J. Mol. Biol. (1998) 275, 731±746
Modified Nucleotides of tRNAPro Restrict Interactions
in the Binary Primer/Template Complex of M-MuLV
Philippe FosseÂ2, Marylene Mougel3, GeÂrard Keith1, Eric Westhof1
Bernard Ehresmann1 and Chantal Ehresmann1*
1
Unite Propre de Recherche
9002 du CNRS, Institut de
Biologie MoleÂculaire et
Cellulaire, 15 rue Descartes
67084, Strasbourg cedex
France
2
Unite de BiochimieEnzymologie, CNRS URA147
Laboratoire de Physicochimie et
Pharmacologie des
MacromoleÂcules Biologiques,
Institut Gustave Roussy, rue
Camille Desmoulins,
94805, Villejuif cedex, France
3
Unite Mixte de Recherches
5535, Institut de GeÂneÂtique
MoleÂculaire, 1919 route de
Mende, 34033, Montpellier
France
In all retroviruses, reverse transcription is primed by a cellular tRNA,
which is base-paired through its 30 -terminal 18 nucleotides to a complementary sequence on the viral RNA genome termed the primer binding
site (PBS). Evidence for speci®c primer-template interactions in addition
to this standard interaction has recently been demonstrated for several
retroviruses. Here, we used chemical and enzymatic probing to investigate the interactions between Moloney murine leukemia virus (M-MuLV)
RNA and its natural primer tRNAPro. The existence of extended interactions was further tested by comparing the viral RNA/tRNAPro complex
with simpli®ed complexes in which viral RNA or tRNA were reduced to
the 18 nt of the PBS or to the complementary tRNA sequence. These
data, combined with computer modeling, provide important clues on the
secondary structure and three-dimensional folding of the M-MuLV
RNA/tRNAPro complex. In contrast with other retroviruses, we found
that the interaction between tRNAPro and the M-MuLV RNA template is
restricted to the standard PBS interaction. In this binary complex, the
viral RNA is highly constrained and the rest of tRNAPro is rearranged,
with the exception of the anticodon arm, leading to a very compact structure. Unexpectedly, when a synthetic tRNAPro lacking the post-transcriptional modi®cations is substituted for the natural tRNAPro primer, the
interactions between the primer and the viral RNA are extended. Hence,
our data suggest that the post-transcriptional modi®cations of natural
tRNAPro prevent additional contacts between tRNAPro and the U5 region
of M-MuLV RNA.
# 1998 Academic Press Limited
*Corresponding author
Keywords: reverse transcription; M-MuLV retrovirus; tRNA primer;
primer/template interactions; conformation
Introduction
A ubiquitous property of retroviruses is the synthesis, by the viral reverse transcriptase (RT), of
the single-stranded viral RNA genome into linear
Abbreviations used: RT, reverse transcriptase; ASLV,
avian sarcoma and leukosis virus; HIV-1, human
immunode®ciency virus type 1; M-MuLV, Moloney
murine leukemia virus; PBS, primer binding site; DMS,
dimethylsulfate; CMCT, 1-cyclohexyl-N0 -[2-(Nmethylmorpholino)]ethyl-carbodiimide-ptoluenesulfonate; DEPC, diethylpyrocarbonate; AMV,
avian myeloblastosis virus; m1A, 1-methyladenosine; D,
dihydrouridine; , pseudouridine; I, inosine; U*, 5carbamoylmethyluridine; Um, 20 -O-methyluridine; m,
20 -O-methylpseudouridine; m7G, 7-methylguanosine;
m2G, N2-methylguanosine; m1G, 1-methylguanosine;
m5C, 5-methylcytosine; nt, nucleotide(s).
0022±2836/98/050731±16 $25.00/0/mb971487
double-stranded DNA prior to integration into the
host cell genome (Baltimore, 1970; Temin &
Mizutani, 1970; Cof®n, 1984). To initiate this process, which is referred to as reverse transcription,
the viral RT utilizes a cellular tRNA as primer.
Different retroviruses use different tRNAs as primers for reverse transcription (for a review, see
Marquet et al., 1995). For example, the avian sarcoma and leukosis viruses (ASLV) use tRNATrp, the
human immunode®ciency viruses type 1 and 2
(HIV-1 and HIV-2) utilize tRNALys
3 , and the human
T-cell leukemia virus and Moloney murine leukemia virus (M-MuLV) use tRNAPro. The 30 -terminal
18 nucleotides (nt) of the tRNA primer is basepaired to the primer binding site (PBS), a complementary sequence near the 50 -end of the viral
genome. Recently, both in vitro and in vivo studies
have supported the presence of interactions besides
# 1998 Academic Press Limited
732
the canonical PBS-tRNA interaction, between genomic RNA and primer tRNAs in the initiation complexes of ASLV and HIV-1 reverse transcription
(Aiyar et al., 1992, 1994; Isel et al., 1993, 1995;
Wake®eld et al., 1996; Zhang et al., 1996; Kang et al.,
1996, 1997). In ASLV, an additional contact
between the retroviral U5 RNA and the TC loop
of tRNATrp is required for ef®cient synthesis of (ÿ)
strong stop DNA (Aiyar et al., 1992, 1994). In HIV1, the additional contacts involve the anticodon
loop of tRNALys
and a conserved adenine-rich loop
3
upstream of the PBS. This interaction requires the
post-transcriptional modi®cations of the tRNA (Isel
et al., 1993) and facilitates the transition from
initiation to elongation during reverse transcription
in vitro (Isel et al., 1996; Lanchy et al., 1996).
