Viroids and the RNA World

Viroids
&
the RNA World
from genomic scale (RNA)
to
atomic scale (ribozyme)
Viroid (ASBVd)
G
G
A A
G
A
G
A
U
U
G
A
A
G
A
C
G
A
G
U
G
A
A
C
UAA
U
U
U
U
U
U
U A A
U
A
A
A
A
G
U
U
C
A
C C
A
C
G
A
C
U
C
C
U
C
C
U
U
C
U
C
U
C A C
A A
G U C
G
A
A
A
C
U
C
A
G
A
G
U
C
G G A A A G
U C
G G A A
C A
G A C C U G G U
U U C
G U C
A A A
C A A
A
G U U U A
A
U
C A
U
A
U C C U C
A
C U U C U U G U U
C
U
A A U
A
A
A C A A G
A
U
UUUGU
A
AAA
A
AACAAUGAAG
AUA
GAGGA
A
UAAAC
C
UUG
CGA
GAC
UC
AUCAGUGUU
C
UUCC
CAU
CUUUCC
C
U
GAA
G
A
GAC
GAA
GUG
A
UC
1
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210220230
240
249
79-nt
249-nt
HHRz
1

Introduction
• evolution (RNA world)& genomics
• RNA biology & RNomics
(molecular and cellular functions)
• structural biology & structural
bioinformatics (structural basis for
functions)
• enzymology & computational enzymology
(catalysis) 2

Prebiotic & RNA Worlds
selﬁsh elements,
viroids,
etc
self-replication replication,
ampliﬁcation
Horning & Joyce, PNAS, 2016.Attwater et al., Nat. Chem., 2013.
ribozymes
Martin et al., Life, 2015.
3

Virus World
&
RNA World
« The ancient Virus World and
evolution of cells »
Pre-archaeal
compartment
Pre-bacterial
compartment
ocean
selfish
ribozymes
(group I
Introns)
positive-
strand,
ds
RNA
viruses
retrons,
group II
introns
crust
dsDNA
and RCR
viruses,
plasmids
Bacteria with
plasmids, retrons,
group I & II introns
Archaea with
plasmids, group
I introns
Escape of cells with
their viruses and other
parasitic elements
RNA World
RNA-DNA Retro
World
inorganic compartments
RNA-Protein World
DNA World
Koonin et al., Biol. Direct, 2006
viroids
4

Viroids: « survivors from
the RNA World »
• small size
• high GC
• circRNA
• periodicity
• no protein-
coding
• ribozyme
• error-prone
replication
• replication
ﬁdelity
• replication
• genome
assembly
• ribosome-
free
• replicationfeatures
functions
Holmes, J. Virol., 2011
5

Viroids: Families
remain to be determined.
A critical issue for all viroids is that bona fide promoter se-
quences have not been characterized fully. This obviously is one
of the most pressing issues that need to be addressed in order
to fully understand how viroid RNA templates are recognized
and transcribed by the cellular machinery.
RNA motifs and protein factors for cleavage and ligation.
The sequence and structural conservation of the CCR of several
members of Pospiviroidae suggests its potential importance in
Weak se
some resea
(Tabler and
thought tha
catalyzes t
1987a,b). R
ing self-cle
to the inter
strates use
Fig. 3. Asymmetric rolling circle replication of Potato spindle tuber viroid (PSTVd) and sym
(ASBVd). The secondary structures of the genomic or circular RNAs are sketched to facilitate i
an in vitro transcription system. With a combination of proto-
cols to remove cellular RNAs and, thereby, enrich the de novo
synthesized (–)-strand PSTVd RNAs from the circular (+)-
RNA templates in potato nuclear extracts and primer extension,
Kolonko and associates (2006) mapped the transcription initiate
site on the circular (+)-RNA to U359/C1 of the left terminal loop
(Fig. 1). Because of the low resolution of sequencing gels, it is
not possible to determine precisely whether U359 or C1 is the
exact initiation site. Site-directed mutagenesis in combination
with infection studies in tomato revealed that the C1G mutation
was maintained stably, whereas U359G reverted to wild type,
suggesting that perhaps U359 is the bona fide initiation site. It
is notable that these data are consistent with previous in vitro
studies showing that Pol II binds to the terminal loop or loops
of PSTVd (Goodman et al. 1984). These observations establish
a basis for further investigations to determine whether the in
vitro transcription initiation site is the same as that used in vivo.
The transcription initiation sites on the (–)-strand template also
remain to be determined.
A critical issue for all viroids is that bona fide promoter se-
quences have not been characterized fully. This obviously is one
of the most pressing issues that need to be addressed in order
to fully understand how viroid RNA templates are recognized
and transcribed by the cellular machinery.
RNA motifs and protein factors for cleavage and ligation.
The sequence and structural conservation of the CCR of several
members of Pospiviroidae suggests its potential importance in
viroid processing during replication (Candresse et al. 1990;
Diener 1986; Hashimoto and Machida 1985; Meshi et al.
1985; Tabler and Sänger 1985; Visvader et al. 1985). Extensive
in vitro studies provided evidence to support this hypothesis
(Baumstark and Riesner 1995). Furthermore, in vitro studies
with longer than unit-length PSTVd transcripts mapped the
cleavage and ligation site to between G95 and G96 (Baumstark
et al. 1997). The first cleavage at the 5′ end of G96 occurs in a
metastable tetraloop motif, which results in a conformational
change to form a stable loop E that drives the second cleavage
at the 3′ end of G95 and subsequent ligation (Baumstark et al.
1997). Recent work with a minicircle RNA showed that the
CCR contains all the necessary elements for cleavage and liga-
tion (Schrader et al. 2003). It is important to note that process-
ing also can occur outside CCR, with the specific sites to be
elucidated (Hammond et al. 1989; Tabler et al. 1992). A key
question that remains to be answered is whether single or mul-
tiple sites are used for processing in vivo.
Weak self-cleavage of PSTVd RNAs has been reported by
some researchers (Robertson et al. 1985) but not by others
(Tabler and Sänger 1985; Tsagris et al. 1987a,b). It generally is
thought that a cellular RNase which remains to be identified
catalyzes the cleavage of concatemeric RNAs (Tsagris et al.
1987a,b). Reasoning that the general difficulty of demonstrat-
ing self-cleavage of RNAs in Pospiviroidae could be attributed
to the interference of nonribozyme RNA sequences in the sub-
strates used during in vitro assays, Liu and Symons (1998)
Flores et al.,
Arch. Virol.,
1998.
6

