., 1993; Brion et al.,
1999a,b). This approach was used by Brion et al.
(1999b) to analyze the effect of mutations affecting
various tertiary interactions on melting pro®les of
the catalytic core of the td intron, synthesized as an
in vitro transcript containing A37 to A1011. The
RNA used here is identical to that used by Brion
et al. (1999b), except for the single nucleotide
change U976G, which introduces an EcoRI site for
cloning of the P4-P6 mutants (see Materials and
Methods and Figure 1).
Figure 5 shows the UV-melting pro®les for the
core regions of the wild-type and selected P4 bp-1
and P6 bp-1 mutant introns in reaction medium
containing 3 mM Mg2‡
, the same Mg2‡
concen-
tration used in the in vitro splicing assays (see
above). The plots show the ®rst derivative of the
absorbance as a function of temperature, and Tm
and ÁTm values derived from the plots are sum-
marized in Table 4. The wild-type intron shows an
initial structural transition at 49.6
C due to disrup-
tion of tertiary structure and a second transition at
67.3
C due to disruption of secondary structure.
The Tm for the tertiary structure transition of the
wild-type intron is somewhat lower than that
measured by Brion et al. (1999a) (52.4
C at 3 mM
Mg2‡
, 56
C at 5 mM Mg2‡
), possibly re¯ecting
subtle differences in conditions, or the single
nucleotide difference in the construct used here. In
our experiments, the in-sample digital temperature
sensor was calibrated independently to obtain
accurate temperature readings.
Compared to the wild-type intron, the amplitude
of the tertiary-structure transition for all of the
mutants was decreased, and the transitions
occurred over a wider temperature range,
suggesting decreased cooperativity. Surprisingly,
however, in most cases, the Tm for the residual ter-
tiary structure was similar to or only slightly lower
(3
C) than that for the wild-type intron. Four of
the P4 bp-1 mutants behaved differently, with the
thermal denaturation pro®les showing two rela-
tively low temperature transitions (Table 4). These
include the two non-splicing mutants Ag and gg,
which were found by chemical structure mapping
to form an alternative secondary structure (see
Figure 3). The other two mutants P4 bp-1 cg and
gc are readily suppressed by CYT-18, but show a
signi®cant proportion of inactive RNA (42-48 %) in
in vitro splicing assays (see above). We cannot dis-
tinguish whether the second transition re¯ects dis-
ruption of secondary or tertiary structure or both.
The thermal denaturation analysis indicates that
even very small changes, such as substitution of a
different base-pair at P4 bp-1 or P6 bp-1, strongly
affect the folding of the group I intron catalytic
core. We could discern no systematic difference in
melting pro®les of mutants that could or could not
be rescued by CYT-18 (Table 4). Thus, the differ-
ence must re¯ect that CYT-18 binding can promote
formation of the active tertiary structure in some
mutant introns but not others.
Fe(II)-EDTA analysis
The decreased amplitude of the tertiary structure
transition in the P4-P6 mutants could re¯ect either
a mixture of folded and unfolded RNAs or a uni-
form population of RNAs that contains a subset of
tertiary-structure interactions. To distinguish these
possibilities, we carried out Fe(II)-EDTA structure
mapping on several non-splicing P4-P6 double
mutants. These mutants are expected to have the
greatest structural disruptions, and two of them,
cU/ug and uU/Gu, have the greatest ÁTm among
those mutants that have single low temperature
transitions (Table 4). Thus, we reasoned that any
residual tertiary structure would have to form
independently of the correct geometry at the junc-
tion of the P4-P6 stacked helices. As shown in
Figure 6, the wild-type intron showed protections

throughout the P4-P6 and P3-P9 domains. By con-
trast, the mutants lost most protections in the P4-
P6 domain (e.g. P4, J4/5, P5, P6, and J6/6a), but
retained protections in the P3-P9 domain (P7.2,
P3[3H
], P7, and P9) at both 3 mM and 8 mM Mg2‡
.
Together with the thermal denaturation analysis,
these ®ndings indicate that the P4-P6 mutations
disrupt tertiary structure that leads to protection of
the P4-P6 domain, but that some regions of the
intron remain at least partially folded.
Equilibrium-binding assays
The inability of CYT-18 to rescue some mutants
could re¯ect either that the folding defect is too
great or that the CYT-18 binding site is impaired.
To investigate how the mutations at the P4/P6
junction affect the interaction with CYT-18, selected
mutant introns were used for protein-binding
assays. Equilibrium-binding experiments are
shown in Figure 7, and the data are summarized in
Table 4. For equilibrium binding, low concen-
trations of 32
P-labeled RNAs containing the core
regions of wild-type or mutant introns were incu-
bated with increasing amounts of CYT-18, and
binding was assayed by retention of the labeled
RNA on a nitrocellulose ®lter. The core region
RNAs were used so that the results could be com-
pared more directly with those from RNA struc-
ture mapping and thermal denaturation analysis.
We con®rmed that the binding characteristics of
the wild-type core region RNA are essentially the
same as that of the precursor RNA containing the
full-length ÁORF td intron and ¯anking exons (koff
values ˆ 0.011 and 0.0086 sÀ1
, respectively; Table 4
and data not shown). The binding of CYT-18 to the
Figure 5. UV-melting pro®les for
wild-type and mutant td introns.
RNAs corresponding to the core
regions of the wild-type and
mutant td introns (0.2 A260;
$0.32 mM) were heated slowly
from 25
C to 80
C, and A260 was
monitored as a function of tem-
perature. The plots show the ®rst
derivative of the absorbance
(dA260/dT) as a function of tem-
perature (T). (a) P4 bp-1 purine-
purine mutants. (b) P4 bp-1 base-
paired and Ac mutants. (c) P6 bp-1
purine-purine mutants. (d) P6 bp-1
pyrimidine-pyrimidine (Pyr-Pyr)
mutants. (e) P6 bp-1 base-paired
mutants. (f) P4-P6 double mutants.
The melting pro®le for the wild-
type td intron is included with each
group for comparison. Each assay
was repeated at least twice with
similar results.

td intron is signi®cantly weaker than its binding to
the N. crassa mt introns (Kds 0.7 pM) and within
a range where Kds can be measured directly by
equilibrium methods (see Materials and Methods).
The equilibrium binding assays show that wild-
type td intron RNA binds CYT-18 with a Kd at
37
C of 0.12 nM, with 19 % of the input RNA
bound at saturating protein concentration. The
relatively small proportion of input RNA bound,
which was unchanged using different renaturation
protocols, may re¯ect a combination of inef®cient
retention of the complex on the nitrocellulose ®lter,
the relatively rapid dissociation rate of CYT-18,
and/or that some proportion of the RNA folds
into an inactive conformation that is unable to
bind CYT-18. Although the in vitro binding assays
monitor only a proportion of the active RNAs,
they nevertheless show a generally good corre-
lation with the in vivo and in vitro splicing pheno-
types (see below).
For P4 bp-1, we analyzed CYT-18 binding of all
mutants that have a purine-purine mismatch (Ag,
gg, ga, and Aa), along with the four CYT-18 sup-
pressible mutants that retain a base-pair at this
position (cg, gc, gU and ug) and the mutant Ac. In
all these cases, the ability to bind CYT-18 mirrored
the ability of the protein to promote splicing.