Although it has been shown that ASLV and HIV-1
can use tRNAs different from their wild-type
tRNA primer to initiate reverse transcription when
the PBS is complementary to other tRNAs, they all
reverted back to the wild-type PBS sequence upon
extended time in cell culture (Li et al., 1994; Das
et al., 1995; Whitcomb et al., 1995; Wake®eld et al.,
1996). However, reversion in HIV-1 could be prevented by mutating the sequence complementary
to the corresponding tRNA anticodon loop
(Wake®eld et al., 1996; Zhang et al., 1996; Kang
et al., 1996, 1997). Therefore, additional interactions
between the tRNA primer and the viral genome
outside the PBS might contribute to the selective
use of a speci®c tRNA to initiate ASLV and HIV-1
reverse transcription. Conversely, M-MuLV can
replicate by using various tRNAs as primers without reversion, provided that the PBS matches the
30 -end of the tRNAs (Lund et al., 1993). M-MuLV
also shows other remarkable particularities as compared to other retroviruses. The mature M-MuLV
RT is a single polypeptide, while those of ASLV
and HIV-1 are heterodimeric proteins (Di Marzo
Veronese et al., 1986; Kacian et al., 1971). The
nucleocapsid protein (NC), that facilitates annealing between the tRNA primer and the viral RNA
template in vitro (Barat et al., 1989; Prats et al.,
1988), contains one conserved motif termed the
Cys-His box in M-MuLV but two in ASLV and
HIV-1. Therefore, it would be interesting to know
whether extended interactions do take place
between viral RNA and tRNAPro in M-MuLV. Up
to now, no data on the structure of the M-MuLV
RNA/tRNAPro binary complex are available. For
this purpose, we used chemical and enzymatic
probing to investigate the secondary structure of
M-MuLV RNA/tRNAPro complex. In order to
further test the presence or the absence of
additional interactions outside of the PBS, we also
compared the structure of both viral RNA and
tRNAPro hybridized to oligonucleotides corresponding to the anti PBS or PBS sequence. The role
of post-transcriptional modi®cations of tRNAPro on
the binary complex formation was also tested by
substituting a synthetic tRNAPro for the natural
tRNAPro primer.
Binary Primer/Template Complex of M-MuLV
Results
Experimental strategy
The mutual adaptation of M-MuLV viral RNA
and tRNAPro induced by formation of the primer/
template complex required for initiation of reverse
transcription was investigated by a variety of
chemical and enzymatic structure probes. The
binary complex contained RNA 1-725 as viral template. This RNA starts at position ‡1 and encompasses the 50 -untranslated region and the 50 ®rst
105 nt of gag sequence (Mougel et al., 1993). It folds
in vitro into independent and highly structured
domains (Tounekti et al., 1992; Mougel et al., 1993).
The conformation of one of these (the domain,
located downstream of the PBS domain) is in close
agreement with that observed within the virions
(Alford et al., 1991). Natural tRNAPro puri®ed from
beef liver was used as primer. The isolated tRNA
contains two isoacceptors which differ only by two
nt located in the anticodon loop (Figure 1). Two
tRNAPro isoacceptors were also found within MMuLV particles (Harada et al., 1979). The bovine
tRNAPro (U*GG) is completely identical to the
mouse tRNAPro (U*GG), whereas the second isoacceptor differs only by one nt (m31 of bovine
tRNAPro (IGG) is replaced by Um31 in mouse
tRNAPro (IGG); Keith et al., 1990).
The existence of possible additional base-pairing
contacts between RNA 1-725 and tRNAPro was
further investigated by analyzing the structural
changes induced in RNA 1-725 upon hybridization
of an oligodeoxyribonucleotide complementary to
the PBS sequence (D18PBS0 ). Similarly, an oligoribonucleotide complementary to the 30 -terminal
18 nt of tRNAPro (R18PBS) was used to follow the
structural changes induced in the tRNA primer by
base-pairing with the PBS sequence. In addition,
the possible role of post-transcriptional modi®cations of tRNAPro in the binary complex structure
was studied by substituting a synthetic tRNAPro
(utRNAPro), lacking post-transcriptional modi®cations.
The different binary complexes were probed
with chemicals that provide precise information on
the reactivity of various atomic positions on the
RNA (Ehresmann et al., 1987). DMS (speci®c for
A(N-1) and C(N-3)) and CMCT (speci®c for G(N-1)
and U(N-3)) modify bases when they are not
involved in Watson-Crick base-pairing. DEPC provides information on the reactivity of position N-7
of adenines. Note that adenines in helices are
unreactive to DEPC, due to the high sensitivity of
this reagent to base stacking. The structure was
also probed with RNase T1 (speci®c for unpaired
guanines) and RNase V1 (speci®c for doublestranded and for stacked regions). However, due
to the bulky size of RNases, the absence of cuts by
these enzymes might result from steric hindrance
rather than speci®c structural features. Chemical
modi®cations and enzymatic cuts were identi®ed
by primer extension on viral RNA, and by direct
733
Binary Primer/Template Complex of M-MuLV
Figure 1. Secondary structure models proposed for the M-MuLV PBS region by Mougel et al. (1993) and cloverleaf
structure of tRNAPro. The PBS and the complementary sequence in tRNA are shown in red ®lled boxes. The structural rearrangements occurring in tRNAPro upon formation of the template/primer binary complex, deduced from
this study, are indicated by blue, green and yellow ®lled boxes. The putative additional interaction between tRNAPro
and M-MuLV RNA, postulated by Aiyar et al. (1992), involves nt GGGUCU (shown in an empty green box) in MMuLV RNA and nt GGGC in tRNA. m and I pointed out by arrows indicate that these nt replace and U,
respectively, in the second isoacceptor tRNAPro (Keith et al., 1990).
detection on 30 -end-labeled tRNAPro. Indeed, the
presence of post-transcriptional modi®cations on
natural tRNA prevents the use of primer extension.
Thus, in tRNAPro only C(N-3) and A(N-7) modi®ed
positions, which can be cleaved by further adapted
chemical treatment, could be tested. In addition,
tRNAPro was probed with Pb(II), which is able to
detect subtle conformational differences in RNAs.
Pb(II) induces two types of cleavages: cleavages
requiring tight binding of Pb2‡ (Krzyzosiak et al.,
1988; Behlen et al., 1990; Zito et al., 1993; Paillart
et al., 1997), and low yield cleavages at multiple
sites in ¯exible and dynamic RNA regions, usually
interhelical and loop regions (Gornicki et al., 1989;
Brunel et al., 1991). Examples of probing gels are
shown in Figures 2 and 3, and results re¯ecting
several independent experiments are summarized
in Figures 4 and 5. The derivation of the secondary
structure models was assisted by computer prediction using the Zuker algorithm (Jaeger et al., 1989)
and allowed the selection of the foldings that were
consistent with experimental data.