Viroids: Plant Parasites
Ding & Itaya, Mol Plant Microbe Interact, 2007.
10 / Molecular Plant-Microbe Interactions
unsuccessful attempts to establish a transcription system for
PLMVd in cell extracts of several plant species, Pelchat and
associates (2002) tested whether the Escherichia coli DNA-
dependent RNA polymerase would transcribe PLMVd in vitro.
The observed transcription led to the suggestion that, in infected
plant cells, the PEP catalyzes transcription of PLMVd. However,
recent work suggests that NEP more likely is involved in the
transcription of PLMVd in vivo (Delgado et al. 2005). Thus,
further biochemical and genetic studies will be necessary to
ments showed that this left loop is the binding site for the β
and β′ subunits of the E. coli enzyme (Pelchat and Perreault
2004). The in vivo significance of these sites remains to be
seen, in light of recent work that mapped the in vivo initiation
sites of PLMVd to C51 in the (+)-strand RNA and A286 in the
(–)-strand RNA, in similar 6- to 7-bp double-stranded motifs
(Fig. 1) (Delgado et al. 2005).
Mapping the transcription initiation sites for members of
Pospiviroidae has been achieved only recently for PSTVd using
Fig. 2. Distinct steps of systemic infection of Avocado sunblotch viroid (ASBVd) and Potato spindle tuber viroid (PSTVd), type members of the two viroid
families. The mechanisms of the different trafficking steps for the family Avsunviroidae remain to be investigated. (Modified from Ding et al. 2005, with
permission from Elsevier Ltd.)
7

Viroids: Replication
Rz: hammerhead ribozyme
Flores et al., Viruses, 20098

Viroids: 2D Structures
Vol. 20, No. 1, 2007
. 1. Secondary structures of representative viroids from the two viroid families, Avsunviroidae: Avocado sunblotch viroid (ASBVd) and Peach latent mo
id (PLMVd), and Pospiviroidae: Potato spindle tuber viroid (PSTVd). The transcription initiation sites on the viroid genomic RNAs are indicated. Note
ASBVd and PSTVd, these sites are mapped to terminal loops. The transcription initiation site for the (–)-PSTVd RNA template remains to be determined.
MVd, the dashed lines indicate kissing-loop interactions. For PSTVd, the five structural domains (Keese et al. 1985) are indicated. TL = left-terminal dom
central domain, and TR = right-terminal domain. HPII′ and HPII indicate nucleotide sequences that base pair to form the metastable hairpin II structure.
Pospiviroids
Avsunviroids
9

Viroids: Pospiviroids
Figure 1. Structure and replication of Pospiviroidae. (A) Schematic representation of the consensus secondary structure of the 359 nt circular (+) PSTVd
with the five functional domains. TL: Left terminal domain, P: pathogenicity-modulating domain, C: conserved central core, V: variable domain, TR:
right terminal domain.11
(B) Replication follows an asymmetric rolling-circle mechanism.12
For details, see text.
Hammann & Steger, RNA Biol., 2012.
10

Viroids: Avsunviroids
Pelamoviroids Avsunviroids
11

ASBVd: 2D Structures
esolving these issues is of great interest to broaden
wledge of the molecular processes in these organelles
rther our understanding of the molecular basis for the
of infectious RNAs.
zyme machinery for transcription. The DNA-depend-
polymerase II (Pol II) is generally accepted to be in-
the transcription of members of Pospiviroidae. Three
plate in vitro (Rackwitz et al. 1981). Second, α-amaniti
the replication of PSTVd (Mühlbach and Säng
Schindler and Mühlbach 1992), Cucumber pale fr
(Mühlbach and Sanger 1979), Hop stunt viroid
(Yoshikawa and Takahashi 1986), and CEVd (Flor
Flores and Semancik 1982; Rivera-Bustamante and S
1989; Semancik and Harper 1984). Low concentrati
13

ASBVd(-): 2D Structures
A
G
G
A A
G
A
G
A
U
U
G
A
A
G
A
C
G
A
G
U
G
A
A
C
UAA
U
U
U
U
U
U
U A A
U
A
A
A
A
G
U
U
C
A
C C
A
C
G
A
C
U
C
C
U
C
C
U
U
C
U
C
U
C A C
A A
G U C
G
A
A A
C
U
C A
G
A
G
U
C
G G A A A G
U C
G G A A
C A
G A C C U G G U
U U C
G U C
A A A
C A A
A
G U U U A
A
U
C A
U
A
U C C U C
A
C U U C U U G U U
C
U
A A U
A
A
A C A A G
A
U
UUUGU
A
AAA
A
AACAAUGAAG
AUA
GAGGA
A
UAAAC
C
UUG
CGA
GAC
UC
AUCAGUGUU
C
UUCC
CAU
CUUUCC
C
U
GAA
G
A
GAC
GAA
GUG
A
UC
1
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210220230240
249
G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A C G A A G
1a 10a 20a 30a 40a 50a 60a 70a 80a 90a 100a 110a 120a 130a 140a 150a 160a 170a 180a 190a 200a 210a 220a 230a 240a
monomer 1
Rz
gguu
c
uucc
cau
cuuucc
c
u
gaa
g
a
gac
ga
a
g
c
a a
g u c
g
a
a a
c
u
c a
g
a
g
u
c
g g a a a g
u c
g g a a
c a
g a c c
u
g g u
u
u
c
g
u
c
1
10
20
30
40
50
60
70
79
intra-molecular base-pairs
tertiary or inter-molecular contacts
nucleotide in tertiary contact
cleavage site
Rz
5'
3'
5' 3'
(-)
Leclerc et al., Sci. Rep., 2016.
(-)
3bps
(+)
2bps
Hammerhead (HHR)
14
Flores et al., Adv, Virus Res., 2000.

HHR motifs in life forms
Perreault et al.,
PLoS Comput. Biol., 2011
Gupta & Swati,
Interdiscip. Sci., 2016.
FIG. 1. Schematic representation of the ASBVd RNAs expressed from plasmids and produced by the replication process. Hammer
ribozymes are represented as black boxes within the pFL61-ASBVd(ϩ) and pFL61-ASBVd(Ϫ) plasmids expressing, respectively, the A
and ASBVd(Ϫ) DNA dimer (dASBVd), and in the different expected transcripts. Plasmid sequences are indicated by gray boxes. The A
and ASBVd(Ϫ) sequences are represented in blue and green, respectively. In the first step, self-cleavage of the ASBVd dimeric form
via the ribozymes lead to the linear monomeric form (lmASBVd) and to the circular monomeric form (cmASBVd). In the second
RNA-dependent replication occurs, linear oligomers (loASBVd), lmASBVd, and cmASBVd of the opposite polarity are produced. Blue
arrows encompassing the linker sequence (LK), indicated as a black line, represent primers used during strand-specific reverse transcrip
During PCR amplification, the LK sequence alone is used with the PCR primers (blue or green arrows), in order to avoid any amplificati
from the plasmid. It is important to note that the cDNA resulting from RT of dASBVd, cmASBVd, and loASBVd can be PCR amplifie
VOL. 85, 2011 A VIROID REPLICATES IN YEAS
Delan-Forino et al.,
J. Virol., 2011.
yeast
15

