Selective isolation and detailed analysis of intra-RNA cross-links induced in the large ribosomal subunit of E. coli: A model for the tertiary structure of the tRNA binding domain in 23S RNA

Abstract

Intramolecular RNA cross-links were induced within the large ribosomal subunit of E. coli by mild ultraviolet irradiation. Regions of the 23S RNA previously implicated in interactions with ribosomal-bound tRNA were
then specifically excised by addressed cleavage using ribonuclease H, in conjunction with synthetic complementary decadeoxyribonucleotides.
Individual cross-linked fragments within these regions released by such ‘directed digests’ were isolated by twodimensional
gel electrophoresis and the sites involved in the cross-links determined using classical oligonucleotide analysis techniques.
Using this approach, seven ‘new’ cross-links could be precisely localised, between positions 1782 and 2608–2609, 1940 and
2554,1941–1942 and 1964–1965,1955 and 2552–2553, 2145–2146 and 2202, 2518–2519 and 2544–2545, and between positions 2790–2791
and 2892–2895 in the 23S RNA sequence. These data, in conjunction with data from RNA-protein cross-linking studies carried
out in our laboratory, were used to define a model for the tertiary organisation of the tRNA binding domain of 23S RNA ‘in situ’, in which the specific nucleotides associated with tRNA binding in the ‘A’ and ‘P’ sites are clustered at the base of the
‘central protuberance’ of the 50S subunit.

Full text

Nucleic
Acids
Research,
Vol.
18,
No.
15
4325
Selective
isolation
and
detailed
analysis
of
intra-RNA
cross-links
induced
in
the
large
ribosomal
subunit
of
E.
coli:
a
model
for
the
tertiary
structure
of
the
tRNA
binding
domain
in
23S
RNA
Philip
Mitchell,
Monika
Osswald,
Dierk
Schueler
and
Richard
Brimacombe*
Max-Planck
Institut
fuer
Molekulare
Genetik,
Abt.
Wittmann,
Berlin-Dahlem,
FRG
Received
June
15,
1990;
Accepted
July
10,
1990
ABSTRACT
Intramolecular
RNA
cross-links
were
induced
within
the
large
ribosomal
subunit
of
E.
coil
by
mild
ultraviolet
irradiation.
Regions
of
the
23S
RNA
previously
implicated
in
interactions
with
ribosomal-bound
tRNA
were
then
specifically
excised
by
addressed
cleavage
using
ribonuclease
H,
in
conjunction
with
synthetic
complementary
decadeoxyribonucleotides.
Individual
cross-linked
fragments
within
these
regions
released
by
such
'directed
digests'
were
isolated
by
two-
dimensional
gel
electrophoresis
and
the
sites
involved
in
the
cross-links
determined
using
classical
oligonucleotide
analysis
techniques.
Using
this
approach,
seven
'new'
cross-links
could
be
precisely
localised,
between
positions
1782
and
2608-2609,
1940
and
2554,
1941
-
1942
and
1964
-
1965,
1955
and
2552
-
2553,
2145
-
2146
and
2202,
2518
-
2519
and
2544
-
2545,
and
between
positions
2790
-
2791
and
2892
-
2895
in
the
23S
RNA
sequence.
These
data,
in
conjunction
with
data
from
RNA-protein
cross-linking
studies
carried
out
in
our
laboratory,
were
used
to
define
a
model
for
the
tertiary
organisation
of
the
tRNA
binding
domain
of
23S
RNA
'in
situ',
in
which
the
specific
nucleotides
associated
with
tRNA
binding
in
the
'A'
and
'P'
sites
are
clustered
at
the
base
of
the
'central
protuberance'
of
the
50S
subunit.
INTRODUCTION
A
number
of
specific
nucleotides
within
the
16S
and
23S
rRNAs
of
E.
coli
have
been
associated
with
the
binding
of
tRNA,
largely
through
photoaffinity
cross-linking
(1-4)
and
chemical
footprinting
studies
(5,6).
The
availability
of
models
for
the
three-
dimensional
organisation
of
16S
RNA
'in
situ'
in
the
30S
subunit
(7,8)
have
allowed
those
nucleotides
in
the
16S
RNA
to
be
mapped
within
the
architecture
of
the
small
subunit.