Among the purine-purine mismatches, the weak
self-splicing mutant Aa bound CYT-18 relatively
strongly (Kd at 37
C ˆ 0.028 nM; 12 % bound), the
CYT-18 suppressible mutant ga bound less well
(Kd ˆ 2.5 nM, 12 % bound), and the two non-spli-
cing mutants (Ag and gg) bound CYT-18 poorly
(2.6 % bound) (Figure 7).
The four CYT-18-suppressible mutants that
retain a base-pair at P4 bp-1 bound CYT-18 with
Kd values that were 17.5- to 75-fold higher than
that of the wild-type intron (2.1 to 9.0 nM com-
pared to 0.12 nM), with the weakest binding dis-
played by the mutant ug, which is the least
Table 4. Thermal denaturation and CYT-18 binding properties of wild-type and selected mutant td introns
Phenotypeb
Mutanta
ÀCYT-18 ‡CYT-18
Tm
(
C)
ÁTm
(
C)c
Kd
d
(% bound RNA)e
(nM) koff
f
(sÀ1
)
Calculated kon
g
(MÀ1
sÀ1
)
WT ‡ ‡ ‡ ‡ ‡ ‡ 49.6 - 0.12 (19%) 0.011 9.2 Â 107
P4 bp-1 mutants
Aa ‡ ‡‡ 50.3 0.7 0.028 (12%) 0.012 4.1 Â 108
Ag À À 42.0/53.9h
À7.6/4.3 8.2 (2.5%) 0.011 1.3 Â 106
ga À ‡‡ 53.2 3.6 2.5 (12%) 0.0085 3.4 Â 106
gg À À 40.7/53.9h
À8.9/4.3 3.8 (2.6%) 0.23 6.1 Â 106
cg À ‡ ‡ ‡ 40.7/53.9h
À8.9/4.3 2.2 (10%) 0.012 5.5 Â 106
gc À ‡ ‡ ‡ 37.8/58.0h
À11.8/8.4 2.1 (9.5%) 0.014 6.7 Â 106
gU À ‡ ‡ ‡ 48.7 À0.9 5.4 (13%) 0.015 2.8 Â 106
Ac À ‡ ‡ ‡ 50.6 1.0 0.46 (10%) 0.014 3.0 Â 107
P6 bp-1 mutants
aa À À 48.6 À1.0 (2%) n.d.i
n.d.i
ag À ‡ 49.0 À0.6 2.9 (3.9%) n.d.i
n.d.i
Ga À ‡‡ 48.6 À1.0 1.2 (11%) n.d.i
n.d.i
Gg À ‡‡ 50.3 0.7 1.0 (3.1%) n.d.i
n.d.i
cC À ‡ 49.6 0.0 (2%) n.d.i
n.d.i
cu À ‡ ‡ ‡ 49.6 0.0 0.11 (6.4%) 0.0087 7.9 Â 107
uc À ‡‡ 50.3 0.7 1.3 (3.5%) 0.0071 5.5 Â 106
uu À ‡ 50.6 1.0 9.4 (3.4%) 0.0090 9.6 Â 105
au ‡ ‡‡ 49.6 0.0 0.080 (21%) 0.0081 1.0 Â 108
cg ‡ ‡ ‡ ‡ 47.7 À1.9 1.5 (28%) 0.0063 4.2 Â 106
Gu À ‡‡ 50.6 1.0 0.29 (29%) 0.0079 2.7 Â 107
ua À ‡‡ 49.5 À0.1 0.67 (18%) 0.0076 1.1 Â 107
ug À ‡‡ 49.6 0.0 1.6 (12%) 0.010 6.7 Â 106
P4-P6 double mutants
Ac/uC À À 48.7 À0.9 (2%) n.d.i
n.d.i
cU/ug À À 47.3 À2.3 (2%) n.d.i
n.d.i
cg/uC À À 50.4 0.8 (2%) n.d.i
n.d.i
gU/uC À À 48.5 À1.1 (2%) n.d.i
n.d.i
uU/Gu À À 47.2 À2.4 (2%) n.d.i
n.d.i
a
Wild-type and mutant td intron sequences are indicated as in Tables 1 and 3.
b
Splicing phenotypes are based on the data in Tables 1 and 3.
c
ÁTm is calculated as ÁTm ˆ Tm (mutant) À Tm (WT).
d
Kd, Equilibrium constants for binding of CYT-18 to the wild-type and mutant td introns. Values are averages for two or more
determinations, with standard deviations less than 10 %.
e
Percentage of input RNA bound at saturating protein concentration.
f
koff, Dissociation rate constants for the stable CYT-18/intron RNA complexes. Values are averages for two or more determina-
tions, with standard deviations less than 10 %.
g
kon, Association rate constant calculated as kon ˆ koff/Kd.
h
Some mutants show two transitions below 60
C.
i
n.d., not determined.

ef®ciently suppressed by CYT-18 (Kd ˆ 9.0 nM,
6.7 % bound) (Figure 7(b)). The mutant Ac, which
is well suppressed by CYT-18, has a relatively low
Kd value (0.46 nM) with $10 % of the RNA bound.
We note that two of the three mutants that retain
the wild-type A residue at P4 bp-1[5H
] have rela-
tively low Kd values, the only exception being the
non-splicing mutant Ag, which forms an alterna-
tive secondary structure. These ®ndings suggest
that the A residue at P4 bp-1 contributes directly
or indirectly to formation of the CYT-18 binding
site. However, this contribution must be dispensa-
ble, since an A is not found at this position in other
CYT-18-suppressible td intron mutants, nor in the
N. crassa CYT-18-dependent introns (Wallweber
et al., 1997).
For P6 bp-1, we tested all purine-purine mis-
matches, all pyrimidine-pyrimidine mismatches,
and all mutants that retain a base-pair. Again,
within groups of mutants, there was a good corre-
spondence between CYT-18 binding and the
degree of suppression of the splicing defect.
Among the purine-purine mismatches, the greatest
extent of binding was displayed by mutant Ga,
which is suppressed moderately by CYT-18
(Kd ˆ 1.2 nM, 11 % bound) (Figure 7(c)). The
mutant Gg, which is also moderately suppressed,
binds with a lower Kd (1.0 nM), but only 3.1 % of
the RNA was bound, suggesting that a smaller
proportion of this intron can fold into the correct
conformation. ag, which is weakly suppressed,
showed weaker binding af®nity (Kd ˆ 2.9 nM,
3.9 % bound), whereas the non-splicing mutant aa,
showed little, if any, binding af®nity (2 % bound).
Likewise for the P6 bp-1 pyrimidine-pyrimidine
mismatches, the fully-rescueable mutant, cu,
showed the best binding af®nity (Kd ˆ 0.11 nM,
6.4 % bound), uC, which is moderately suppressed,
showed intermediate binding (Kd ˆ 1.3 nM, 3.5 %
bound), and the weakly rescued mutants, cC and
uu, showed the weakest level of binding (2 %
bound and Kd ˆ 9.4 nM, 3.4 % bound, respectively)
(Figure 7(d)).