A prototype for RNA
Catalysis
Scott et al., Science, 1996Scott, Q. Rev. Biophys., 1999
substrate/enzyme cleavage site
Canonical 2D Structure folded 2D Structure
“Minimum” Hammerhead Ribozyme
41-nt
16

Asp424
Glu357
O
NiO
O O
P
OR
OS
O
H
5'
3'
2'
5'
4'
O
Ni+1
OO
H
3'2'
4' R
RO(-)H
Mg2+
(OH-)
Mg2+(H2O)
Mg2+
Self-Cleaving / Self-Splicing
O
NiO
O O
P
OR
OS
O
H
5'
3'
2'
5'
4'
O
Ni+1
OO
H
3'2'
4' R
(H2O)
Mg2+
(OH-)
Mg2+
O
NiO
O O
P
OR
OS
O
H
5'
3'
2'
5'
4'
O
Ni+1
OO
H
3'2'
4' R
RO(-)H
Mg2+
(OH-)
Mg2+(H2O)
Mg2+
O
NiO
O O
P
OR
OS
O
H
5'
3'
2'
5'
4'
O
Ni+1
OO
H
3'2'
4' R
AH(+)
B(-) NHN
His12
HN
N
His119
H
ribozyme ribozyme
RNase A 3’-5’-exonuclease
SN2(P) reaction
(“in-line”)
17

Catalytic Metal Ions: One/
Two
ound water in the fully hydrated La3ϩ
ion, the low kobs for
cleavage reaction involving the La3ϩ
ion in both positions
not compatible with the observed correlation between the
a of a water bound to a metal ion and the kobs produced by
ferent divalent metal ions. That correlation has been inter-
ted in the metal hydroxide model (Fig. 4) as an effect on
concentration of the aqueous metal hydroxide, which then
ves as a Brønsted base in the abstraction of the proton from
2Ј-oxygen. We have argued (12) that this logic is flawed,
ause the metal hydroxide complexes formed with metal
s with lower pKa values are weaker bases and, therefore,
uld be less able to abstract the 2Ј-OH proton, despite their
ater concentration. This conclusion is supported by the data
sented in Fig. 3 because the pKa of the 2Ј-OH is two or more
a units higher than those of any of the aqueous metal ions
died, making the metal hydroxide poorly suited to the task
deprotonating the 2Ј-OH. It has been convincingly shown
t proton transfer does not occur in the rate-determining
p of the ribozyme cleavage reaction (30). The observed pH
pendence and the correlation between the pKa values of the
ueous metal ions and kobs must, therefore, reflect the effects
Mg2+/Mg2+
Mg2+/La3+
La3+/La3+
experimental: Pontius et al., 1997; Lott et al., 1998
theoretical: Boero et al., JCTC, 2005
X-ray (active)
O
NiO
O O
P
OR
OS
O
H
5'
3'
5'
4'
O
Ni+1
OO
H
3'2'
4' RAH(+)
B(-)O
Mg2+
O
Mg2+
41-nt
18 theoretical: Leclerc & Karplus, JPC, 2006

Contribution of Metals to
Catalysis
-30
-25
-20
-15
-10
-5
0
5
10
15
20
25
30
I II III IV V VI VII VIII IX
no metal
1 metal
2 metals
RelativeFreeEnergy(kcal/mol)
Reaction Coordinate
B3LYP/6-31+G(d,p)
B3LYP/6-311+G(2d,2p)//B3LYP/6-31G(d,p)
B3LYP/6-31+G(d,p)//HF/3-21+G(d)
22.9 kcal/mol
20.8 kcal/mol
19.3 kcal/mol
Lopez et al., 2006
Torres et al., 2003
Leclerc & Karplus, 2006
ΔG
exp
= 20.1 kcal/mol
dianionic mechanism
19

Reaction Path Following: B3LYP/6-31+G(d,p)//HF/3-21+G(d)
HHR: reaction mechanism
20

longest time courses (48–96 h). Each phase of the time course was
10-fold faster at pH 7.5 than at pH 6.5, as expected if each process
were limited by the chemical step (15). Finally, purification of this
phosphorothioate-substituted HH16 by anion exchange HPLC (8) re-
sulted in partial separation of ribozyme forms such that the two phases
had identical rate constants to those observed in the racemic mixture
but different relative amplitudes (one fraction gave 0.8 of the fast
component and 0.2 of the slow, whereas a second fraction gave 0.2 of the
fast and 0.8 of the slow).
Rates and relative amplitudes of the two phases for reactions in 10
mM Mg2⌅
did not change upon addition of 0.2 mM EDTA or 2 mM
dithiothreitol to the reaction mixture, suggesting that neither kinetic
process depended on the presence of contaminating metal ions. In
reactions with added Cd2⌅
, the concentration of EDTA carried over
from the ribozyme and substrate stocks was ⌃15 nM.
RESULTS
We have used two different hammerhead ribozyme con-
structs, HH⇥1 and HH16 (Scheme 1), in testing the role and
A Specific Metal Ion in the Hammerhead Ribozyme 26823
Folding Metal Ions Models
experimental:
Peracchi et al., 1997
X-ray (active)
20Å
41-nt
21

Metal Binding Sites in the
Hammerhead Ribozyme ?
unable to rescue activity for the A13 or A14 phosphoro-
thioate substitutions (Ruffner & Uhlenbeck, 1990; Knoll
et al+, 1997; Peracchi et al+, 1997; Scott, 1997)+ The A9
phosphate is part of a metal-binding site observed in
the original X-ray structure of the hammerhead (Pley
et al+, 1994), where a Mn2ϩ
ion is ligated by the pro-RP
high negative potentia
also modeled metal b
stead of the metal inte
posed here (Fig+ 4), the
with the N1 of G8+ We
Brownian-dynamics sim
FIG
dem
the
high
ing s
ture
The
met
pha
to m
pha
gan
resid
colo
illus
and
Hansen et al., RNA, 2008Chartrand et al., RNA, 1997
22

Minimum/Full-Length
HHR
Khvorova et al., Nat. Struct. Biol., 2003
de la Peña et al., EMBO J., 2003 Canny et al., JACS, 2004
Wang et al., Biochem., 1999
41-nt 50-nt 56-nt 50-nt
23