Although
well
separated
in
the
primary
sequence
and
secondary
structure
of
the
16S
RNA,
these
nucleotides
are
localised
in
two
clusters
in
the
three-dimensional
model,
one
on
each
side
of
the
'head'
of
the
30S
particle
(9).
On
the
other
hand,
the
lack
of
knowledge
concerning
the
detailed
three-dimensional
arrangement
of
the
23S
RNA
has
meant
that
nucleotides
within
the
RNA
of
the
large
subunit
identified
as
being
proximal
to
tRNA
sites
can
only
be
discussed
with
respect
to
the
phylogenetically
established
secondary
structure
models
(10,1
1).
The
majority
of
these
latter
sites
are
clustered
in
a
region
of
the
secondary
structure
associated
with
the
peptidyl
transferase
activity
through
biochemical
and
genetic
studies
(12),
although
a
number
of
other
nucleotides
that
have
been
implicated
in
tRNA
binding
(e.g.
4,6)
lie
in
distinct
regions
of
the
secondary
structure.
Clearly,
a
better
understanding
of
the
structural
organisation
of
the
23S
RNA
is
needed
to
gain
further
insight
into
the
interaction
of
tRNA
with
the
ribosome
and
to
provide
a
basis
for
more
detailed
mechanistic
studies.
The
precise
analysis
of
intra-RNA
cross-links
generated
'in
situ'
is
the
most
direct
method
for
obtaining
information
concerning
the
topographical
folding
of
rRNA
within
the
ribosome.
In
previous
studies,
we
have
reported
the
analyses
of
a
number
of
intra-RNA
cross-linked
complexes
within
23S
RNA
generated
through
partial
digestion
with
the
double
strand-specific
cobra
venom
nuclease
(CSV)
(13,14).
Recently,
we
have
developed
a
more
suitable
approach
for
the
isolation
of
individual
intra-RNA
cross-links
by
addressed
cleavage
using
ribonuclease
H.
This
enzyme
is
known
to
induce
specific
cuts
in
RNA
molecules
where
the
RNA
is
involved
in
a
hybrid
helix
with
DNA
(15).
Thus,
by
carefully
designing
deoxyribonucleotide
templates
and
hybridising
them
with
cross-linked
RNA
under
controlled
conditions,
ribonuclease
H
partial
digests
can
be
tailored
so
as
to
excise
individual
cross-links
in
regions
of
the
RNA
molecule
chosen
at
will.
Because
only
a
very
few
cuts
are
introduced
into
the
RNA,
the
cross-linked
complexes
can
be
isolated
with
high
purity
in
a
semi-quantitative
manner,
using
our
established
gel
separation
techniques.
Moreover,
this
method
can
be
efficiently
used
to
probe
selected
areas
of
the
molecule
for
additional
cross-
linking
data.
In
the
series
of
experiments
reported
here,
we
have
utilised
this
'directed
digest'
approach,
combined
with
additional
developments
in
our
methods
of
oligonucleotide
analysis,
to
investigate
the
tertiary
structure
of
23S
RNA
regions
implicated
in
the
binding
of
tRNA;
these
include
the
'peptidyl
transferase
*
To
whom
correspondence
should
be
addressed
k.)
1990
Oxford
University
Press

4326
Nucleic
Acids
Research,
Vol.
18,
No.
15
ring'
(12)
and
the
binding
domains
of
ribosomal
proteins
LI
(16)
and
L2
(17).
Using
this
approach,
seven
new
cross-links
could
be
precisely
localised,
including
three
long
range
'tertiary'
cross-
links
between
regions
widely
separated
in
the
secondary
structure.
These
new
data,
along
with
previously
established
intra-RNA
and
RNA-protein
cross-links
(reviewed
in
ref.
11),
have
been
used
to
derive
a
three-dimensional
model
for
the
organisation
of
E.
coli
23S
RNA
'in
situ'
in
the
50S
subunit
(see
ref.
18
for
a
preliminary
description).
Here
we
present
a
detailed
arrangement
for
helices
64-93
of
the
23S
RNA
secondary
structure
(1
1),
which
are
involved
in
tRNA
binding,
and
which
are
constrained
by
the
cross-linking
data
into
a
distinct
domain
in
the
tertiary
structure.