As a group, the mutants that retain a base-pair
at P6 bp-1 showed the highest proportion of
bound RNA (12 % to 29 %) (Figure 7(e)). All these
mutants are moderately (‡‡) or well (‡ ‡ ‡) sup-
pressed by CYT-18. The mutants, au and Gu,
which retain a purine at the 5H
position of P6 bp-1,
have Kd values comparable to wild-type (0.080 nM
and 0.29 nM, respectively), while the other three
mutants, cg, ua and ug, have somewhat increased
Figure 6. Fe(II)-EDTA cleavage
patterns of wild-type and selected
non-splicing P4-P6 mutant td
introns. 5H
end-labeled in vitro tran-
scripts containing wild-type or
mutant td introns were incubated
with (‡) or without (À) Fe(II)-
EDTA in reaction media containing
0, 3 or 8 mM Mg2‡
, as indicated.
Cleavage products were analyzed
in a denaturing 9 % polyacrylamide
gel, which was dried, autoradio-
graphed, and quantitated with a
phosphorImager. Wild-type (left)
and mutant sequences (right) are
shown above the lanes, with wild-
type nucleotide residues indicated
in uppercase letters and mutant
nucleotide residues indicated in
lowercase letters. Ladders: OH,
partial alkaline hydrolysis; G, par-
tial hydrolysis with RNase T1; A,
partial hydrolysis with RNase U2.
Intron regions are demarcated to
the right. The open arrows indicate
regions that are protected in both
wild-type and mutant RNAs, and
the ®lled arrows indicate regions
that are protected in wild-type but
not in mutant RNAs.

Kd values (0.67 to 1.6 nM). The non-splicing double
mutants Ac/uC, cU/ug, cg/uC, gU/uC, and uU/
Gu all showed little if any CYT-18 binding (2 % of
input RNA bound), consistent with their inability
to be suppressed by CYT-18 (Figure 7(f)). Across
groups, some mutants showed similar degrees of
CYT-18 binding, but different degrees of CYT-18
suppression, suggesting that some mutations may
affect steps after CYT-18 binding. Nevertheless, all
P4 bp-1 or P6 bp-1 mutants that are non-splicing
or weakly suppressed by CYT-18 showed at most
low levels of CYT-18 binding.
koff measurements
To supplement the equilibrium-binding assays,
we carried out koff measurements, which provide
information about the strength of the complex once
it is formed. In these experiments, pre-formed
CYT-18/intron RNA complexes were diluted into
reaction medium containing 1 mg/ml heparin, and
dissociation of the complex was measured as a
function of time (Figure 8). As found previously
for other CYT-18-dependent group I introns
(Wallweber et al., 1997), the dissociation curves
were biphasic with the initial fast phase represent-
ing loosely associated protein and the second,
slower phase representing the stable complex. The
few cases where there appear to be discrepancies
with the proportion of bound RNA in the equili-
brium binding assays (e.g. P4 bp-1 Ag, ga, and gg,
and P6 bp-1 uC and uu) likely re¯ect differences in
the amount of loosely bound RNA for the different
mutants. The koff values for the stable complex are
summarized in Table 4. Since the Kd values were
measured independently, the koff values could be
Figure 7. Equilibrium-binding
assays for representative td intron
mutants. 32
P-labeled RNAs (4 pM)
containing wild-type or mutant td
introns were incubated with
increasing concentrations of CYT-
18 protein dimer (0 pM-34 nM) for
20 minutes at 37
C, and binding
was assayed by the retention of the
32
P-labeled RNA on a nitrocellulose
®lter. (a) P4 bp-1 purine-purine
mutants. (b) P4 bp-1 base-paired
and Ac mutants. (c) P6 bp-1 pur-
ine-purine mutants. (d) P6 bp-1
pyrimidine-pyrimidine (Pyr-Pyr)
mutants. (e) P6 bp-1 base-paired
mutants. (f) P4-P6 double mutants.
Each assay was repeated at least
twice with similar results.

used to calculate kon. Signi®cantly, the koff values
for all of the mutants were within a factor of two
of the wild-type value. Thus, for those mutants
that show elevated Kd values, the decreased bind-
ing appears to be due largely to substantially
increased kon values. Considered together with the
structural analysis, these ®ndings suggest that the
mutations cause structural disruptions that impair
binding of CYT-18. Once bound, however, CYT-18
folds the intron into the required tertiary structure,
and the resulting complex has a stability similar to
that of the complex with the wild-type intron.
Discussion
Our results show that most mutations at the
junction of the P4-P6 stacked helices lead to loss of
self-splicing activity, but can be suppressed by
CYT-18. Considering the P4 bp-1 and P6 bp-1
mutants together, only four of 30 possible non-
wild-type nucleotide combinations at either pos-
ition retained any ability to splice in E. coli in the
absence of CYT-18, and even in these cases, spli-
cing was not as ef®cient as for the wild-type intron
(Table 1). Mutations at the P4-P6 junction that dis-
rupt base-pairing or base-stacking interactions or
the base triple between P6 bp-1 and J3/4, presum-
ably lead to altered geometry at the junction of the
P4-P6 stacked helices. Thermal denaturation and
Fe(II)-EDTA analysis indicate that this altered geo-
metry in turn leads to grossly impaired tertiary-
structure formation. In the Fe(II)-EDTA analysis,
P4-P6 double mutants, which are expected to have
the most severe structural disruptions, lost most
protections in the P4-P6 domain, but retained some
protections indicative of tertiary-structure for-
mation in the P3-P9 domain. The loss of protec-
tions in the P4-P6 domain likely re¯ects loss of
inter-domain contacts, which cannot be made
properly when the geometry at the junction of the
P4-P6 stacked helix is distorted.
The deleterious effects of nucleotide substi-
tutions at the P4/P6 junction are consistent with
comparative sequence analysis, which shows that
P4 bp-1 is remarkably constrained, corresponding
to either AU or UA in 98 % of all naturally occur-
ring group I introns (Table 5). Among subgroup IA
introns, P4 bp-1 is UA in 85 % and AU in 12 %,
including the td intron. Of the 15 mutant nucleo-
tide combinations analyzed here, only Aa and ua
could splice to some extent in the absence of CYT-
18. In general, the constraints on P4 bp-1 could
re¯ect either a need for a speci®c geometry or ther-
modynamic stability of this base-pair, or that
P4 bp-1 participates in an as yet unknown tertiary
interaction, perhaps involving A263 in the 5H
strand
of P7, which is in proximity to P4 bp-1 in the struc-
tural models (cf. Michel Westhof, 1990; Golden
et al., 1998). An additional possibility is that the
strong selection for either AU or UA re¯ects a
requirement that the conformation of the base-pair
be isosteric on reversal. For an AU pair, this would
require the adoption of a trans Watson-Crick con-
®guration, with hydrogen bonds between the ade-
nine N1 and uridine N3, and between the adenine
N6 and uridine O2 (Leontis Westhof, 1998). To
Figure 8. koff measurements for
representative td intron mutants.
32
P-labeled RNAs (100 nM) contain-
ing wild-type or mutant td introns
were incubated with 100 nM CYT-
18 dimer in 100 ml TMKBDG buffer
for 30 minutes at 37
C, then mixed
with 900 ml of TMKBDG containing
1 mg/ml heparin. At the indicated
times, 100 ml portions were with-
drawn and the amount of complex
remaining was assayed by nitrocel-
lulose ®lter binding. (a) P4 bp-1
purine-purine mutants. (b) P4 bp-1
base-paired and Ac mutants. (c)
P6 bp-1 pyrimidine-pyrimidine
(Pyr-Pyr) mutants. (d) P6 bp-1
base-paired mutants. Each assay
was repeated at least twice with
similar results.