Variations in HHR motifs
Perreault et al., PLoS Comput. Biol., 2011
24

p
F
l
r
e
s
o
t
d
d
p
e
t
l
h
t
FIGURE 7. A folding scheme for the hammerhead ribozyme. Schematic to show the two-stage
folding scheme previously proposed for the hammerhead ribozyme. State U exists in the
absence of added metal ions, in which the three helical arms extend from an open central core.
Penedo et al.
Cold Son November 10, 2016 - Published byrnajournal.cshlp.orgDownloaded from
Penedo et al., RNA, 2004.
25
Loop-Loop interactions in
HHR folding

Loop-Loop interactions in
HHR catalysis
26

Minimum HHR, Tertiary
Contact and Catalysis
45-nt
O’Rourke et al., JMB, 2015 27

HHR morphing
O’Rourke et al., JMB, 2015
28

O
C17O
O O
P
OR
OS
OR
H
5'
3'
2'
5'
4'
N1
N
N
N7
O6
H2N
H
G12
O
G8
O
O
O
H
5'
O-
H
2' Mg2+
A Nucleobase
Catalyst
experimental: Chi et al., PLoS Biol., 2008
theoretical: Lee et al., JACS, 2008
X-ray (active)
O
C17O
O O
P
OR
OS
OR
H
5'
3'
2'
5'
4'
N1
N
N
N7
O6
H2N
H
G12
O
G8
O
O
O
H
5'
OH
H
O-
H
2'
29

Metal Catalysts in the
2’OH activation ?
Chval et al., JPC, 2011
<
O
O O
P
OR
OS
OH3C
H3'
2'
5'
4'
N
N
N
NO
H2N
H
H
O
H
O
O O
P
OR
OS
OH3C
H3'
2'
5'
4'
N
N
N
N
O
H2N
H
H
O
H
H
O
O O
P
OR
OS
OH3C
H3'
2'
5'
4'
N
N
N
N
O
H2N
H
H
OHMg2+(VI)
<
<
30

Metal Catalysts in the
Hammerhead Ribozymes ?
O
NiO
O O
P
OR
OS
O
H
5'
3'
2'
5'
4'
O
Ni+1
OO
H
3'2'
4' RAH(+)
B(-)
Mg2+
N1
N
N
N7
O6
H2N
H
G12
O-
H
Osborne et al., Biochem., 2009
Osborne et al.
e
n
e
r
n
d
+
U
al
z
2
Scheme 1
31

Cooperative Models in
Self-Cleaving ?
O
Ni
O
O
O
P
O
HRNA5'
3'
2'
4'
O
Ni+1O
O OH
RNA3'
O
B(-)
AH(+)
M/H-R
R-H/M 5'
32

Cooperative Models in
Self-Cleaving ?
Leclerc, Molecules, 2010
O
G
O
O
O H
2'
G-8
O
C17
O
O
O
P
O
HRNA5'
3'
2'
4'
O
N1.1
O
O OH
RNA3'
O
5'
N N
N
N
-O
NH2
R
G-12
Mg2+
RNA3'
RNA5'
Mg2+
O
G
O
O
O H
2'
G-8
O
C17
O
O
O
P
O
HRNA5'
3'
2'
4'
O
N1.1
O
O OH
RNA3'
O
5'
N
N
N
NO-
H2N
R
G-12
Mg2+
Mg2+
RNA3'
RNA5' 33

Metal Ions back in the
Hammerhead Catalysis
Ward & DeRose, RNA, 2011
Cold Spring Harbor Laboratory Press11 - Published by
and DeRose 2000; Boots et al. 2008). Moderate rates of
catalysis can also be achieved in molar concentrations of
monovalent cations, an important property that helped to
uncover the critical roles of nucleobases in the HHRz re-
action mechanism (Murray et al. 1998; O’Rear et al. 2001;
Bevilacqua et al. 2004). At physiological ionic strengths, the
HHRz requires divalent ions for appreciable rates of catal-
ysis; therefore, it is reasonable to assume that the divalent
metal-dependent channel is the primary mode of catalysis in
nature (Khvorova et al. 2003).
The HHRz was studied for years in its simplest active
form, as three short helices meeting at a junction of con-
served nucleotides that form the active site of the ribozyme
(for review, see Blount and Uhlenbeck 2005). Studies using
this ‘‘truncated’’ form of the HHRz (trHHRz) led to a
model of catalysis in which a catalytic metal in the P9/
G10.1 site coordinates the pro-R oxygen of the scissile
phosphate, presumably to stabilize the negative charge of
the phosphorane transition state (Peracchi et al. 1997;
Wang et al. 1999). Based on detailed metal-rescue exper-
iments, Wang et al. (1999) predicted that the metal ion
coordinates to the P9/G10.1 site in the ground state and
bridges to the scissile phosphate in the transition state of
the trHHRz reaction. A ground state that is very different
from the transition state is consistent with structural
studies of the truncated HHRz, which in general did not
show catalytically relevant atoms within appropriate dis-
tances of the active site (Blount and Uhlenbeck 2005). In
these structures, the P9/G10.1 metal ion site is z20 A˚ away
from its predicted ligand during catalysis, the pro-R oxygen
of the scissile phosphate (Pley et al. 1994; Scott et al. 1995).
FIGURE 1. (A) Secondary structure of the modified Schistosoma
mansoni HHRz (MSL1L2) (Osborne et al. 2005) used in these studies.
(B) Crystallographic active site of the S. mansoni HHRz (2OEU)
34

HHR: Active Conformation
and Metal Ions
66-nt Insert Table of Contents artwork here
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17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
Mir et al., Biochem., 201535

HHR: Metal Ions and
Catalysis
Mir & Golden, Biochem., 201536Leclerc, Molecules, 2010

ASBVd(-)/HHR(-): SANS
StudyASBVd(-) HHR(-)
Leclerc et al., Sci. Rep., 2016.37

HHR(-) dimerization/
catalysis
38 Leclerc et al., Sci. Rep., 2016.