Limited interactions between viral RNA
and tRNAPro
The U5-PBS domain was found to fold into two
mutually exclusive conformations, an extended
734
Binary Primer/Template Complex of M-MuLV
Figure 2. Chemical probing of the PBS region of M-MuLV RNA. Modi®cations of G(N-1) and U(N-3) with CMCT
were carried out under conditions described in Materials and Methods. A, Free RNA 1-725 (lanes 1 to 4) or RNA 1725 hybridized to tRNAPro (lanes 5 to 8), D18PBS0 (lanes 9 to 12) or utRNAPro (lanes 13 to 18) were incubated with
CMCT for ®ve minutes (lanes 2, 6, 10 and 14), ten minutes (lanes 3, 7, 11 and 15) or 20 minutes (lanes 4, 8, 12 and
16). Lanes 1, 5, 9 and 13 are controls without CMCT. B, RNA 1-725 hybridized to utRNAPro (lanes 1 to 4), tRNAPro
(lanes 5 to 8) or D18PBS0 (lanes 9 to 12) were incubated with DMS for two minutes (lanes 2, 6 and 9), four minutes
(lanes 3, 7 and 10) or seven minutes (lanes 4, 8 and 11). Lanes 1, 5 and 12 are controls without DMS. Sequence lanes
(A, C, G and U) were run in parallel. Arrows point out the chemically modi®ed bases.
helical structure (E form) and a three-branch
structure (B form), depending on ionic strength
and thermal renaturation conditions (Mougel et al.,
1993) (Figure 1). In particular, the dimeric RNA
was found in the B form. According to the probing data, a control RNA subjected to the hybridization procedure (in the absence of tRNA)
shows structural heterogeneity, with some preference for the B form. The following results focus
on the structure of the viral RNA in the various
binary complexes.
Probing of viral RNA 1-725 hybridized to tRNAPro
Reactivity changes induced by hybridization of
tRNAPro to RNA 1-725 are mostly located in the
close vicinity of the PBS. As expected, hybridization of tRNAPro to the PBS induces a strong
decrease in reactivity to chemical probes of the nt
in the PBS which were reactive in the free B form.
For example, U153, U156, and U163 which are reactive towards CMCT in the free RNA become
unreactive in the binary complex (Figure 2A, compare lanes 2 to 4 to lanes 6 to 8). In addition, a new
RNase V1 cut is observed at G160 (not shown). Conversely, reactivity increases are observed on the
opposite strand (see U109, Figure 2A), re¯ecting the
disruption of base-pairing between nt 105 to 110
and 146 to 151 of the free B form. However, the
absence of reactivity of CC106 and the decreased
reactivity of U104 and U107 towards CMCT
(Figure 2A) most likely re¯ect a local rearrangement resulting in the extension of helix 99-104/
168-173 present in B form (Figure 1) by three Watson-Crick base-pairs involving nt CCU107 and
GGG167 (Figure 4A). This scheme accounts for the
non-reactivity of GG166. Besides, the adjacent C164,
unreactive in both free B and E forms becomes
reactive to DMS (Figure 2B, lanes 6 to 8). It should
be noted that the non-reactivity of nt GGGUCU104
Binary Primer/Template Complex of M-MuLV
could also be explained by an interaction with
GGGC of tRNAPro, as proposed on the basis of
the interactions demonstrated in avian retroviruses
735
(Aiyar et al., 1992). However, it does not explain
the non-reactivity of CC106. On the other hand,
reactivity increases are observed just upstream of
Figure 3. Chemical and enzymatic probing of 30 -end-labeled tRNAPro and utRNAPro. Modi®cations and enzymatic
cleavages were carried out as described in Materials and Methods. A, Enzymatic probing of tRNAPro and utRNAPro
with RNase T1. Lanes 1 to 3 and 13 to 14 show free tRNAPro and free utRNAPro, respectively. tRNAPro and utRNAPro
were hybridized to R18PBS (lanes 4 to 6 and 16 to 18) or to RNA 1-725 (lanes 7 to 9 and 10 to 12). Free or hybridized
tRNAs were incubated with 0.05 unit (lanes 2, 5, 8, 11, 14 and 17) and 0.1 unit (lanes 3, 4, 9, 12, 15 and 16) of RNase
T1. Lanes 1, 6, 7, 10, 13 and 18 are controls without enzyme. Arrows point out guanines cleaved by RNase T1.
B, Modi®cations of A(N-7) with DEPC. Lanes 1 to 5 correspond to free tRNAPro, lanes 6 to 10 and 11 to 15 correspond to tRNAPro hybridized to RNA 1-725 and R18PBS, respectively. DEPC treatment was for 20 minutes (lanes 3, 8
and 13), 40 minutes (lanes 4, 7 and 14) or 60 minutes (lanes 5, 6 and 15). Lanes 1, 10 and 11 are controls without any
chemical treatment. Lanes 2, 9 and 12 are controls without DEPC treatment but with aniline treatment. Bc, T1 and L
refer to RNases Bacillus cereus, T1 sequencing and to alkaline ladder, respectively. Chemically modi®ed adenines are
pointed out by arrows. Control experiments showed that the third band above A58 and observed in lanes 13 to 15
does not correspond to a modi®ed adenine.
736
the PBS (see UUU146, Figure 2A), probably resulting from disruption of base-pairing between nt
GAG112 and UUU146 in both B and E forms. Note
the high reactivity of U146, which suggests that it
does not form a stable interaction with the 30 -terminal A of tRNAPro. Taken together, these results
strongly suggest that the viral RNA adopts a
Binary Primer/Template Complex of M-MuLV
unique conformation derived from the B form
(Figure 4A), constrained by a helical and irregular
structure (nt 77 to 107/165 to 199) similar to that
found in the B form RNA, with an unchanged top
hairpin (nt 114 to 142). At a ®rst glance, these data
do not suggest additional interactions between
viral RNA and tRNAPro.
Figure 4. Proposed secondary structures for the PBS region of M-MuLV RNA hybridized to tRNAPro (A), D18PBS0 (B)
or utRNAPro (C). Part of the tRNA molecules that does not interact with viral RNA is schematized by a continuous
green line. Secondary structure diagrams display the relative reactivities of nt 69 to 203 of M-MuLV RNA. The color
codes used for the reactivities (estimated between 1 and 4 from marginal to strong) are indicated in the inset. The
reactivity changes induced by D18PBS0 and utRNAPro hybridization versus tRNAPro hybridization are indicated by
asteriks. Cleavage induced by RNase V1 was not determined when RNA 1-725 was hybridized to utRNAPro. Numbering of RNA 1-725 is relative to the genomic cap site (‡1).
Binary Primer/Template Complex of M-MuLV
Probing of viral RNA 1-725 hybridized to D18PBS0
The reactivity pattern of viral RNA hybridized
to D18PBS0 does not signi®cantly differ from that
737
obtained with tRNAPro (Figure 4B), consistent with
the view that interactions between viral RNA and
tRNAPro are mainly restricted to the canonical PBS
interaction. Only minor differences in reactivity
Figure 5. Proposed secondary structures for tRNAPro (A, B) and utRNAPro (B, C) hybridized to RNA 1-725 (A,C) or
R18PBS (B, C). The viral RNA that does not interact with tRNA is schematized by a continuous green line. Secondary
structure diagrams display the relative reactivities of nt in tRNAPro and utRNAPro to DMS, DEPC, lead(II), RNases T1
and V1. The color codes used for the reactivities (estimated between 1 and 4 from marginal to strong) are indicated
in the inset. Cleavage induced by lead(II) was not determined between nt 1 to 10 of tRNAPro and utRNAPro when
these tRNAs were hybridized to R18PBS. Numbering of tRNAs does not correspond to the conventional numbering
system (Gauss & Sprinzl, 1984).