maintain such a con®guration, the adenosine sugar
moiety would have to adopt a syn conformation in
order to keep the local stands anti-parallel
(Westhof, 1992). The effect would be to widen the
minor groove in this region, perhaps facilitating
formation of the base-triple interaction between
J3/4 and P6 or some other interaction required for
group I intron function.
Our results indicate that P6 bp-1 is constrained
not only by its base-triple interaction with J3/4,
but also by its base-stacking interaction with
P4 bp-1. Comparative analysis con®rms that
P6 bp-1 covaries with P4 bp-1 (Gautheret et al.,
1995). Among subgroup IA introns, only ®ve
combinations have been found in nature, UA/GU
(75 %), UA/GC (12 %), AU/GC (6 %), AU/GU
(5 %), and UG/GC (2 %) (Table 5), with AU/GC
found in the td intron. Of the subgroup IA combi-
nations tested here in the td intron, ua/GC splices
weakly in the absence of CYT-18, while ug/GC is
not self-splicing but is moderately suppressed by
CYT-18 (Table 1). Other combinations that give
some self-splicing are Aa/GC and AU/au, which
are not found in naturally-occurring group I
introns, and AU/cg, which is the predominant
combination in subgroup ID introns (Tables 1A
and B).
Remarkably, CYT-18 can suppress mutations
having nearly all possible combinations of nucleo-
tides at either P4 bp-1 (13/15) or P6 bp-1 (14/15).
These include mutations that affect base-pairing or
base-stacking interactions, as well as mutations at
P6 bp-1 that affect formation of the normal base
triple with J3/4-3. The few non-suppressible
mutations at one or the other positions are all pur-
ine-purine mismatches, and in the case of P4 bp-1,
these lead to formation of an alternate, presumably
non-productive secondary structure. The ability of
CYT-18 to suppress nearly all nucleotide substi-
tutions indicates that there are no indispensable
base-speci®c contacts between CYT-18 and P4 bp-1
or P6 bp-1. This conclusion is consistent with stu-
dies indicating that CYT-18 makes primarily phos-
phodiester backbone contacts in this region (see
Introduction). Mutants that retain the wild-type A-
residue at P4 bp-1[5H
] and purine at P6 bp-1 have
lower Kd values, but the effect is due primarily to
an increased kon rather than increased stability of
the complex. Although it remains possible that
base-speci®c contacts make some contribution,
these ®ndings suggest that the preferred nucleo-
tides act primarily by making it easier for the
intron to fold into the productive RNA structure.
In most cases, suppression of the splicing defect
by CYT-18 correlates with its ability to bind to the
mutant intron. The CYT-18-suppressible introns
bind the protein with Kd values up to 79-fold high-
er than that for the wild-type intron, but in all
cases the koff for the complex remains within two-
fold of the wild-type value, and the increased Kd
re¯ects primarily an increased kon for the binding
of CYT-18 to the misfolded intron. These ®ndings
suggest that the CYT-18 binding site is not formed
properly in the misfolded intron, but is induced by
Table 5. Base-pair frequencies at P4 bp-1 and P6 bp-1 in naturally occurring group I introns
Base-pair frequenciesc
(%)
Subgroupa
Number of
sequencesb
GC CG UA AU GU UG AA AC CA UC UU
P4 bp-1
A 87 85 12 2 1
B 117 85 7 2 1 5
C1-2 318 99 0.5 0.5
C3 321 98 2
D 17 100
E 59 32 68
P6 bp-1
A 87 20 1 79
B 117 79 2 4 13 2
C1-2 318 0.5 5 1 92 1.5
C3 321 1 99
D 17 12 76 6 6
E 59 62 2 36
UA/GU UA/GC AU/GC AU/GU AU/CG UA/UA UG/GC Othersd
P4-P6 combinations
A 87 75 12 6 5 2
B 117 9 73 4 14
C1-2 318 92 5 3
C3 321 97 3
D 17 12 76 12
E 59 25 5 56 10 4
a
Intron classi®cation based on Michel Westhof (1990) and Suh et al. (1999).
b
The number of group I intron sequences in each subgroup publically available as of January 2000.
c
Percentage of introns having indicated combinations. Combinations not found in naturally occurring group I introns are not
listed.
d
All other combinations.

binding of CYT-18, so that the resulting complex
has nearly the same stability as the wild-type com-
plex. Since the primary high-af®nity binding site
for CYT-18 is around the junction of the P4-P6
stacked helices (see Introduction), the most likely
possibility is that CYT-18 binds initially to this
region to induce formation of the correct geometry
at the junction of the P4-P6 stacked helix. The
resulting complex can then form the required
scaffold for RNA and protein interactions with the
P3-P9 domain.
Those mutants that cannot be suppressed by
CYT-18 presumably have structural defects that
make it dif®cult for CYT-18 to induce the correct
geometry at the junction of the P4-P6 stacked
helices. The analysis of double mutants at P4 bp-1
and P6 bp-1 shows that CYT-18 has dif®culty sup-
pressing mutations that simultaneously affect both
base-pairing and base-stacking interactions, par-
ticularly when P6 bp-1 is unpaired. A prediction
from our results is that naturally occurring group I
introns that have disfavored nucleotide combi-
nations at the P4-P6 junction may be non-self-spli-
cing, protein-dependent introns. Although
biochemical data are lacking for most introns in
the database, two sea anemone mtDNA group I
introns (Genebank accession numbers U36783 and
U36784), which have a UC mismatch at P4 bp-1,
were found to be non-self-splicing in vitro (Beagley
et al., 1996), and the Saccharomyces douglasii aI1
intron (Genebank accession number M97514),
which also has a UC mismatch at P4 bp-1 also
appears to be protein dependent (Tian et al., 1993).
Furthermore, consistent with our ®ndings that pur-
ine-purine mismatches at P4 bp-1 or P6 bp-1
impede splicing, GA, GG, and AG are not found at
either position in naturally-occurring group I
introns, while AA, which we ®nd to be a relatively
permissible substitution only at P4 bp-1, is present
at P4 bp-1 in two group IB introns but is not found
at P6 bp-1 (Table 5).
Together with previous work, our ®ndings for
the suppression of td intron mutants suggest a
model in which CYT-18 contacts both sides of the
P4-P6 stacked helix and promotes formation of the
correct geometry of the phosphodiester backbone
in this region. Although the CYT-18 binding site
may be preformed in the wild-type td intron,
which is self-splicing, the same induced-®t model
likely applies to the N. crassa mt introns, which are
not self-splicing and do not fold into the catalyti-
cally active RNA tertiary structure in the absence
of CYT-18 (see Introduction). Indeed, recent studies
directly demonstrate a CYT-18-induced confor-
mational change in the isolated P4-P6 domain of
the N. crassa mt LSU intron (M.G. Caprara
A.M.L., unpublished results). Finally, we noted
that the P4-P6 stacked helix is structurally analo-
gous to the anticodon arm/D-arm stacked helix of
tRNATyr
, which is recognized by the C-terminal
tRNA-binding domain of bacterial TyrRSs
(Bedouelle et al., 1993). The ability of CYT-18 to
induce the correct structure of the P4-P6 domain
could re¯ect that it uses the same binding site
adapted to recognize the phosphodiester backbone
conformation of the anticodon/D-arm stacked
helix. This structure may be conformationally rigid
in the tRNA, but can be induced in the group I
intron mutants studied here.