HHR(-): 2D structures
monomer 1 monomeHI HII HIII
A
E
E
Egguu
c
uucc
cau
cuuucc
c
u
gaa
g
a
gac
ga
a
g
c
a a
g u c
g
a
a a
c
u
c a
g
a
g
u
c
g g a a a g
u c
g g a a
c a
g a c c
u
g g u
u
u
c
g
u
c
1
10
20
30
40
50
60
70
79
g g u u c u u c c c a u c u u u c c c u g a a g a g a c g a a g c a a g u c g a a a c u c a g a g u c g g a a a g u c g g a a c a g a c c u g g u u u c g u c g g u u c u u c
1a 10a 20a 30a 40a 50a 60a 70a 79a 1b
monomer 1 monomer 2HI HII HIII
A
B
Emonomer
(10ºC) = -26.9 kcal/mol
Emonomer
(25ºC) = -19.4 kcal/mol
Emonomer
(45ºC) = -9.6 kcal/molgguu
c
uucc
cau
cuuucc
c
u
gaa
g
a
gac
ga
a
g
c
a a
g u c
g
a
a a
c
u
c a
g
a
g
u
c
g g a a a g
u c
g g a a
c a
g a c c
u
g g u
u
u
c
g
u
c
1
10
20
30
40
50
60
70
79
g g u u c u u c c c a u c u u u c c c u g a a g a g a c g a a g c a a g u c g a a a c u c a g a g u c g g a a a g u c g g a a c a g a c c u g g u u u c g u c g g u u c u u c c c a u c u u u c c c u g a a g a g a c g a a g c a
1a 10a 20a 30a 40a 50a 60a 70a 79a 10b 20b 30b1b
A
Emonomer
(10ºC) = -26.9 kcal/mol
Emonomer
(25ºC) = -19.4 kcal/mol
Emonomer
c
uucc
cau
cuuucc
c
u
gaa
g
a
gac
ga
a
g
c
a a
g u c
g
a
a a
c
u
c a
g
a
g
u
c
g g a a a g
u c
g g a a
c a
g a c c
u
g g u
u
u
c
g
u
c
1
10
20
30
40
50
60
70
79
cleavage site
g g u u c u u c c c a u c u u u c c c u g a a g a g a c g a a g c a a g u c g a a a c u c a g a g u c g g a a a g u c g g a a c a g a c c u g g u u u c g u c g g u u c u u c c c a u c u u u c c c u g a a g a g a c g a a g c a a g u c g a a a c u c a g a g u c g g a a a g u c g g a a c a g a c c u g g u u u c g u c
1a 10a 20a 30a 40a 50a 60a 70a 79a 10b 20b 30b 40b 50b 60b 70b 79b1b
39

HHR folding
p
F
l
r
e
s
o
t
d
d
p
e
t
l
h
t
s
FIGURE 7. A folding scheme for the hammerhead ribozyme. Schematic to show the two-stage
folding scheme previously proposed for the hammerhead ribozyme. State U exists in the
absence of added metal ions, in which the three helical arms extend from an open central core.
On addition of metal ions, the minimal ribozyme undergoes folding in two steps, correspond-
Penedo et al.
Rg(I)/(F) ~ 1.1
ASBVd: Rg(I)/(F) ~ 1.241
Penedo et al., RNA, 2004.

HHR morphing
Rg(I)/(F) ~ 1.1 ASBVd: Rg(I)/(F) ~ 1.2
O’Rourke et al., JMB, 2015
42

Modeling/SANS
dexp = 96.0Å
dcalc = 96.7Å
Rg
exp = 31Å
Rg
*calc = 31 (26)Å
43

HHR (-): dimerization
A
B
Eint
(10ºC) = -9.3 kcal/mol
Eint
Eint
Edimer
(10ºC) = -47.1 kcal/mol
Edimer
(25ºC) = -33.6 kcal/mol
Edimer
(45ºC) = -15.7 kcal/mol
Emonomer
(10ºC) = -26.9 kcal/mol
Emonomer
(25ºC) = -19.4 kcal/mol
Emonomer
c
uucc
cau
cuuucc
c
u
gaa
g
a
gac
ga
a
g
c
a a
g u c
g
a
a a
c
u
c a
g
a
g
u
c
g g a a a g
u c
g g a a
c a
g a c c
u
g g u
u
u
c
g
u
c
1
10
20
30
40
50
60
70
79
gguu
c
uucc
cau
cuuucc
c
u
gaa
g
a
gac
ga
a
g
c
a a
g u c
g
a
a a
c
u
c a
g
a
g
u
c
g g a a a g
u c
g g a a
c a
g a c c
u
g
g
u
u u c g
u
c
g g u u
c u u c c
c
a u
c u u u c c
c
u
g
a
a
g a g a
c
g
a
a
g
c
a
a
g
u
c
gaaa
c
u
ca
g
a
g
u
c
ggaaag
uc
ggaa
ca
gacc
u
g
g
u
u
ucgu
c
1a
10a
20a
30a
40a
50a
60a 70a
79a
10b
20b
30b
40b
50b
60b
70b
79b
1b
cleavage site
44

HHR (-): dimerizationmonomer 1 monomer 2HI HII HIII
B
Eint
Eint
Eint
Edimer
(10ºC) = -47.1 kcal/mol
Edimer
(25ºC) = -33.6 kcal/mol
Edimer
(45ºC) = -15.7 kcal/mol
Edimer
(10ºC) = -53.7 kcal/mol
Edimer
(25ºC) = -38.9 kcal/mol
Edimer
(45ºC) = -19.2 kcal/mol
gguu
c
uucc
cau
cuuucc
c
u
gaa
g
a
gac
ga
a
g
c
a a
g u c
g
a
a a
c
u
c a
g
a
g
u
c
g g a a a g
u c
g g a a
c a
g a c c
u
g
g
u
u u c g
u
c
g g u u
c u u c c
c
a u
c u u u c c
c
u
g
a
a
g a g a
c
g
a
a
g
c
a
a
g
u
c
gaaa
c
u
ca
g
a
g
u
c
ggaaag
uc
ggaa
ca
gacc
u
g
g
u
u
ucgu
c
1a
10a
20a
30a
40a
50a
60a 70a
79a
10b
20b
30b
40b
50b
60b
70b
79b
1b
gguu
c
uucc
cau
cuuucc
c
u
gaa
g
a
gac
ga
a
g
c
a a
g u c
g
a
a a
c
u
c a
g
a
g
u
c
g g a a a g
u c
g g a a
c a
g a c c
u
g
g
u
u u c g
u
c
g g u u
c u u c c
c
a u
c u u u c c
c
u
g
a a
g
a
g a c
g a
a
g
c
aa
guc
g
a
a
a
c
u
c
a
g
a
g
u
c
ggaaag
uc
ggaa
ca
gacc
u
g
g
u
u
ucgu
c
1a
10a
20a
30a
40a
50a
60a 70a
79a
10b
20b
30b
40b50b
60b
70b
79b
1b
C
45

Mir et al., Biochem., 2015
HHR: Monomer & Dimer
Figure S2. Crystal contacts involve intermolecular base pairs. A. In crystals of the RzB
PDB ID:
51

Rg & self-association
HIII
HII
HI/HII
core
R(exp)g ~ 50Å
Rg*=50.4Å
Rg*=44.6Å
Rg*=46.1Å52
HI
HI/HI