MATERIALS
AND
METHODS
The
preparation
of
ribonuclease
H
and
DNA
oligonucleotide
templates,
and
the
isolation
of
uniformally
32P-labelled
23S
RNA
from
'in
situ'
cross-linked
50S
subunits
were
carried
out
as
described
in
(19).
Ribonuclease
H
digests
of
the
cross-linked
RNA
were
performed
as
in
(19)
with
the
following
modifications:
the
RNA
was
preincubated
with
the
appropriate
decadeoxy-
ribonucleotide
templates
at
55°C
for
10
min.,
after
which
the
mixture
was
made
1
mM
in
MgOAc,
0.1
mM
in
DTT,
and
a
suitable
quantity
of
ribonuclease
H
was
added.
Incubation
was
continued
for
a
further
30
min.
at
55°C.
This
modification
has
the
dual
advantage
of
a
higher
specificity
of
the
digest
and
an
increased
accessibility
of
the
target
sites
in
the
RNA.
The
digestion
was
stopped
by
the
addition
of
1/10
vol.
of
1
%
SDS,
20
mM
EDTA-KOH,
pH
7.0.
The
digest
was
treated
with
1
mg/ml
proteinase
K
(55°C,
30
min.)
and
the
RNA
fragments
recovered
by
phenol
extraction
and
ethanol
precipitation
(19).
The
two
dimensional
gel
system
used
to
resolve
the
partial
digests
was
a
modification
of
the
one
given
in
(13):
a
5
%
gel
was
used
in
the
first
dimension,
from
which
a
single
gel
strip
was
excised
and
directly
polymerised
(without
prior
washing)
into
a
12%
second
dimension
gel.
Cross-linked
complexes
were
recovered
from
the
gels
and
digested
totally
with
ribonuclease
T1
or
A,
as
in
(13).
The
oligonucleotides
released
were
separated
by
two
dimensional
chromatography
on
polyethyleneimine (PEI)
cellulose
plates,
using
the
system
of
Volckaert
and
Fiers
(20)
or
the
'alternative'
chromatographic
procedure
(56%
formic
acid,
5
M
urea;
7.6%
formic
acid/pyridine
pH
4.3,
4
M
urea
(19)).
Secondary
digestions
and
analyses
of
oligonucleotides
were
performed
as
in
(19).
In
some
instances,
ribonuclease
T1
end-products
were
further
characterised
by
digestion
with
ribonuclease
U2.
These
digestions
were
performed
with
5
units
of
ribonuclease
U2
(Sankyo,
Tokyo)
in
IOtI
of
10
mM
NH4OAc,
pH
4.5,
1
mM
EDTA,
with
5
,tg
carrier
tRNA
for
30
min.
at
37°C,
followed
by
slow
warming
(15
min.)
to
60°C.
The
digests
were
lyophilised,
taken
up
in
5
itl
0.1
%
SDS
containing
a
little
xylene
cyanol,
and
resolved
using
the
standard
fingerprint
system
(20).
After
recovery
from
the
PEI
cellulose
plates,
oligonucleotides
were
digested
with
ribonuclease
T2
(Sankyo,
Tokyo)
as
in
(21)
and
the
resulting
3'-nucleotide
phosphates
resolved
on
PEI
cellulose
plates
using
the
system
of
Bernardi
(22).
In
all
cases,
the
oligonucleotide
data
were
fitted
to
the
23S
RNA
sequence
of
Brosius
et
al.
(23).
A
'wire
and
tube'
model
for
the
'in
situ'
structure
of
23S
RNA
was
constructed
as
described
in
(7).
Coordinates
of
RNA
helices
measured
from
the
model
were
fed
into
a
VAX
8000
computer
graphics
system,
again
as
described
in
(7).
RESULTS
AND
DISCUSSION
Directed
digests
of
cross-linked
rRNA
with
ribonuclease
H
Fig.
1
shows
a
two
dimensional
gel
separation
of
fragments
generated
by
addressed
cleavage
using
ribonuclease
H.
In
this
example,
the
digest
was
performed
using
4
decadeoxyr-
ibonucleotide
templates
complementary
to
positions
1808-1817,
1808-2084
2670-2904
....