Materials and Methods
E. coli strains and growth media
E. coli strain 1904, a thyA::Kanr
derivative of C600
(Bell-Pederson et al., 1991), was used for td assays.
DH5aFH
was used for plasmid ampli®cation and cloning,
and BL21(DE3; plysS) was used for expression of CYT-18
protein. Bacteria were grown in LB or minimal medium
(MM) supplemented with 0.1 % Norit-A-treated Casami-
no acids and 0.2 % (w/v) glucose (Belfort et al., 1983).
Thymine was added at 50 mg/l. Antibiotics were added
at the following concentrations: ampicillin, 100 mg/l;
kanamycin, 40 mg/l; chloramphenicol, 25 mg/l; and tri-
methoprim, 20 mg/l.
Construction of td intron mutants
The wild-type construct, pTZtd1304 (Myers et al.,
1996), contains a 265 nt ÁORF-derivative of the full-
length td intron (Galloway Salvo et al., 1990), with
additional sequence changes U34A and U976G that cre-
ate SpeI and EcoRI sites, respectively, for the introduction
of mutations in the P4-P6 region. pTZtdÁP4-7, used as a
negative control, has an internal deletion (Á69-875) in
the intron core and does not splice in the presence or
absence of CYT-18 protein (Mohr et al., 1992).
The P4 bp-1 mutants were generated via two overlap-
ping primers, TDP4 bp-1A (5H
TTA TAC TAG TAA TCT
ATC TAA NCG GGG AAC CTC TCT AGT AGA C 3H
)
and TDP4 bp-1B (5H
GAA CCC GGG CAG TCC TAC
AAT TTA GCN CGG GAT TGT CTA CTA GAG AGG
3H
). First, the primers were annealed and ®lled in with
phage T7 DNA polymerase to generate a double-
stranded DNA fragment that corresponds to intron pos-
itions 27 through to 854 and covers the SpeI and SmaI
sites in the intron. This segment was then ampli®ed by
PCR with primers XC1 (5H
ACT TAT ACT AGT AAT C
3H
) and XC2 (5H
GAA CCC GGG CAG TCC TAC 3H
) (25
cycles, 30 seconds at 94
C, 30 seconds at 55
C and 90
seconds at 72
C, followed by extension for ten minutes
at 72
C). The resulting PCR product was puri®ed in a
1 % agarose gel, digested with SpeI and SmaI, and cloned
between the corresponding sites of pTZtd1304 (Figure 1).
To construct the P6 bp-1 mutants, two overlapping
segments of the intron were generated by PCR using
pTZtd1304 as template with primers XC5 (5H
GAC TGC
CCG GGT TCT ACA TAA ATG NCT AAC G 3H
) plus
NBS2 (5H
GAC GCA ATA TTA AAC GGT 3H
), and XC6
(5H
AAC CCG GGC AGT CCT ACA ATT TAG NAC
GGG T 3H
) plus YK2 (5H
GTA GAT GTT TTC TTG GGT
3H
). The PCR products were puri®ed in a 1 % agarose gel,
and 0.1 mg of each independently ampli®ed segment was
mixed, and ampli®ed in a single PCR reaction with the
outside primers YK2 and NBS2. The PCR product was
gel-puri®ed, digested with EcoRI and SpeI, and cloned
between the corresponding sites of pTZtd1304 (Figure 1).
To construct the P4-P6 double mutants, PCR was car-
ried out using pTZtd1304 as template with the junction
primer (5H
TTA TAC TAG TAA TCT ATC TAA NCG
GGG AAC CTC TCT AGT AGA CAA TCC CGN NCT

AAA TTG TAG GAC TGC CCG GGT TCT ACA TAA
ATG NCT AAC GAC TAT CCC T 3H
) and reverse primer
(5H
CTC TGC GCG CAG CTG CCA GT 3H
). The PCR pro-
duct was then digested with EcoRI and SpeI and cloned
between the corresponding sites of pTZtd1304.
Colony assays of CYT-18-dependent splicing of
mutant td introns
td plating assays were carried out as described (Mohr
et al., 1992; Myers et al., 1996). Libraries of pTZ plasmids
containing mutant td introns were transformed into
E. coli DH5aFH
. Plasmid DNAs were isolated from ran-
domly chosen colonies and transformed into E. coli 1904
(C600 thyA::Kanr
) in combination with either the vector
pACYC184 (Chang Cohen, 1978) or with pA550,
which expresses the wild-type CYT-18 protein (Mohr
et al., 1992). Cells were grown overnight at 37
C on LB
plates containing kanamycin for selection of the thyAÀ
host strain, ampicillin for selection of the pTZtd plasmid,
and chloramphenicol for selection of pA550 or
pACYC184. For plating assays, 1 ml of cells were spotted
on plates containing either minimal medium (MM), mini-
mal medium plus thymine (MMT), or minimal medium
plus thymine plus trimethoprim (TTM), in the presence
of the above antibiotics. Splicing phenotypes were
characterized after overnight incubation at 37
C and are
based on analysis of four to ten colonies in at least two
independent experiments (Mohr et al., 1992). Phenotypes
are de®ned as follows: ‡ ‡ ‡ , maximal splicing, growth
on MM comparable to MMT and no growth on TTM;
‡ ‡ , moderate splicing, growth on MM comparable to
MMT and incomplete inhibition on TTM; ‡, weak spli-
cing, growth on MM less than or equal to growth on
MMT and growth on TTM comparable to MMT; À, no
splicing, no growth on MM and growth on TTM com-
parable to MMT. After scoring the phenotypes,
mutations in the targeted regions were identi®ed by
sequencing using primers YK2 or NBS2 (see above). The
mutant td introns were then sequenced completely to
insure that no adventitious mutations had been intro-
duced during the constructions.
In vitro transcription
In vitro transcription was for two hours at 37
C in
100 ml of reaction medium containing 500 units phage T7
RNA polymerase (Life Technologies, Gaithersburg, MD),
2 to 5 mg of DNA template, 1 mM of each rNTP, 40 mM
Tris-HCl (pH 8.0), 25 mM NaCl, 8 mM MgCl2, 2 mM
spermidine-(HCl)3, 5 mM dithiothreitol (DTT), 0.1 mCi
[a-32
P]UTP (3000 Ci/mmole; New England Nuclear, Bos-
ton, MA) and 1 ml human placental ribonuclease inhibi-
tor (Amersham, Arlington Heights, IL). 32
P-labeled
transcripts used in in vitro splicing experiments and 5H
end-labeled transcripts used in Fe(II)-EDTA cleavage
experiments were synthesized as described (Myers et al.,
1996). Higher speci®c activity 32
P-labeled transcripts
used in equilibrium binding and kon experiments were
synthesized in a reaction mixture containing 0.2 mM
ATP, GTP, CTP, 0.02 mM UTP, and 0.35 mCi
[a-32
P]UTP. RNA transcripts for in vitro splicing were
generated from PCR-ampli®ed DNA templates as
described (Myers et al., 1996). Unlabeled transcripts used
for thermal denaturation experiments were synthesized
from gel-puri®ed, PCR-generated DNA templates using
a MEGAscriptTM
kit (Ambion, Inc., Austin TX).