MD setup for HHR dimer
53
•~ 300 000 atoms
• K+ & Cl- (0.15M)
• T = (283K, 313K)
• NAMD

Dynamics of HHR(-) dimer
RMSD vs time (ns)
Rg vs time (ns)
Histogram of Rg
Histogram of RMSD
Rg (monomer 1) vs time (ns)
Rg (monomer 2) vs time (ns)
Histogram of Rg (monomer 1)
Histogram of Rg (monomer 2)
RMSD(Å)
0
5
10
15
Rg(Å)
25
30
35
40
45
time (ns)
0 10 20 30 40 50 0 2500 5000 7500 10000125001500017500
54

G
G
A A
G
A
G
A
U
U
G
A
A
G
A
C
G
A
G
U
G
A
A
C
UAA
U
U
U
U
U
U
U A A
U
A
A
A
A
G
U
U
C
A
C C
A
C
G
A
C
U
C
C
U
C
C
U
U
C
U
C
U
C A C
A A
G U C
G
A
A A
C
U
C A
G
A
G
U
C
G G A A A G
U C
G G A A
C A
G A C C U G G U
U U C
G U C
A A A
C A A
A
G U U U A
A
U
C A
U
A
U C C U C
A
C U U C U U G U U
C
U
A A U
A
A
A C A A G
A
U
UUUGU
A
AAA
A
AACAAUGAAG
AUA
GAGGA
A
UAAAC
C
UUG
CGA
GAC
UC
AUCAGUGUU
C
UUCC
CAU
CUUUCC
C
U
GAA
G
A
GAC
GAA
GUG
A
UC
1
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210220230240
249
G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A
1a 10a 20a 30a 40a 50a 60a 70a 80a 90a 100a 110a 120a 130a 140a 150a 160a 170a 180a 190a 200a 210a 220a 230a
monomer 1
A
Emon
Emon
Emon
G
G
A A
G
A
G
A
U
U
G
A
A
G
A
C
G
A
G
U
G
A
A
C
UAA
U
U
U
U
U
U
U A A
U
A
A
A
A
G
U
U
C
A
C C
A
C
G
A
C
U
C
C
U
C
C
U
U
C
U
C
U
C A C
A A
G U C
G
A
A A
C
U
C A
G
A
G
U
C
G G A A A G
U C
G G A A
C A
G A C C U G G U
U U C
G U C
A A A
C A A
A
G U U U A
A
U
C A
U
A
U C C U C
A
C U U C U U G U U
C
U
A A U
A
A
A C A A G
A
U
UUUGU
A
AAA
A
AACAAUGAAG
AUA
GAGGA
A
UAAAC
C
UUG
CGA
GAC
UC
AUCAGUGUU
C
UUCC
CAU
CUUUCC
C
U
GAA
G
A
GAC
GAA
GUG
A
UC
1
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210220230240
249
G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A C G A A G U G A U C
1a 10a 20a 30a 40a 50a 60a 70a 80a 90a 100a 110a 120a 130a 140a 150a 160a 170a 180a 190a 200a 210a 220a 230a 240a 249a
monomer 1
A
B
Eint
Eint
Eint
Edimer
(10ºC)
Edimer
(25ºC)
Edimer
(45ºC)
Emonomer
(10ºC) = -26.9 kcal/mol
Emonomer
(25ºC) = -19.4 kcal/mol
Emonomer
c
uucc
cau
cuuucc
c
u
gaa
g
a
gac
ga
a
g
c
a a
g u c
g
a
a a
c
u
c a
g
a
g
u
c
g g a a a g
u c
g g a a
c a
g a c c
u
g g u
u
u
c
g
u
c
1
10
20
30
40
50
60
70
79
gguu
c
uucc
cau
cuuucc
c
u
gaa
g
a
gac
ga
a
g
c
a a
g u c
g
a
a a
c
u
c a
g
a
g
u
c
g g a a a g
u c
g g a a
c a
g a c c
u
g
g
u
u u c g
u
c
g g u u
c u u c c
c
a u
c u u u c c
c
u
g
a
a
g a
aa
c
u
ca
g
a
g
u
c
ggaaag
uc
ggaa
ca
gacc
u
g
g
u
u
ucgu
c
1a
10a
20a
30a
40a
50a
60a 70a
79a
10b
20b
50b
60b
70b
79b
1b
g g u u c u u c c c a u c u u u c c c u g a a g a g a c g a a g c a a g u c g a a a c u c a g a g u c g g a a a g u c g g a a c a g a c c u g g u u u c g u c g g u u c u u c c c a u c u u u c c c u g a a g a g a c g a a g c a a g u c g a a a c u c a g a g u c g g a a a g u c g g a a c a g a
1a 10a 20a 30a 40a 50a 60a 70a 79a 10b 20b 30b 40b 50b 60b1b
intra-molec
tertiary or i
nucleotide
cleavage si
g g u u c u u c c c a u c u u u c c c u g a a g a g a c g a a g c a a g u c g a a a c u c a g a g u c g g a a a g u c g g a a c a g a c c u g g u u u c g u c g g u u c u u c c c a u c u u u c c c u g a a g a g a c g a a g c a a g u c g a a a c u c a g a g u c g g a a a g u c g g a a c a g a
1a 10a 20a 30a 40a 50a 60a 70a 79a 10b 20b 30b 40b 50b 60b1b
HHR (-)
ASBVd(-): self-association
56