I
I44
28
2084
-
2231
Figure
1.
Two
dimensional
gel
electrophoresis
of
cross-linked
23S
RNA,
following
addressed
cleavage
with
ribonuclease
H.
The
digest
was
performed
using
decadeoxyribonucleotides
complementary
to
positions
1808-1817,
1904-1913,
2084-2093
and
2231-2240
in
the
23S
RNA
sequence
(numbered
from
the
5'-end).
Separation
of
the
fragments
was
from
right
to
left
in
the
first
dimension
and
from
top
to
bottom
in
the
second
dimension.
The
specifically
excised
single
stranded
fragments,
lying
in
a
'diagonal'
on
the
gel,
are
labelled
according
to
their
positions
in
the
23S
RNA
sequence.
Cross-linked
complexes
(shown
with
arrows)
are
retarded
in
the
second
dimensional
separation,
appearing
as
spots
above
the
corresponding
fragments
in
the
diagonal
(connected
by
the
broken
lines).
1.
#:
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I
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I
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I
::....

Nucleic
Acids
Research,
Vol.
18,
No.
15
4327
001I
9
3
.._
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2
+
B
up
0
0
9x
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4,
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Figure
2.
Ribonuclease
T1
fingerprints
of
a
non-cross-linked
RNA
fragment
(panels
A
and
B)
and
the
corresponding
cross-linked
complex
(panels
C
and
D)
from
positions
ca.
1911-2054
in
the
23S
RNA
sequence.
Panels
A
and
C
show
the
'standard'
chromatographic
system,
panels
B
and
D
the
'alternative'
system
(see
Materials
and
Methods).
Direction
of
chromatography
is
from
right
to
left
in
the
first
dimension
and
from
bottom
to
top
in
the
second
dimension.
Sample
application
points
in
panels
B
and
D
are
denoted
by
'+'.
The
asterisks
at
the
sample
application
points
in
panels
A
and
C
indicate
oligonucleotides
which
resolve
into
pairs
of
spots
(also
asterisked)
in
panels
B
and
D,
respectively.
Oligonucleotides
1,
2
and
3
from
the
non-cross-linked
fragment
(indicated
by
arrows
in
panels
A
and
B)
are
'missing'
in
the
cross-linked
samples,
where
instead
the
cross-linked
oligonucleotide
'X'
can
be
seen.
(Oligonucleotide
1
co-elutes
with
an
isomeric
oligonucleotide,
also
present
in
the
fragment;
hence
the
spot
is
still
present,
albeit
weaker,
in
panel
C).
1904-1913,
2084-2093
and
2231-2240
in
the
23S
RNA
sequence
(cf.
Fig.
3
below)
The
gel
has
the
characteristic
features
(cf.
refs.
13,14)
of
a
'diagonal'
of
non-cross-linked
fragments,
above
which
are
spots
(indicated
with
arrows)
corresponding
to
the
cross-linked
complexes.
In
all
cases,
the
gel
patterns
for
a
particular
digest
were
highly
reproducible.
The
fragment
pattern
is
very
simple,
in
contrast
to
the
patterns
obtained
from
CSV
nuclease
digests
(Fig.
1,
ref.
13),
or
ribonuclease
H
partial
digests
using
sets
of
deoxyoligonucleotides
complementary
to
sequences
at
approximately
50
nucleotide
intervals
along
the
complete
length
of
the
23S
RNA
(Fig.
2,
ref.
19).
The
diagonal
is
dominated
by
fragments
generated
by
specific
cleavage
at
the
targeted
sites,
with
the
noteable
exception
in
this
case
of
a
single
strong
spot
corresponding
to
the
last
ca.
230
nucleotides
at
the
3'-end
of
the
molecule
(positions
2670-2904).
Moreover,
the
pattern
of
cross-
linked
complexes
is
equally
simplified.
The
cross-links
are
present
for
the
most
part
as
discrete
spots
running
directly
above
the
corresponding
single-stranded
fragment
in
the
diagonal,
and
are
free
of
contamination
from
complexes
within
other
regions
of
the
molecule.