After transcription, the DNA template was digested
with DNase I (Pharmacia, Piscataway, NJ; 50 units for 20
minutes at 37
C), and the transcripts were extracted
with phenol-CIA. Unlabeled transcripts used in thermal
denaturation experiments and 32
P-labeled transcripts
used in Fe(II)-EDTA experiments and in vitro splicing
experiments were puri®ed in denaturing 4 or 6 % poly-
acrylamide gels, ethanol precipitated, dissolved in dis-
tilled water, and centrifuged (3000 g, four minutes 20
C)
through a Sephadex G-50 spun column (Sigma, St. Louis,
MO). Other 32
P-labeled transcripts were centrifuged
through a Sephadex G-50 spun column and ethanol pre-
cipitated. The quality of transcripts was monitored by
gel electrophoresis. Prior to use, all transcripts were
renatured by heating to 60
C in reaction medium with-
out Mg2‡
for ten minutes, then adding MgCl2 to give the
desired Mg2‡
concentration and slowly cooling to room
temperature. This renaturation protocol was found to
give maximal extents of self-splicing for the wild-type
td intron construct pTZtd1304 compared to other
renaturation protocols.
In vitro splicing assays
In vitro splicing reactions were carried out
as described by Myers et al. (1996). Brie¯y, renatured,
32
P-labeled precursor RNA was preincubated with or
without 200 nM CYT-18 protein dimer in the splicing
reaction medium (100 mM KCl, 3 mM MgCl2, 20 mM
Tris-HCl (pH 7.5)) on ice for 30 to 45 minutes, then
warmed to 37
C before initiating splicing by addition of
5 mM prewarmed GTP. The reaction was incubated at
37
C, and time points were taken at different intervals
from 0 minutes to two hours. The products were
analyzed in a denaturing 6 % polyacrylamide gel, which
was dried and quantitated with a phosphorImager.
Kinetic parameters were calculated as described (Myers
et al., 1996).
RNA structure mapping
RNA structure mapping experiments with DMS and
Fe(II)-EDTA were as described (Caprara et al., 1996a;
Myers et al., 1996). DNA templates used to generate
in vitro transcripts were derived from the corresponding
wild-type or mutant pTZtd plasmids by PCR with pri-
mers TDSXM5H
(5H
GGT ACC TAA TAC GAC TCA CTA
TAG GGC CTG AGT ATA AGG TGA C 3H
), which con-
tains a sequence corresponding to intron positions 8 to
26 and adds a phage T7 promoter, and TDSXM3H
(5H
TTT
AAA TGT TCA GAT AAG GTC GTT AA 3H
), which con-
tains a sequence complementary to intron positions 992-
1012. Transcription with phage T7 RNA polymerase
yields 260 nt RNAs that begin at intron position G8 and
end at A1012, 5 nt upstream of the 3H
splice site. The
transcripts have an extra 5H
G-residue derived from the
T7 promoter, and 5 extra nt (5H
AAUUU) at their 3H
end
derived from the PCR primer used to generate the DNA
template.
Thermal denaturation analysis of mutant td introns
Thermal denaturation analysis was as described by
Brion et al. (1999b). DNA templates used to obtain in vitro
transcripts were generated by PCR of wild-type or
mutant pTZtd plasmids using the primers XC19 (5H
TGT
TCA GAT AAG GTC GT 3H
), which is complementary to
intron positions 995-1011, and one of four primers

XC18A, XC18G, XC18C and XC18T (5H
ATT TAA TAC
GAC TCA CAT TAG AAT CTA TCT AAN CGG 3H
) that
contain a T7 promoter sequence and a variable nucleo-
tide N that depends on the identity of the nucleotide at
P4 bp-1 in the mutant being studied. Transcription with
T7 RNA polymerase yields 224 nt RNAs corresponding
to the catalytic core of the ÁORF intron from position
A37 in J2/3 to A1011 in J9.2/10, with an extra 5H
G resi-
due from in vitro transcription. The wild-type transcript
is identical to that used by Brion et al. (1999b), except for
the mutation U976G introduced to create an EcoRI site
for cloning.
Thermal denaturation was carried out in a Perkin-
Elmer Bio20 UV/Vis spectrometer equipped with water-
thermostatable automatic six-cell changer controlled by a
PTP-6 temperature controller (Perkin-Elmer). RNA (0.2
A260, $0.32 mM) was dissolved in 3 ml of reaction med-
ium (50 mM Na cacodylate (pH 7.5), 50 mM NH4Cl,
3 mM MgCl2 ) and renatured by heating to 60
C for ®ve
minutes and then slowly cooled to room temperature.
After de-gassing, A260 was measured as a function of
temperature from 25
C to 80
C, with a heating rate of
0.2
C/minute. Temperature was monitored by an in-
sample digital temperature sensor (Perkin-Elmer), which
had been calibrated with a digital circulation water bath,
as well as a thermometer. Plots of the ®rst derivative
of A260 versus temperature were generated using the
TempLab software provided by Perkin-Elmer.
Equilibrium binding
RNA binding assays were carried out with the same
in vitro transcripts used for RNA structure mapping
experiments (see above). For equilibrium binding assays,
4 pM 32
P-labeled RNA (11,500 Ci/mmol) was incubated
with increasing amounts of CYT-18 protein in 500 ml of
TMKBDG buffer (10 mM Tris-HCl (pH 7.5), 5 mM
MgCl2, 100 mM KCl, 100 mg/ml acetylated bovine serum
albumin, 5 mM dithiothreitol and 10 % (v/v) glycerol)
for 20 minutes at 37
C, then quickly mixed (5 seconds)
with an equal volume of pre-warmed reaction medium
containing 2 mg/ml heparin and ®ltered through nitro-
cellulose prewetted with TMK (10 mM Tris-HCl (pH 7.5),
5 mM MgCl2, 100 mM KCl). The ®lters were washed
three times with 1 ml of TMK buffer, dried, and counted
using a Beckman LS6500 scintillation counter (Beckman
Instruments, Fullerton, CA). Since the 20 minute equili-
bration time is more than ten half-lives of the RNA-pro-
tein complex, 99.9 % of the complexes formed should
have reached equilibrium. The brief (5 seconds) incu-
bation with heparin was found to be essential to elimin-
ate non-speci®c binding at high CYT-18 concentrations.
Based on the measured koff values (Table 4), 5 % of
the speci®c complex with the wild-type intron
(t1/2 ˆ 62.2 seconds) would dissociate during the heparin
incubation, and 11 % of the least stable of the com-
plexes with the mutant introns (Table 4). Data were ®t to
the equation ([P] ‡ [R] ‡ Kd)/2 À (([P] ‡ [R] ‡ Kd)2
/
4 À [P].