738
levels are observed (marked with asterisks in
Figure 4B). The only remarkable difference is the
non-reactivity of C164 (Figure 2B, lanes 9 to 11),
accompanied by two new strong RNase V1 cuts at
the 30 end of the PBS (A162 and U163). These differences probably re¯ect steric constraints caused by
the presence of other parts of the bound tRNA
when tRNAPro is hybridized. As a consequence,
the orientation of C164 might differ in the two
binary complexes, accounting for the reactivity
difference. These data argue against extended
interactions between viral RNA and tRNAPro, but
the presence of additional minor contacts might
not be totally excluded. Therefore, it was necessary
to investigate the conformation of tRNAPro in the
binary complex.
Probing of tRNAPro hybridized to viral RNA 1-725
Due to its compact tertiary folding, the free
tRNAPro shows only limited accessibility to both
enzymatic and chemical probes. Indeed, the only A
and C residues reactive to DEPC or DMS are
CCC74 near the 30 -end, and A58 in the loop
(Figure 3B). RNase T1 exclusively cleaves in the
anticodon loop (Figure 3A) and RNase V1 in the
anticodon stem and acceptor stems (not shown).
One main lead cleavage occurs in the anticodon
loop at U33 (not shown). A direct consequence of
the hybridization of tRNAPro to the PBS is the loss
of reactivity of CCC74 accompanied by a displacement of the RNase V1 cuts from positions 68 to 70
to 69 to 74. Base-pairing between the 30 -terminal
18 nt of tRNAPro (in red in Figure 1) and the PBS
was expected to disrupt acceptor and arms, as
well as the tertiary interactions between the D and
the loops. Accordingly, A14, A20 in the D loop,
and A56 in the loop, become strongly reactive
towards DEPC upon formation of the tRNAPro/
RNA 1-725 complex (Figure 3B, compare lanes 3 to
5 to lanes 6 to 8). Similarly, C55 unreactive towards
DMS in the free state, as already observed in
tRNALys
(Isel et al., 1995) and tRNAAsp (Romby
3
et al., 1987), become reactive in the bound state
(data not shown). However, the 50 parts of both
acceptor and stems, which should be liberated
upon PBS binding, remain unreactive towards
RNase T1 and DMS in the binary complex
(Figure 5A). A possible rearrangement accounting
for these observations is base-pairing between the
50 -terminal pGGCmUC5 sequence and GGG54
(in green in Figure 1). This pairing is further supported by a RNase V1 cut between residues C3 and
Um4 (Figure 5A).
Otherwise, guanine bases 15 to 18 in the D loop,
expected to become accessible after disruption of
the tertiary interactions, remain unaccessible to
RNase T1 (Figure 3A). This absence of reactivity
may be explained by base-pairing of GGGGD19
with m7GDm5Cm5CC49, the only potentially complementary sequence (shown in blue in Figure 1).
Such pairing would account for the absence of
reactivity of m5Cm5CC to DMS and the presence
Binary Primer/Template Complex of M-MuLV
of RNase V1 cleavage at m5C47 and C49. However,
stable pairing is probably limited to the three adjacent G-C pairs, which are favored by the presence
of the two m5C at position 47 and 48. Indeed, the
presence of two dihydrouridines (D) and a m7G in
the ¯anking region is expected to destabilize basepairing between G and D, since the non-planar D
base is known to decrease stacking and to increase
RNA ¯exibility (Dalluge et al., 1996). Nevertheless,
the question on whether D is involved in hydrogen
interactions is unresolved and the presence of
these three modi®ed bases might result in a particular three-dimensional conformation. The postulated interactions are accompanied by the
disruption of stem D, as shown by the accessibility
of G10 and G22 to RNase T1 (Figure 3A) and that
A23 is modi®ed by DEPC (Figure 3B). On the other
hand, the anticodon arm does not appear to be
affected by the formation of the binary complex,
since G34 and G35 are still cleaved by RNase T1,
and A41 (in the anticodon stem) remains unreactive
to DEPC (Figure 3). Moreover, lead still induces a
strong cleavage at U33 (Figure 5A). The anticodon
arm can be extended by two Watson-Crick basepairs (U.24G44 and U.25A43, shown in yellow in
Figure 1), acccounting for the non-reactivity of A43
to DEPC and by RNase V1 cleavage (Figure 5A).
Altogether our data are consistent with a model
in which tRNA rearranges upon formation of basepairing with PBS (Figure 5A). These intramolecular
rearrangements involve base-pairing between the
50 strand of the acceptor stem and part of the arm, as well as between the D loop and part of the
stem and variable loop. The remaining interhelical regions 6 to 14 and 20 to 23 are fully accessible
to chemicals and RNase T1. These regions also
become very sensitive to lead cleavage, as expected
for single-stranded and ¯exible regions. It is noteworthy that nt linking the PBS stem to the rest of
the tRNA molecule are reactive to DMS and DEPC
but are not cleaved by lead, indicating that they
are highly exposed to the solvent but cannot move
freely. A surprising observation is the reactivity of
m1G9 and m1G36 to DMS in the binary complex,
while only the latter was reactive in the naked
tRNA. A possible explanation is that DMS methylates the N-7 position of G, which would be cleaved
by the hydrazine/aniline treatment when position
1 is methylated. Consistent with this assumption,
position N-7 of residue 9 is involved in a tertiary
interaction in the consensus tRNA structure
(Saenger, 1984). The proposed model supports the
previous assumption that the interaction between
tRNAPro and the M-MuLV RNA template is
restricted to base-pairing between the PBS and the
30 -terminal 18 nt of tRNAPro (Figure 5A). If this
assertion is true, then tRNAPro should fold into the
same secondary structure when it is annealed to
R18PBS.