In
this
particular
example,
we
investigated
the
two
distinct
regions
in
the
23S
RNA
secondary
structure
previously
associated
with
protein
LI
through
binding
studies
(16)
and
our
own
RNA-
protein
cross-linking
experiments
(reviewed
in
ref.
II
and
cf.
Fig.
3,
below).
Although
cross-links
were
found
within
each
of
the
excised
LI-regions
(and
in
the
resulting
fragment
between
the
two
regions),
no
intra-RNA
cross-links
connecting
these
two
distinct
portions
of
the
molecule
were
observed.
Such
cross-links
would
be
expected
to
run
at
positions
in
the
first
dimensional
gel
corresponding
to
the
combined
molecular
weight
of
a
pair
of
the
excised
fragments.
However,
using
other
similar
'directed
digests',
long
range
tertiary
cross-links
of
this
type
were
indeed
found
(cross-links
3,
4
and
6,
see
Table
1
below).
Most
of
the
individual
cross-links
identified
in
this
series
of
experiments
were
isolated
within
a
set
of
overlapping
fragments
by
using
varying
combinations
of
deoxyoligonucleotides.
This
flexibility
of
the
directed
digest
approach
permits
the
location
of
undefined
cross-link
sites
to
be
gradually
narrowed
down
until
they
can
be
unequivocally
assigned.
In
this
manner,
the
digests
can
be
tailored
to
avoid
complications
in
the
oligonucleotide
analyses,
by
avoiding
regions
containing
non-characteristic
oligonucleotides
or
isomeric
oligonucleotides
which
co-elute
A
..
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ai

4328
Nucleic
Acids
Research,
Vol.
18,
No.
15
under
the
standard
chromatographic
conditions.
However,
addressed
cleavage
of
the
RNA
was
not
observed
in
some
particular
cases,
presumably
due
to
competition
of
the
RNA
secondary
or
tertiary
structure
with
the
hybridisation
of
the
DNA
template,
or
inaccessibility
of
the
RNA-DNA
hybrid
for
the
enzyme.
A
useful
side
product
of
the
directed
digest
approach
is
the
cleavage
of
RNA
at
sites
of
partial
complementarity
with
the
DNA
templates,
such
as
the
release
of
the
3'-end
fragment
of
23S
RNA
in
the
example
given
in
Fig.
1.
This
'semi-specific'
hydrolysis
can
provide
hints
of
new
cross-links,
allowing
a
'stepping stone'
approach
to
the
accumulation
of
cross-link
data.
The
isolation
of
homogeneous
fragments
from
the
diagonal
of
the
two-dimensional
gel
(cf.
Fig.
1)
allows
the
direct
comparison
in
the
same
experiment
of
oligonucleotides
generated
from
the
cross-linked
complexes
with
the
equivalent
non-cross-linked
fragment(s).
This
provides
an
important
internal
control
that
can
avoid
misinterpretations
due
to
anomalies
in
the
sequence,
such
as
strain
or
cistronic
heterogeneities,
or
sites
of
nucleoside
or
sugar
methylations.
In
conjunction
with
the
use
of
the
'alternative'
chromatographic
elution
system
(see
Materials
and
Methods
and
ref.
(19)),
this
comparison
allows
a
direct
unequivocal
assignment
of
the
sites
involved
in
cross-links.
An
example
of
this
method
is
given
in
Fig.
2,
which
shows
'standard'
and
'alternative'
ribonuclease
T,
fingerprints
of
the
cross-linked
complex
5
(see
Table
1:
Locations
of
intra-RNA
cross-link
sites
in
the
23S
RNA.
The
characteristic
ribonuclease
T1
or
A
oligonucleotides
missing
in
each
cross-link
analysed
are
listed,
together
with
their
positions
in
the
23S
sequence
(numbered
from
the
5'-end)
and
the
helical
element
in
the
secondary
structure
(Fig.
3),
in
which,
or
adjacent
to
which,
they
are
located.
In
cases
where
the
cross-link
site
could
be
further
defined
within
the
given
oligonucleotide,
the
nucleotides
concerned
are
underlined.
Where
appropriate,
the
5'-nucleotides
of
the
23S
RNA
sequence
complementary
to
the
deoxydecanucleotide
templates
used
to
direct
exemplary
ribonuclease
H
digests
are
given.