[R])1/2
, where [P] is the concentration of CYT-18
dimer used in the experiment and [R] represents pM
RNA bound by CYT-18 at the saturating protein concen-
tration.
koff measurements
The dissociation rate constants (koff values) of the
CYT-18/intron RNA complex were measured as
described by Wallweber et al. (1997), with minor modi®-
cations. Brie¯y, complexes were formed by incubating
100 nM CYT-18 dimer with 100 nM 32
P-labeled RNA in
100 ml of TMKBDG buffer (see above) for 20 minutes at
37
C. The complexes were then mixed with 900 ml of
TMKBDG containing 1 mg/ml heparin to bind unasso-
ciated CYT-18 protein. At different times, 100 ml aliquots
were removed and the amount of complex remaining
was assayed by nitrocellulose ®lter binding, as described
above. Data were ®t to an equation with two exponen-
tials, A1(eÀk1t
) ‡ A2(eÀk2t
), where k1 and k2 are the koff
values for the stable and rapidly dissociating RNP com-
plex, and A1 and A2 are the amplitudes, representing the
proportion of RNA in each complex.
Comparative sequence analysis of the group I intron
P4-P6 stacked helical junction
Comparative sequence analysis was performed as
described (Gautheret et al., 1995) using a database, which
as of January 2000, contained more than 900 group I
intron sequences assigned to the major structural sub-
groups A, B, C1-2, C3, D and E (Michel Westhof, 1990;
Suh et al., 1999). Within each subgroup, group I intron
sequences were aligned manually with the program AE2
(T. Macke, The Scripps Research Institute, La Jolla, CA).
Positions in the sequence alignment approximate hom-
ologous secondary and tertiary structure elements
among the different group I introns in the database. We
have developed a computer program to obtain single
analysis of the sequence alignments (R.G., unpublished).
Information pertaining to each of the sequences in the
database is available at our new comparative RNA web
site (http://www.rna.icmb.utexas.edu).
Acknowledgments
We thank Christopher Myers and John Stryker for
preparation of CYT-18 protein, and Drs Mark Caprara
and Georg Mohr for comments on the manuscript. This
work was supported by NIH grant GM37951 to A.M.L.
and GM48207 to R.G.
References
Aboul-ela, F., Koh, D., Tinoco, I. Martin, F. H. (1985).
Base-base mismatches. Thermodynamics of double
helix formation for dCA3XA3G ‡ dCT3YT3G (X,
Y ˆ A, C, G, T). Nucl. Acids Res. 13, 4811-4824.
Akins, R. A. Lambowitz, A. M. (1987). A protein
required for splicing group I introns in Neurospora
mitochondria is mitochondrial tyrosyl-tRNA synthe-
tase or a derivative thereof. Cell, 50, 331-345.
Banerjee, A. R., Jaeger, J. A. Turner, D. H. (1993).
Thermal unfolding of a group I ribozyme: the low-
temperature transition is primarily disruption of
tertiary structure. Biochemistry, 32, 153-163.
Beagley, C. T., Okada, N. A. Wolstenholme, D. R.
(1996). Two mitochondrial group I introns in a
metazoan, the sea anemone Metridium senile: one
intron contains genes for subunits 1 and 3 of
NADH dehydrogenase. Proc. Natl Acad. Sci. USA,
93, 5619-5623.
Bedouelle, H., Guez-Ivanier, V. Nageotte, R. (1993).
Discrimination between transfer-RNAs by tyrosyl-
tRNA synthetase. Biochimie, 75, 1099-1108.

Belfort, M., Moelleken, A., Maley, G. F. Maley, F.
(1983). Puri®cation and properties of T4 phage thy-
midylate synthetase produced by the cloned gene
in an ampli®cation vector. J. Biol. Chem. 258, 2045-
2051.
Belfort, M., Chandry, P. S. Pedersen-Lane, J. (1987).
Genetic delineation of functional components of the
group I intron in the phage T4 td gene. Cold Spring
Harbor Symp. Quant. Biol. 52, 181-192.
Bell-Pederson, D., Galloway Salvo, J. L. Belfort, M.
(1991). A transcription terminator in the thymidy-
late synthase (thyA) structural gene of Escherichia
coli and construction of a viable thyA::Kmr
deletion.
J. Bacteriol. 173, 1193-1200.
Brion, P., Michel, F., Schroeder, R. Westhof, E.
(1999a). Analysis of the cooperative thermal unfold-
ing of the td intron of bacteriophage T4. Nucl. Acids
Res. 27, 2494-2502.
Brion, P., Schroeder, R., Michel, F. Westhof, E.
(1999b). In¯uence of speci®c mutations on the ther-
mal stability of the td group I intron in vitro and on
its splicing ef®ciency in vivo: a comparative study.
RNA, 5, 947-958.
Caprara, M. G., Mohr, G. Lambowitz, A. M. (1996a).
A tyrosyl-tRNA synthetase protein induces tertiary
folding of the group I intron catalytic core. J. Mol.
Biol. 257, 521-531.
Caprara, M. G., Lehnert, V., Lambowitz, A. M.
Westhof, E. (1996b). A tyrosyl-tRNA synthetase
recognizes a conserved tRNA-like structural motif
in the group I intron catalytic core. Cell, 87, 1135-
1145.
Cech, T. R. Golden, B. L. (1999). Building a catalytic
active site using only RNA. In The RNA World
(Gesteland, R. F., Cech, T. R. Atkins, J. F., eds),
2nd edit., pp. 321-349, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York.
Cech, T. R., Damberger, S. H. Gutell, R. R. (1994).
Representation of the secondary and tertiary struc-
ture of group I introns. Nature Struct. Biol. 1, 273-
280.
Chang, A. C. Y. Cohen, S. N. (1978). Construction
and characterization of ampli®able multicopy DNA
cloning vehicles derived from the P15A cryptic
miniplasmid. J. Bacteriol. 134, 1141-1156.
Chou, S.-H., Zhu, L. Reid, B. R. (1997). Sheared
purine-purine pairing in biology. J. Mol. Biol. 267,
1055-1067.
Coetzee, T., Herschlag, D. Belfort, M. (1994). Escheri-
chia coli proteins, including ribosomal protein S12,
facilitate in vitro splicing of phage T4 introns by act-
ing as RNA chaperones. Genes Dev. 8, 1575-1588.
Duckett, D. R. Lilley, D. M. J. (1991). Effects of base
mismatches on the structure of the four-way DNA
junction. J. Mol. Biol. 221, 147-161.
Duckett, D. R., Murchie, A. I. H. Lilley, D. M. J.
(1995). The global folding of four-way helical junc-
tions in RNA, including that in U1 snRNA. Cell, 83,
1027-1036.
Ehrenman, K., Schroeder, R., Chandry, P. S., Hall, D. H.
Belfort, M. (1989). Sequence speci®city of the P6
pairing for splicing of the group I td intron of
phage T4. Nucl. Acids Res. 17, 9147-9163.
Galloway Salvo, J. L., Coetzee, T. Belfort, M. (1990).
Deletion-tolerance and trans-splicing of the bacterio-
phage T4 td intron. Analysis of the P6-L6a region.
J. Mol. Biol. 211, 537-549.
Garriga, G. Lambowitz, A. M. (1986). Protein-depen-
dent splicing of a group I intron in ribonucleopro-
tein particles and soluble fractions. Cell, 46, 669-680.