739
Binary Primer/Template Complex of M-MuLV
Probing of tRNAPro hybridized to R18PBS
As postulated above, the reactivity pattern of
tRNAPro remains mostly the same when R18PBS is
hybridized instead of viral RNA (Figures 3 and
5B). One subtle difference is seen at nt CAm1AA58,
linking the PBS helix to the rest of the tRNA molecule. Their reactivity to DEPC and DMS is
strongly reduced when R18PBS is substituted for
tRNAPro. Surprisingly, these nt also become
strongly cleaved by lead. A possible explanation is
that the PBS helix and helix 1 to 5/50 to 54 may be
coaxially stacked when hybridized to R18PBS, with
nt 55 to 58 in a stacked con®guration, thus explaining their reduced reactivity. The appearance of a
strong lead cleavage between A56 and m1A57 might
re¯ect the presence of a magnesium binding site
stabilizing this particular conformation. Such a
coaxial conformation would be most likely prevented by the bulk viral RNA. Likewise, the bulk
viral RNA might reduce the accessibility of G10 to
RNase T1 in the viral/tRNA complex by steric hindrance.
Extended interactions between viral RNA
and utRNAPro
Unexpectedly,
signi®cant
differences
are
observed at discrete positions of viral RNA when
utRNAPro is substituted for natural tRNA. The
most striking differences are the enhanced reactivity of UCCU107 and the loss of reactivity of UG176
(Figure 2A, compare lanes 6 to 8 to lanes 14 to 16).
Other minor differences occur in the reactivity
level of several nt (slight reduction at A98, U163 and
AUUU146). The results are summarized in
Figure 4C. These results suggest that nt UCCU107
are not paired, while nt downstream of the PBS are
rearranged. In particular, nt GGGA168 do not
become reactive, suggesting they are no more
paired with UCCU107, which become reactive. In
addition, reactivity changes were found far from
the PBS within the region spanning nt 69 to 78. For
example, nt UUGU78 are more reactive towards
CMCT (Figure 4C). At that level, it was dif®cult to
understand the nature of the rearrangements
occurring upon hybridization of utRNAPro and it
was necessary to investigate the conformation of
the hybridized utRNAPro.
A ®rst general observation is the increased susceptibility of utRNAPro to lead cleavage and RNase
T1 hydrolysis, compared to the natural tRNA, in
the free or bound states. One example is the
enhanced reactivity of the anticodon loop. This is
not surprising, since unmodi®ed tRNAs are known
to have a relaxed structure and speci®city (Perret
et al., 1990b). However, despite this overall increase
of reactivity, several striking differences can be
noted between the different binary complexes. In
the viral RNA/utRNAPro complex, G10 is not
cleaved by RNase T1 (Figure 3A, lanes 11 and 12),
substantial differences of sensitivity to lead cleavage are observed in the 50 -end region, and a new
RNase T1 cut appears at G52, accompanied by an
increased sensitivity to lead cleavage in the surrounding region (Figure 5C).
In the R18PBS/utRNAPro complex, the 50 -end
becomes more reactive to DMS (see C5) and to
RNase T1 (new cuts at G2, G6, G9, G17, G18) while
the susceptibility to lead cleavage is reduced, and
C55 becomes unreactive to DMS (Figure 5D). Thus,
the folding of utRNAPro not only differs depending
on whether it is hybridized to viral RNA or
R18PBS, but also from the folding of the natural
tRNAPro bound to R18PBS. Therefore, utRNAPro
can adopt various conformations that are normally
prevented by post-transcriptional modi®cations.
For instance, the absence of methylation at position
N-1 of G9 and A57 allows Watson-Crick base-pairing and might favor extended intramolecular interactions such as those shown in Figure 5D (namely
UUGG10 and UCAA57). Note that 50 -terminal nt
appear to be free in utRNAPro, while they are
trapped by intramolecular base-pairing in the natural tRNA when hybridized to R18PBS (Figure 5B
and D). However, the central core of tRNA (nt 15
to 49) remains almost unchanged in both tRNAPro
and utRNAPro irrespective of the nature of the
RNA bound (viral RNA or R18PBS). Note that the
stem involving GGGGU19/ GUCCC49 is probably
strenghthened and extended, due to the absence of
the natural modi®cations (D and m7G), as
suggested by the occurence of new RNase V1 cuts
in the GGGU strand (Figure 5C).
Taking into account different possibilities of
intramolecular rearrangements of viral RNA and
utRNAPro, as well as potential intermolecular interactions, we propose a possible secondary structure
for the binary complex formed by RNA 1-725 and
utRNAPro, accounting for the probing data
(Figures 4C and 5C). In this model, nt 104 to 113
are single-stranded and the GGGUC103 sequence of
RNA 1-725 interacts with the 50 terminal end of
utRNAPro (GGCUC5). The increased reactivity of
upstream nt (C71, U75, U76, U78 and UU94;
Figure 4C) might re¯ect alternative conformations
of the region spanning nt 70 to 94 or/and a
reduced stability of the irregular stem resulting
from the particular structure caused by utRNAPro
annealing.
Three-dimensional modeling of the viral/
tRNAPro interaction
Since interactions between viral RNA and
tRNAPro are restricted to base-pairing between the
viral PBS (nt 146 to 163) and nt 58 to 75 of tRNA,
the complex obtained by hybridization of D18PBS0
can be considered a good model for describing the
interaction. The question we address now is how
helix 99 to 107/165 to 173 (HU5-1), helix 114 to
126/131 to 142 (HU5-2) and the hybrid helix
formed between the PBS and the 30 -end of tRNA
(HPBS) are spatially arranged. Only short singlestranded regions connect these three helices: 6 nt
between HU5-1 and HU5-2, 3 nt between HU5-2
740
Binary Primer/Template Complex of M-MuLV
Figure 6. Stereo view of the three-dimensional model of the D18PBS0 /RNA 1-725 binary complex. The drawings
were made using the software DRAWNA (Massire et al., 1994). The 18 nt of the D18PBS0 corresponding to the terminal sequence of tRNAPro are in green and M-MuLV RNA in orange (only residues 99 to 173 are shown). Some residues are represented: A75 (in green), A143 to U146 (in red), and C164 (in orange). One can expect mobility in the
position of hairpin HU5-2. The RNase V1 cleavages occurring in the viral RNA (shown in blue arrows in Figure 4A)
are represented by purple spheres, and the RNase V1 cleavages ocurring in tRNA (shown by blue arrows in
Figure 5A), by white spheres.