The
class
denotes
wether
the
cross-link
analysed
represents
a
'secondary
structural'
(S)
or
'tertiary
structural'
(T)
contact.
Cross-links
indicated
with
an
asterisk
were
unobserved
in
previous
studies.
Y
denotes
psuedouridine.
The
salient
features
of
each
cross-link
site
analysis
(cf.
refs.
13,14)
are
given
as
footnotes.
Oligonucleotides
Position
Helices
Templates
Class
UAUAAUGp:
AmAGp
ACUAAUGmYTG§:
UCCCUAUCUGp
UUWAUUAAAAACACAGp:
G
AAATUCCUUGp
:
UMG'Ip
AAATUCCWUGp
ACCUG.CACGp
AAGip:
UMGUp
CCAGp
:
GGUp
UCUCCUCCUAAAGp:
CUCAAgp
CUCAUCACAUCCUGp
:
AAGGGUp
UUCUCCCGULGp
:
GAGGCD
571-577
:
2030-2032
739-748
:
2609-2618
1777-1792
2607-2609
1936-1945
:
2552-2554
1936-1945
:
1960-1968
1952-1955
2552-2554
2145-2148
:
2201-2203
2257-2269
:
2422-2428
2510-2523
:
2541-2546
2783-2791
:
2892-2896
25:
72
35:
73
65:
93
70:
92
70
:71
71:
92
78
:
79
80:
88
91:
91
98
:1
T
T
T*
T*
T*
1703,
1767,
2568,
2619
1904,
1957,
2506,
2568
1904,
2050
1904,
1957,
2506,
2568
2084,
2231
T
2438,
2568
1808,
1904,
2084,
2231
Cross-links
and
2
were
previously
reported
(14)
but
could
be
further
localised
by
isolation
of
the
cross-linked
residue
using
the
'alternative'
fingerprint
system.
1:
Secondary
analysis
with
ribonuclease
A
of
the
cross-linked
T,
oligonucleotide
gave
Gp,
AUp
and
AAUp
but
no
Up.
2:
Secondary
analysis
of
the
cross-linked
T1
oligonucleotide
gave
ACp,
Up,
AAUp,
and
GmYp
but
the
AUp
was
either
weak
or
absent.
Ribonuclease
U2
digestion
of
the
cross-linked
residue
gave
UCUGp.
The
5'-site
was
given
by
the
absence
of
the
overlapping
ribonuclease
A
oligonucleotide
GAAAAAUp
(positions
748-754).
3:
UUUAUUAAAAACACAGp
was
clearly
absent
from
the
'alternative'
ribonuclease
T,
fingerprint
(cf.
Fig.
2D).
Secondary
analysis
gave
Up,
AUp,
ACp,
AGp
with
a
weak
AAAAACp.
GGUp
was
missing
in
the
ribonuclease
A
fingerprint
analyses,
whereas
the
overlapping
T1
oligonucleotide
UUCGp
(2604-2607)
was
present.
The
T1
oligonucleotide
UCCCUAUCUGp
(2609
-2618)
was
sometimes
present
and
sometimes
absent,
indicating
a
microheterogeneity
of
the
3'-end
of
the
cross-link
site
between
positions
2608-2609.
4:
AAATUCCUUGp
was
clearly
absent
from
the
'alternative'
ribonuclease
T,
fingerprint.
CUmGp
was
present
in
the
ribonuclease
T,
fingerprint
but
UmGUp
was
absent
from
the
ribonuclease
A
fingerprint,
hence
the
3'-site
was
localised
to
U2554.
The
5'-site
is
tentatively
allocated
to
U1940
due
to
the
submolar
AAATp
spot
in
the
secondary
analysis.
5:
AAATUCCUUGp,
ACCUGp
and
CACGp
were
absent
from
the
fibonuclease
T,
fingerprint
analyses
(see
Fig.
2).
Secondary
analysis
with
ribonuclease
A
of
the
cross-linked
complex
gave
AAATp,
Up,
Gp,
ACp
and
a
weak
Cp
spot.
6:
AAGUp,
UmGUp,
and
the
overlapping
oligoiiucleotides
UUCCGp
and
CUmGp
(positions
1955-
1959
and
2551
-2553
respectively)
were
clearly
absent
from
the
ribonuclease
A
and
T1
fingerprints
respectively.