Gautheret, D. Gutell, R. R. (1997). Inferring the con-
formation of RNA base-pairs and triples from pat-
terns of sequence variation. Nucl. Acids Res. 25,
1559-1564.
Gautheret, D., Konings, D. Gutell, R. R. (1994). A
major family of motifs involving GA mismatches in
ribosomal RNA. J. Mol. Biol. 242, 1-8.
Gautheret, D., Damberger, S. H. Gutell, R. R. (1995).
Identi®cation of base-triples in RNA using com-
parative sequence analysis. J. Mol. Biol. 248, 27-43.
Golden, B. L., Gooding, A. R., Podell, E. R. Cech, T. R.
(1998). A preorganized active site in the crystal
structure of the Tetrahymena ribozyme. Science, 282,
259-264.
Guo, Q. Lambowitz, A. M. (1992). A tyrosyl-tRNA
synthetase binds speci®cally to the group I intron
catalytic core. Genes Dev. 6, 1357-1372.
Ho, Y., Kim, S. J. Waring, R. B. (1997). A protein
encoded by a group I intron in Aspergillus nidulans
directly assists RNA splicing and is a DNA endonu-
clease. Proc. Natl Acad. Sci. USA, 94, 8994-8999.
Jaeger, L., Westhof, E. Michel, F. (1993). Monitoring
of the cooperative unfolding of the sunY group I
intron of bacteriophage T4. The active form of the
sunY ribozyme is stabilized by multiple interactions
with 3H
terminal intron components. J. Mol. Biol.
234, 331-346.
Laggerbauer, B., Murphy, F. L. Cech, T. R. (1994).
Two major tertiary folding transitions of the Tetra-
hymena catalytic RNA. EMBO J. 13, 2669-2676.
Lambowitz, A. M. Perlman, P. S. (1990). Involvement
of aminoacyl-tRNA synthetases and other proteins
in group I and group II intron splicing. Trends Bio-
chem. Sci. 15, 440-444.
Lambowitz, A. M., Caprara, M. G., Zimmerly, S.
Perlman, P. S. (1999). Group I and group II ribo-
zymes as RNPs: clues to the past and guides to the
future. In The RNA World (Gesteland, R. F., Cech,
T. R. Atkins, J. F., eds), 2nd edit., pp. 451-485,
Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York.
Leontis, N. B. Westhof, E. (1998). Conserved geometri-
cal base-pairing patterns in RNA. Quart. Rev.
Biophys. 31, 399-455.
Lusk, J. E., Williams, R. J. P. Kennedy, E. P. (1968).
Magnesium and the growth of Escherichia coli. J. Mol.
Biol. 243, 2618-2624.
Mannella, C. A., Collins, R. A., Green, M. R.
Lambowitz, A. M. (1979). Defective splicing of
mitochondrial rRNA in cytochrome-de®cient
nuclear mutants of Neurospora crassa. Proc. Natl
Acad. Sci. USA, 76, 2635-2639.
Michel, F. Westhof, E. (1990). Modeling of the three-
dimensional architecture of group I catalytic introns
based on comparative sequence analysis. J. Mol.
Biol. 216, 585-610.
Mohr, G., Zhang, A., Gianelos, J. A., Belfort, M.
Lambowitz, A. M. (1992). The Neurospora CYT-18
protein suppresses defects in the phage T4 td intron
by stabilizing the catalytically active structure of the
intron core. Cell, 69, 483-494.
Mohr, G., Caprara, M. G., Guo, Q. Lambowitz, A. M.
(1994). A tyrosyl-tRNA synthetase can function
similarly to an RNA structure in the Tetrahymena
ribozyme. Nature, 370, 147-150.

Murphy, F. L. Cech, T. R. (1993). An independently
folding domain of RNA tertiary structure within
the Tetrahymena ribozyme. Biochemistry, 32, 5291-
5300.
Myers, C. A., Wallweber, G. J., Rennard, R., Kemel, Y.,
Caprara, M. G., Mohr, G. Lambowitz, A. M.
(1996). A tyrosyl-tRNA synthetase suppresses struc-
tural defects in the two major helical domains of
the group I intron catalytic core. J. Mol. Biol. 262,
87-104.
Quigley, G. J. Rich, A. (1976). Structural domains of
transfer RNA molecules. Science, 194, 796-806.
Saldanha, R. J., Ellington, A. Lambowitz, A. M.
(1996). Analysis of the CYT-18 protein binding site
at the junction of stacked helices in a group I intron
RNA by quantitative binding assays and in vitro
selection. J. Mol. Biol. 261, 23-42.
Sclavi, B., Sullivan, M., Chance, M. R., Brenowitz, M.
Woodson, S. A. (1998). RNA folding at millisecond
intervals by synchrotron hydroxyl radical footprint-
ing. Science, 279, 1940-1943.
Shaw, L. C. Lewin, A. S. (1995). Protein-induced fold-
ing of a group I intron in cytochrome b pre-mRNA.
J. Biol. Chem. 270, 21552-21562.
Suh, S. O., Jones, K. G. Blackwell, M. (1999). A group
I intron in the nuclear small subunit rRNA gene of
Cryptendoxyla hypophloia, an ascomycetous fungus:
evidence for a new major class of group I introns.
J. Mol. Evol. 48, 493-500.
Tanner, M. A. Cech, T. R. (1997). Joining the two
domains of a group I ribozyme to form the catalytic
core. Science, 275, 847-849.
Tian, G. L., Michel, F., Macadre, C. Lazowska, J.
(1993). Sequence of the mitochondrial gene encod-
ing subunit I of cytochrome oxidase in Saccharo-
myces douglasii. Gene, 124, 153-163.
van der Horst, G., Christian, A. Inoue, T. (1991).
Reconstitution of a group I intron self-splicing reac-
tion with an activator RNA. Proc. Natl Acad. Sci.
USA, 88, 184-188.
Wallweber, G. J., Mohr, S., Rennard, R., Caprara, M. G.
Lambowitz, A. M. (1997). Characterization of
Neurospora mitochondrial group I introns reveals
different CYT-18 dependent and independent spli-
cing strategies and an alternative 3H
splice site for
an intron ORF. RNA, 3, 114-131.
Weeks, K. M. Cech, T. R. (1996). Assembly of a ribo-
nucleoprotein catalyst by tertiary structure capture.
Science, 271, 345-348.
Werntges, H., Steger, G., Riesner, D. Fritz, H.-J.
(1986). Mismatches in DNA double strands: thermo-
dynamic parameters and their correlation to repair
ef®ciency. Nucl. Acids Res. 14, 3773-3790.
Westhof, E. (1992). Westhof's rule. Nature, 358, 459-460.
Wu, M. Turner, D. H. (1996). Solution structure of
(rGCGGACGC)2 by two-dimensional NMR and the
iterative relaxation matrix approach. Biochemistry,
35, 9677-9689.
Zarrinkar, P. P. Williamson, J. R. (1994). Kinetic inter-
mediates in RNA folding. Science, 265, 918-924.
Zhang, A., Derbyshire, V., Galloway Salvo , J. L.
Belfort, M. (1995). Escherichia coli protein StpA
stimulates self-splicing by promoting RNA assem-
bly in vitro. RNA, 1, 783-793.
Edited by D. Draper
(Received 29 March 2000; received in revised form 14 June 2000; accepted 16 June 2000)

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