Edimer
(10ºC) = -163 kcal/mol
Edimer
Edimer
(45ºC) = -63.6 kcal/mol
Eint
(10ºC) = -9.35 kcal/mol
Eint
(25ºC) = -7.27 kcal/mol
Eint
(45ºC) = -5.61 kcal/mol
G
G
A
A
G
A
G
A
U
UG
A
AG
A
C
G
A
G
U
G
A
A
C
U
A
A
U
U
U
U
U
UU
A
A
U
A
A
A
A
G
U
U
C
A
C
C
A
C
G
A
C U
C
C U
C
C
U
U
C
U
C
U
CAC
AA
GUC
G
A
AA
C
U
CA
G
A
G
U
C
GGAAAG
UC
GGAA
CA
GACCUGGU
UU
C
GUC
AA
A
CAAA
GUUUA
A
U
CA
U
A
UCC
U
C
AC
U
U
C
UUGUU
C
UAAU
A
A
ACAAG
A
U
U
U U G U
A
A A
A
A
A A C A A
U
G
A A G A
U
A
G
A
G G A
A
U A A A C C
U U G
C G
A
G A C
U C
A U C A G U
G U U
C
U U C C
C A
U
C U U U C C
C
U
G A
A
G
A
G A C
G A
A
G U G
A
U
C
G
G A A
G
A
G
A
U
U
G
A
A
G
A
C
G
A
G
U
G
A
A
C
U
AAU
U
U
U
U
U
U
A A U
A
A
A
A
G
U
U
C
A
C C
A
C
G
A
C
U
C
C
U
C
C
U
U
C
U
C
U
C
A
C
A
A
G U C
G
A
A A
C
U
C A
G
A
G
U
C
G G A A A G
U C
G G A A
C A
G A C C U G G U
U
U C
G U C
A
A A
C A A
A G U U U A
A
U
C A
U
A
U C C U C
A
C U U C U U G U U
C
U
A A U
A
A
A C A A
G
A
U
UUUGU
A
A
AA
A
AACAA
UGAAG
AUA
GAGGA
A
UAAAC
CUUG
C
GA
GAC
UC
AUCAG
UGUU
C
UUCC
C
AU
CUUUCC
C
U
GAA
G
A
GAC
G
A
A
G
U
G
AUC1a
10a
20a
30a
40a
50a
60a
70a
80a
90a
100a
110a
120a
130a
140a
150a
60a
170a 180a
190a
200a
210a
220a
230
240
249a
10b
20b
30b
40b
50b
60b
70b
80b
90b
100b
110b
120b
130b
140b
150b
160b
170b
180b
190b
200b
210b
220b
230b
240b
249b
1b
C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A C G A A G U G A U C G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C
20a 30a 40a 50a 60a 70a 80a 90a 100a 110a 120a 130a 140a 150a 160a 170a 180a 190a 200a 210a 220a 230a 240a 249a 10b 20b 30b 40b 50b 60b 70b 80b 90b 100b 110b 120b 130b 140b 150b 160b 170b 180b 190b1b
C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A C G A A G U G A U C G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C
20a 30a 40a 50a 60a 70a 80a 90a 100a 110a 120a 130a 140a 150a 160a 170a 180a 190a 200a 210a 220a 230a 240a 249a 10b 20b 30b 40b 50b 60b 70b 80b 90b 100b 110b 120b 130b 140b 150b 160b 170b 180b 190b1b
omer 1 monomer 2
B
monomer
Emonomer
(25ºC) = -68.7 kcal/mol
Emonomer
(45ºC) = -37.9 kcal/mol
G
G
C A C
A A
G U C G G A A A G G G A A G A C C U G G U G U C C A A G U U U A U C C U C C U U C U U G U U A C A A
A
U
UUUGU
A
AAA
A
AACAAUGAAG
AUA
GAGGA
A
UAAAC
C
UUG
CGA
GAC
UC
AUCAGUGUU
C
UUCC
CAU
CUUUCC
C
U
GAA
G
A
GAC
GAA
GUG
A
UC
1
170
180
190
200
210220230240
249
Edimer
Edimer
Edimer
(45ºC) = -63.6 kcal/mol
Eint
(10ºC) = -9.35 kcal/mol
Eint
(25ºC) = -7.27 kcal/mol
Eint
(45ºC) = -5.61 kcal/mol
monomer 1 monomer 2
G
G
A
A
G
A
G
A
U
UG
A
AG
A
C
G
A
G
U
G
A
A
C
U
A
A
U
U
U
U
U
UU
A
A
U
A
A
A
A
G
U
U
C
A
C
C
A
C
G
A
C U
C
C U
C
C
U
U
C
U
C
U
CAC
AA
GUC
G
A
AA
C
U
CA
G
A
G
U
C
GGAAAG
UC
GGAA
CA
GACCUGGU
UU
C
GUC
AA
A
CAAA
GUUUA
A
U
CA
U
A
UCC
U
C
AC
U
U
C
UUGUU
C
UAAU
A
A
ACAAG
A
U
U
U U G U
A
A A
A
A
A A C A A
U
G
A A G A
U
A
G
A
G G A
A
U A A A C C
U U G
C G
A
G A C
U C
A U C A G U
G U U
C
U U C C
C A
U
C U U U C C
C
U
G A
A
G
A
G A C
G A
A
G U G
A
U
C
G
G A A
G
A
G
A
U
U
G
A
A
G
A
C
G
A
G
U
G
A
A
C
U
AAU
U
U
U
U
U
U
A A U
A
A
A
A
G
U
U
C
A
C C
A
C
G
A
C
U
C
C
U
C
C
U
U
C
U
C
U
C
A
C
A
A
G U C
G
A
A A
C
U
C A
G
A
G
U
C
G G A A A G
U C
G G A A
C A
G A C C U G G U
U
U C
G U C
A
A A
C A A
A G U U U A
A
U
C A
U
A
U C C U C
A
C U U C U U G U U
C
U
A A U
A
A
A C A A
G
A
U
UUUGU
A
A
AA
A
AACAA
UGAAG
AUA
GAGGA
A
UAAAC
CUUG
C
GA
GAC
UC
AUCAG
UGUU
C
UUCC
C
AU
CUUUCC
C
U
GAA
G
A
GAC
G
A
A
G
U
G
AUC1a
10a
20a
30a
40a
50a
60a
70a
80a
90a
100a
110a
120a
130a
140a
150a
160a
170a 180a
190a
200a
210a
220a
230
240
249a
10b
20b
30b
40b
50b
60b
70b
80b
90b
100b
110b
120b
130b
140b
150b
160b
170b
180b
190b
200b
210b
220b
230b
240b
249b
1b
G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A C G A A G U G A U C G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A C G A A G U G A U C
1a 10a 20a 30a 40a 50a 60a 70a 80a 90a 100a 110a 120a 130a 140a 150a 160a 170a 180a 190a 200a 210a 220a 230a 240a 249a 10b 20b 30b 40b 50b 60b 70b 80b 90b 100b 110b 120b 130b 140b 150b 160b 170b 180b 190b 200b 210b 220b 230b 240b 249b1b
G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A C G A A G U G A U C G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A C G A A G U G A U C
1a 10a 20a 30a 40a 50a 60a 70a 80a 90a 100a 110a 120a 130a 140a 150a 160a 170a 180a 190a 200a 210a 220a 230a 240a 249a 10b 20b 30b 40b 50b 60b 70b 80b 90b 100b 110b 120b 130b 140b 150b 160b 170b 180b 190b 200b 210b 220b 230b 240b 249b1b
monomer 1 monomer 2
57