and HPBS and only one between HPBS and
HU5-1. The short length of these connecting
regions restricts the relative orientations of the
three helices in space. We began by constructing
the three helical elements and the possible stacking
possibilities were screened by interactive modeling
until the best agreement between the model and
probing data was reached. The selected orientation
leads to a topology in which HPBS is coaxialy
stacked upon HU5-1, with the connecting C164
stacked inside the helix, whereas HU5-2 is tilted
away (Figure 6). HU5-2 and the connecting singlestranded region span a length corresponding to the
distance of two deep grooves and one shallow
groove. An almost perfect coaxiality between
HPBS and HU5-1 probably occurs in the viral
RNA/D18PBS0 , as suggested by the non-reactivity
of C164 and the presence of a tract of RNase V1 cuts
overlapping both helices. However, the binding of
tRNAPro to the viral RNA probably induces some
bending at the junction of the two helices, as
suggested by the increased reactivity of C164. Moreover, the reactivity of A162 and U163 and the
absence of the two strong RNase V1 cuts at these
residues suggest a local melting of the extremity of
HPBS stem, probably resulting from steric constraints existing in the complex with tRNAPro and
absent with D18PBS0 . In the model, A75 from
tRNAPro is constructed in helical continuity, thus
allowing base-pairing with the viral U146, since this
pair has to be formed during initiation of reverse
transcription.
Discussion
Here, we investigated the conformation of the
viral M-MuLV primer/template binary complex
Binary Primer/Template Complex of M-MuLV
formed in vitro by heat-hybridization. Although
NC is believed to be involved in tRNA annealing
(Prats et al., 1988; Barat et al., 1989), the heatannealed complex is most likely functionally relevant, since thermal renaturation as well as NC
both help nucleic acids to adopt their thermodynamically most stable conformation. Furthermore,
in vitro formed binary complexes are fully competent for reverse transcription (Isel et al., 1996, 1997;
Lanchy et al., 1996; Oude Essink et al., 1996; data
not shown). Probing experiments of the viral MMuLV RNA/tRNAPro binary complex, in comparison with simpli®ed binary complexes in which
viral RNA or tRNA were reduced to the 18 nt of
the PBS or to the complementary tRNA sequence,
led to the conclusion that natural tRNAPro only
interacts with the viral PBS. In particular, the interaction between the 50 part of the stem-loop of
tRNAPro (GGGC) and nt GGGUCU104 of MMuLV RNA, proposed on the basis of the analogous interaction demonstrated in avian retroviruses (Aiyar et al., 1992) does not take place in
the primer/template complex formed in vitro. This
result contrasts with other retroviral systems, in
which virus-speci®c primer/template interactions
have been shown between the primer tRNA and
sequences of the RNA template, outside the standard PBS interaction. Strikingly, these interactions
do not involve the same RNA regions. In ASLV,
additional base-pairing involves the arm of the
tRNATrp primer and a sequence in U5 (see above),
while in HIV-1 the anticodon loop and the 30 strand of the anticodon arm of tRNALys
are paired
3
with a conserved A-rich loop and two upstream
sequences in U5, respectively (Isel et al., 1993,
1995). However, it should be mentioned that the 50
part of the acceptor and the stems of tRNALys
or
3
tRNAPro form intramolecular interactions in both
HIV-1 and M-MuLV binary complexes. In Ty1 retrotransposon, the and D stem-loop of the
initiator tRNAMet
interact with three regions of the
i
Ty1 RNA (Friant et al., 1996). This absence of a
common structural organization, beside the PBS
interaction, indicates that each retroviral system
developed speci®c features for optimizing reverse
transcription.
This view is in agreement with recent studies
which indicate that initiation of reverse transcription is a highly speci®c process, whose ef®ciency
requires homologous RNA template, tRNA primer and RT. Indeed, it was shown that tRNALys
3
bound to its cognate HIV-1 RNA template was
less ef®ciently extended by AMV and M-MuLV
RTs (M-MuLV RT being the least ef®cient) than
with the homologous one in in vitro assays (Isel
et al., 1996; Oude Essink et al., 1996). Moreover,
RTs from lentiviruses that utilize tRNALys
as pri3
mer are not able to synthesize (ÿ) strong-stop
complex (Arts
DNA from the HIV-1/ tRNALys
3
et al., 1996), and substituting tRNAPro for tRNALys
3
does not improve initiation on a HIV-1 template
with a PBS matching tRNAPro (Oude Essink et al.,
1996). Thus, a speci®c conformation of the viral
741
RNA template is required for an ef®cient
initiation, consistent with the idea that a proper
orientation of the RNA template, resulting from
its association with the primer tRNA, facilitates
initiation of reverse transcription (Aiyar et al.,
1994). Accordingly, sequences GGGUCUCC106
and GGAGACCC173 of M-MuLV, proposed to be
involved in an intramolecular base-pairing that
constrains the spatial arrangement of the binary
complex (Figure 4A), are conserved in other murine retroviruses (Mougel et al., 1993). Despite the
absence of additional interactions with tRNAPro,
the viral M-MuLV is compactly organized and
highly constrained (Figure 6). It should be
stressed that the 3 nt directly adjacent to the 50
end of the PBS (the ®rst to be used as template)
are single-stranded and highly exposed in the
proposed 3D model. It is interesting to note that
the corresponding nt of HIV-1 RNA, linking the
PBS helix to the next helical element, are also
single-stranded and reactive in the viral RNA/
tRNALys
complex (Isel et al., 1995). This structural
3
feature might be important for a correct and ef®cient initiation of polymerization. However, since
we have only detailed topographical information
on two systems (HIV-1 and M-MuLV), further
investigations will be necessary to establish
whether this might represent a general requirement.
The functional implications of speci®c interactions between the viral RNA template and its
cognate tRNA primer represent a fundamental
concern. Thus, an interesting question to address
is how the absence of additional interactions
between M-MuLV and tRNAPro might correlate
with aspects unique to the murine system. In the
and RT form
case of HIV-1, viral RNA, tRNALys
3
a speci®c initiation complex that is functionally
distinct from the elongation complex (Isel et al.,
1996; Lanchy et al., 1996). The transition from
initiation to elongation that occurs after extension
of tRNALys
by ®ve deoxyribonucleotides, was
3
shown to be facilitated by the additional interactions (Isel et al., 1996). The existence of a twostep mechanism for the synthesis of (ÿ) strongstop DNA synthesis was also shown in both
AMV and M-MuLV systems (Isel et al., 1997).
Thus, in all three cases, the initiation mode corresponds to the initial non-processive addition of nt
(‡1 to ‡8 in the case of M-MuLV, ‡1 to ‡6 in
the case of AMV), and the elongation mode to a
more processive and non-speci®c process. Strikingly, the short pausing products that transiently
accumulate during the initiation phase, even
though differing by their length depending of the
retroviral system, are more ef®ciently elongated
when natural tRNAs are used as primers than
when using oligoribonucleotides complementary
to the PBS (Isel et al., 1996, 1997). This indicates
that the three tested reverse transcriptases are
able to recognize speci®c features provided by
the annealed tRNAs. Therefore, in the case of MMuLV, speci®c interactions between the annealed
742
tRNAPro and M-MuLV reverse transcriptase,
rather than additional primer/template interactions (as in the case of HIV-1 and most likely
AMV) might facilitate the transition to
elongation.
However, one major difference between MMuLV and HIV-1 is the fact that murine reverse
transcription can be initiated from tRNAs different
from the cognate tRNAPro. Indeed, Colicelli & Goff
(1987) isolated a replication-competent recombinant of M-MuLV whose PBS sequence perfectly
matched for tRNAGln (differing by 5 nt from the
wild-type PBS). This recombinant virus was
reported to replicate in a manner indistinguishable
from that of the wild-type virus. It should be noted
that the GAGC sequence of tRNAGln displays a
base change compared to tRNAPro that is not compensated in the postulated complementary viral
GGGUCU sequence (Aiyar et al., 1992). Furthermore, Lund et al. (1993) altered the wild-type PBS
of an Akv M-MuLV-derived vector to sequences
matching either tRNAGln
or tRNALys
1
3 . The resulting
retroviral vectors were able to replicate as ef®ciently as with the wild-type primer tRNAPro, at
least for a single replication cycle. However, a preference for tRNAPro over other tRNAs cannot be
totally excluded, since growth over repeated cycles
of selection of these genomes with altered PBSs has
not yet been described. On the other hand, stable
utilization of tRNAs different from the wild-type
tRNALys
requires the mutation of the A-rich loop
3
sequence complementary to the anticodon loop of
the corresponding tRNA in HIV-1 (Wake®eld et al.,
1996; Zhang et al., 1996; Kang et al., 1996, 1997).
Moreover, a recent report indicates that the deletion of the A-rich loop results in decreased viral
infectivity, due to a defect in viral DNA synthesis,
and that partial restoration of the A-rich loop and
ef®cient DNA synthesis occurred after long-term
culture (Liang et al., 1997).
Otherwise, it appears that some tRNAs are
enriched relative to their concentration in the host
cell, while others are selectively excluded from
murine retroviral particles (Waters & Mullin, 1977).
Although tRNAPro is relatively abundant in MMuLV virions (13.3% of the total packaged tRNA),
it is surprisingly not the predominant tRNA
(Waters & Mullin, 1977). Indeed, tRNALys and
tRNAArg represent 22% and 24.3% of the total
packaged tRNA, respectively. Taken together, our
study and the various observations discussed
above suggest that the PBS/primer tRNA interaction is the primary determinant for the selective
use of tRNAPro in M-MuLV reverse transcription.
A further con®rmation was recently provided by
the fact that alteration of the PBS designed not to
match any naturally occurring tRNA molecule
could be complemented by an arti®cial tRNA-like
primer matching the mutated PBS in single cycle
replication experiments (Lund et al., 1997). In
addition, these experiments indicate that helical
structure rather than actual sequence of the PBS is
recognized by the replication machinery. Since the
Binary Primer/Template Complex of M-MuLV
utilization of tRNAPro is not an absolute requirement for the virion and since the PBS is regenerated from the tRNA itself during reverse
transcription (Gilboa et al., 1979), how is the
tRNAPro PBS maintained in murine retroviruses?
One possible explanation may be that sequences in
this PBS are important for functions other than primer binding. For example, nt of the PBS may interact with the U5 region which plays a role in
encapsidation of viral RNA (Murphy & Goff,
1989). The PBS sequence is also included within
the repressor-binding site (RBS) element which
regulates the M-MuLV expression in embryonal
carcinoma cells (Petersen et al., 1991).
A surprising observation is that the unmodi®ed
tRNAPro is able to form additional interactions
(involving the 50 -terminal nt of the utRNA and
the complementary sequence GGGUC103 of viral
RNA) which do not take place with the natural
tRNA. Again, this contrasts with HIV-1, in which
post-transcriptional modi®cations of tRNALys
3
were shown to stabilize extended interactions
with the viral RNA (Isel et al., 1993, 1995). On
the contrary, post-transcriptional modi®cations of
tRNAPro appear to favor intramolecular interactions within tRNAPro rather than intermolecular
interactions. Base modi®cations appear to play
versatile roles in modulating RNA structure and
function. Indeed, they are known to be involved
in stabilizing RNA structures (Perret et al., 1990b;
Houssier et al., 1988; this study). On the contrary,
some modi®ed nucleotides have been described
that prevent possible alternative conformations
(Steinberg & Cedergren, 1995; Florentz et al.,
RNA Workshop in Tokyo, 1997). They can also
act as anti-determinants by preventing mischarging of tRNAs by non-cognate aminoacyl-tRNA
synthetases (Perret et al., 1990a; PuÈtz et al., 1994).
Hence, the post-transcriptional modi®cations of
natural tRNAPro may play a role during the formation of the template/primer complex by preventing additional contacts outside the PBS, and
allowing the tRNAPro primer to adopt a stable
structure. At the present time, it is dif®cult to
indicate which particular modi®ed nucleotide(s)
may play a role in preventing (or favoring) a
particular conformation. One can also imagine an
indirect effect of modi®ed bases. Indeed, the
naturally modi®ed tRNA may adopt a de®ned
and rigid conformation incompatible with the formation of extended interactions, which are
allowed by the ¯exibility of the unmodi®ed
tRNA. Thus M-MuLV developed a unique strategy for the formation of a binary primer/template complex competent for initiation of reverse
transcription.
Materials and Methods
Chemicals and enzymes
Aniline, dimethylsulfate (DMS) and lead(II) acetate
trihydrate were from Merck (Germany). 1-Cyclohexyl-
743
Binary Primer/Template Complex of M-MuLV
N0 -[2-(N-methylmorpholino)]ethyl-carbodiimide-p-toluenesulfonate (CMCT) and diethylpyrocarbonate (DEPC)
were from ICN Biomedicals Inc. (USA). Hydrazine
anhydrous was from Sigma Chemical Co. (USA).
[a-32P]ATP(3000Ci/mmol) and [g-32P]ATP (3000Ci/
mmol) were from Amersham (France). Acrylamide
and N,N0 -methyl bisacrylamide were from Roth
(Germany). RNases Bacillus cereus, T1, U2 and V1
were from Pharmacia Biotech (Sweden), cobra venom
phosphodiesterase from Worthington (USA), T4 polynucleotide kinase from USB (USA), avian myeloblastosis virus (AMV) RT from Life Science (USA). T7
RNA polymerase was puri®ed from the overproducing strain BL21/pAR1219, kindly supplied by F. W.