G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A C G A A G U G A U C G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A C G A A G U G A U C G G A A G A G A U U G A A G A C G A G U G A A C U A A U U U U U U U A A U A A A A G U U C A C C A C G A C U C C U C C U U C U C U C A C A A G U C G A A A C U C A G A G U C G G A A A G U C G G A A C A G A C C U G G U U U C G U C A A A C A A A G U U U A A U C A U A U C C U C A C U U C U U G U U C U A A U A A A C A A G A U U U U G U A A A A A A A C A A U G A A G A U A G A G G A A U A A A C C U U G C G A G A C U C A U C A G U G U U C U U C C C A U C U U U C C C U G A A G A G A C G A A G U G A U C
1a 10a 20a 30a 40a 50a 60a 70a 80a 90a 100a 110a 120a 130a 140a 150a 160a 170a 180a 190a 200a 210a 220a 230a 240a 249a 10b 20b 30b 40b 50b 60b 70b 80b 90b 100b 110b 120b 130b 140b 150b 160b 170b 180b 190b 200b 210b 220b 230b 240b 249b 10c 20c 30c 40c 50c 60c 70c 80c 90c 100c 110c 120c 130c 140c 150c 160c 170c 180c 190c 200c 210c 220c 230c 240c 249c1b 1c
GG
A
A
G
A
G
A
U
U
G
A
A
G
A
C
G
A
G
U
G
A
A
C
U A A
U
U
U
U
U
U
UA
A
U
A
A
A
A
G
U
U
C
A
C
C
A
C
G
A
C
U
C
C
U
C
C
U
U
C
U
C
U
C
A
C
A
A
G
U
C
G
A
A
A
C
U
C
A
G
A
G
U
C
G
G
A
A
A
G
U
CG
G
A
A
C
AG
A
C
C
U
G
G
U
U
U
CG
U
C
A
AA
C
A
A
AG
U
U
U
A
A
U
CA
U
A
U
C
C
UCA
C
U
U
C
U
U
G
U
U
C
UA
A
U
A
A
A
C
A
A
G
A
U U
U
U
G
U
A A
A
A
A
A
A
C
A
A
U
G
A A G
A
U
A
G
A
G
G
A A
U
A
A
A
C
C U
U
G
C
G A
G
A
C
U
C A
U
C
A
G
U G
U
U
C U
U
C
C
C
A U
C
U
U
U
C
C
C
U
G
A A G
A
G
A
C
G
A A G
U
G
A
U
C
G
G
A
A
G
A
G
A
UU
G
AA
G
AC
G
A
G
U
G
A
A
C
U
A
A
U
U
UUU
U
U
A
A
U
A
A A A
G
U
U
C
A
C
C
A C
G
A C
U
C C
U
C
C U
U
C
U
C
U
C
A
C
A
A
G
U
CGA
A
ACUC
A
G A G U
C
G
G
A
A
A
G
U
C
G
G
A
AC
A
G
A
C
C
U
G
G
UU
U
C
G
U
CA
A
A
C
A
A
A
G
U
U
U
AA
U
C
A
U
A U
C
C
U
C
A
C
U
U
C U
U
G
U
U
C
U
A
A
U
A
A A
C
A
A
G
A
U
U
U
U
G
U
A
A
A
A
A
A
A
C
A
A
U
G
A
A
G
A
U
A
GAG
G
A
A
U
A
A
A
C
C
U
U
G C
G
A
G
A
C U
C
A
U
C
A
G
U
G
U
U
C
U
U
C
C C
A
U
C
U
U
U
C
C C U
G
A
A
G
A
G
A
C
G A A G U G A
U
C
G
G
A
A
G
A
G
A
U
U G
A
A G
A
C
G
A
G
U
G
A
A
C
U
A
A
U
U
U
U
U U U
A
A
U
A
A
A
A
G
U
U
C
A
C
C
A
C
G
A
CU
C
CU
C
C
UU
C
U
C
U
C
A
C
A
A
G
U
C
G
A A A
C
U
C A
G
A
G
U
C
G
G
A
A
A
G
U C
G G A A
C A
G
A
C C U G G U
U U
C
G U C
A A
A
C A A A
G U U U A
A U
C
A
U
A
U
C
C U
C
A
C
U
U
C
U
U
G
U
U C
U
A
A
U
A
A
A
C
A
A
G
AU
U
U
U
G
U
A
A
A
A
A
A
A
C
A
AUG
A
A
G
A
U
A
G
A
G
G
A
A
UAAACC
UUG
CG
A
GAC
UC
AUCAGU
G
U
UC
UUCC
CA
U
C
U
U
U
C
C
C
UGA
A
G
A
G
A
C
G
A
A
G
U
G
A
UC
1a
10a
20a
30a
40a
50a
60
70a
80a
90a
100a
110a
120a
130a
140a
150a
160a
170a
180a
190a
200a
210a
220a
230a
240a
249a
10b
20b
30b
40b
50b 60b
70b
80b
90b
100b
110b
120b
130b
140b
150b
160b
170b
180b
190b
200b
210b
220b
230b
240b
249b
10c
20c
30c
40c
50c
60c
70c
80c
90c
100c
110c
120c
130c
140c
150c
160c
170c
180c
190c
200c
210c
220c
230c
240c
249c
inter-molecular contacts
HHR motif
cleavage site
58

What did we learn ?
• for RNA and ribozymes: « too short » may be bad
• don’t forget about dynamics
• SANS & modeling approaches to infer self-
association modes
• theoretical approaches to explore reaction
mechanisms and pathways

Viroids: Plant Parasites
Genus Pospiviroids:
PSTVd (potato spindle tuber)
Genus Hostuviroids:
HSVd (hop stunt)
Genus Cocadviroids:
CCCVd (coconut cadang-cadang)
Genus Apscaviroids:
ASSVd (apple scar skin)
Genus Coleviroids:
CbVd 1 (coleus blumei 1)
Genus Avsunviroids:
ASBVd (avocado sunblotch)
Genus Pelamoviroids:
PLMVd (peach latent mosaic)
60

Acknowledgments
•Zdenek Chval (University of South Bohemia, CK)
•Daniela Chvalová (University of South Bohemia, CK)
•Xavier Lopez (Euskal Herriko Unibertsitatea, SP)
•Annick Dejaegere (ESBS Strasbourg)
•Darrin M. York (Rutgers University, USA)
•Martin Karplus (Harvard University, USA)
•Giuseppe Zaccai (IBS, Grenoble)
•Jacques Vergne (MNHN, Paris)
•Anne Martel (ILL, Grenoble)
•Martina Rihova (Institute of Physics, Prague, CK)
•Marie-Christine Maurel (MNHN, Paris)
•William G. Scott (UCSC, Santa-Cruz, USA)
62
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!
!

NITA SAHAI and HUSSEIN KADDOUR, Guest Editors
63
Fabrice Leclerc, Ph.D.
I2BC /Dept. de Biologie des Génomes,
« Séquence Structure Fonction des ARN »
SSFA (D. Gautheret)
fabrice.leclerc@u-psud.fr

Viroids and the RNA World

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