Studier (Upton, NY, USA). tRNA nucleotidyltransferase was prepared from baker's yeast according to the
procedure of Rether et al. (1974).
Preparation of nucleic acids
RNA 1-725 corresponding to nt 1 to 725 of M-MuLV
was obtained by in vitro transcription and puri®ed as
previously described (Roy et al., 1990). Natural tRNAPro
was puri®ed from beef liver using published procedures
(Keith et al., 1983). ptRNABA-1, kindly supplied by J.-L.
Darlix (Lyon, France), was used to generate by in vitro
transcription a tRNAPro lacking post-transcriptional
modi®cations (utRNAPro; Darlix et al., 1992). In vitro transcription reaction contained 16 mM GMP in order to
obtain 50 -monophosphorylated utRNAPro. Labeling of
tRNAPro and utRNAPro at their 30 -end was achieved by
reconstitution of the 30 -terminal CCA with [a-32P]ATP
and unlabeled CTP using tRNA nucleotidyltransferase,
after limited hydrolysis with cobra venom phosphodiesterase, as described by Vlassov et al. (1981). R18PBS
(an oligoribonucleotide complementary to the 30 -terminal
18 nt of tRNAPro) and D18PBS0 (an oligodeoxyribonucleotides complementary to the viral PBS) were synthesized using the phosphoramidate method on an
Applied Biosystems synthesizer. DNA oligonucleotides
were labeled at their 50 -end using T4 polynucleotide
kinase and [g-32P]ATP. Unlabeled and labeled nucleic
acids were puri®ed by electrophoresis on 12% to 15%
denaturing polyacrylamide gels in 89 mM Tris-borate,
2 mM EDTA. Nucleic acids were checked for purity and
integrity on denaturing polyacrylamide gels.
Chemical and enzymatic probing of the PBS region
of M-MuLV RNA
Viral RNA (3 pmol) and 9 pmol of either tRNAPro,
utRNAPro or D18PBS0 were incubated in 13.5 ml of
double-distilled water at 90 C for two minutes, and
chilled on ice for two minutes. Four ml of standard buffer
(375 mM KCl, 250 mM sodium cacodylate (pH 7.5))
were added and the sample was incubated for 20 minutes at 70 C. The sample was then incubated for ®ve
minutes at 20 C before the addition of 2 ml of 50 mM
MgCl2 and was further incubated for ten minutes at 20 C
before the addition of 2 mg of total carrier tRNA. This
procedure allowed us to obtain more than 90% of the
template bound to the primer. The primer/template
binary complex (20 ml) was treated with 2ml of DMS
diluted 20-fold in ethanol for two, four and seven minutes at 20 C or with 5 ml of CMCT (42 mg/ml) for ®ve,
ten and 20 minutes at 20 C. In the case of probing with
CMCT, standard buffer was substituted by 375 mM KCl
and 250 mM sodium borate (pH 8.0). Reactions were
stopped by addition of 70 ml ethanol and 2.5 ml 3 M
sodium acetate. Cleavage with RNase V1 was at 20 C for
ten minutes with 0.025 and 0.050 unit of enzyme. Reactions were stopped by a double phenol-chloroform
extraction followed by ethanol precipitation. The precipitated RNA was dissolved in double-distilled water.
Modi®ed bases and enzymatic cleavages were analyzed
by primer extension with AMV RT as previously
described (Baudin et al., 1993). An oligodeoxyribonucleotide complementary to nt 186 to 200 of RNA 1-725 was
used as primer. The samples were analyzed on 8% (w/
v) denaturing polyacrylamide gels.
Chemical and enzymatic probing of tRNA
Labeled tRNAPro or utRNAPro (100,000 cts/min, corresponding to 0.4 pmol) was hybridized to 3 pmol of
viral RNA 1-725 or R18PBS as described above. More
than 90% of the tRNA was hybridized under the conditions used. Modi®cation of C(N-3) was with 2 ml of
DMS diluted 20 times in ethanol, for two, four, or
eight minutes at 37 C. Modi®cation of A(N-7) was
with 10 ml of DEPC for 20, 40, or 60 minutes at 37 C.
Reactions were stopped as above. Aniline treatment
subsequent to DEPC modi®cation was performed as
previously described (Peattie & Gilbert, 1980). Strand
cleavage at modi®ed C residues was achieved by incubation with 10 ml of 50% hydrazine for ®ve minutes at
0 C, followed by aniline treatment (Peattie & Gilbert,
1980). Lead(II) induced-cleavage was adapted from
Gornicki et al. (1989). In this case, KCl and MgCl2 in
the standard buffer were replaced by potassium acetate
and magnesium acetate, respectively. Two ml of lead(II)
acetate trihydrate (3.3, 11, 33, 110 and 220 mM) were
added to the sample (20 ml) and incubation was for
®ve minutes at 20 C. Reactions were stopped by adding 10 ml of 0.16 M EDTA, 250 ml ethanol and 100 ml
0.3 M sodium acetate. Cleavage with RNase T1 was
with 0.05 and 0.1 unit of enzyme, for ten minutes at
37 C. Cleavage with RNase V1 was at 20 C for ten
minutes with 0.012 and 0.025 unit of enzyme. Cleavage
reactions were stopped by phenol-chloroform extraction
followed by ethanol precipitation. Samples were analyzed by electrophoresis on 10% to 15% denaturing
polyacrylamide gels. Cleavage positions were identi®ed
by running in parallel RNase Bacillus cereus, T1 and U2
digests plus ladder obtained by limited alkaline
hydrolysis of tRNA in 50 mM sodium bicarbonate buffer (pH 8.9) at 90 C for 15 minutes.
Computer modeling
The 3D model integrating stereochemical constraints
and experimental data was constructed with the help of
several computer programs (Westhof, 1993) and tested
by comparing the theoretical accessibility of atoms with
the observed experimental reactivity, as described earlier
(Westhof et al., 1989).
Acknowledgements
We greatly acknowledge Drs C. Isel and P. Romby for
useful technical advice and stimulating discussions. S.
Lodmell and R. Marquet are thanked for critical reading
of the manuscript. We are endebded to N. Motte for skil-
744
ful technical assistance and C. Allmang for the synthesis
of R18PBS.
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Edited by J. Karn
(Received 25 July 1997; received in revised form 16 October 1997; accepted 24 October 1997)