7:
CCAGp
and
GGUp
were
not
present
in
the
ribonuclease
T,
and
A
fingerprints
respectively.
Position
2202
is
heterogenous,
with
G
not
U
as
in
Fig.
3.
The
cross-linked
ribonuclease
T,
residue
gave
AGp
on
secondary
analysis.
8:
CUCAACGp
and
UCUCCUCCUAAAGp
were
clearly
absent
from
the
ribonuclease
T,
fingerprints.
The
cross-linked
ribonuclease
T,
residue
resolved
into
two
spots
by
'alternative'
fingerprint
system
which
gave
identical
secondary
analyses
with
strong
Cp
and
Up
spots
and
AAAGp
but
no
AACp
or
Gp.
9:
CUCAUCACAUCCGp
and
AAGGGUp
were
absent
from
the
ribonuclease
T1
and
A
fingerprints
respectively.
UAUGp
(positions
2546-2549)
was
present,
as
was
UCCCAAGp
(positions
2537-2543).
Secondary
analysis
of
the
cross-linked
ribonuclease
T,
residue
gave
submolar
AUp,
and
digestion
with
ribonuclease
U2
did
not
generate
UCCUGp.
10:
This
cross-link
was
isolated
fortuitously,
due
to
hydrolysis
of
the
RNA
by
ribonuclease
H
at
a
site
of
partial
complementarity
with
the
templates
indicated
(see
text
for
discussion).
UUCUCCCUGp
and
GAGGCp
were
clearly
absent
from
the
ribonuclease
T,
and
A
fingerprint
analyses
respectively.
Secondary
analysis
of
the
cross-linked
ribonuclease
T,
residue
yielded
strong
Up
and
Cp
spots
and
a
cross-linked
residue,
but
no
Gp.
No.
1
2
3
4
5
6
7
8
9
10

Nucleic
Acids
Research,
Vol.
18,
No.
15
4329
Table
1
below)
and
the
corresponding
non-cross-linked
RNA
fragment
between
positions
ca.
1911-2054
of
the
23S
RNA
sequence.
Three
oligonucleotides
which
are
present
in
the
control
fragment
(indicated
by
arrows)
but
which
are
absent
from
the
cross-linked
complex,
constitute
the
cross-linked
ribonuclease
T1
residue.
This
gives
an
additional
characteristic
spot
(X)
in
the
fingerprints
of
the
cross-linked
sample,
comprising
the
adjacent
oligonucleotides
ACCUGp
(spot
2)
and
CACGp
(spot
1)
covalently
linked
to
AAATUCCUUGp
(spot
3)
(cf.
Table
1).
We
have
previously
reported
the
use
of
immobilised
cDNA
clones
complementary
to
sequences
within
the
16S
RNA
to
hybridise
specific
populations
of
RNA
fragments
after
a
partial
digest
with
CSV
nuclease
(24).
In
this
manner,
the
complexity
of
the
gel
patterns
from
CSV
digests
could
be
markedly
reduced,
and
the
technique
proved
a
successful
method
to
isolate
cross-
linked
fragments
located
in
selected
regions
of
the
RNA.
However,
this
approach
cannot
overcome
the
inherently
heterogeneous
nature
of
the
partial
digestion
with
CSV
and,
moreover,
it
is
not
suitable
for
the analysis
of
low-yield
cross-
links,
due
to
the
losses
incurred
during
the
hybridisation
step.
In
contrast,
directed
digests
with
ribonuclease
H
are
semi-
quantitative
and
the
product
fragments
can
be
tailored
to
suit
the
particular
requirements
of
the
experiment.
Localisation
of
cross-links
within
the
23S
RNA
secondary
structure
The
sites
of
intramolecular
cross-links
within
the
23S
RNA
which
were
localised
by
a
large
number
of
directed
digests
in
these
studies
are
summarised
in
Table
1,
and
are
superimposed
on
the
updated
version
of
our
model
for
the
secondary
structure
of
23S
RNA
(11)
in
Fig.
3.
Also
included
are
three
tertiary
cross-links
(numbered
1,
2
and
8
in
Table
1)
which
were
isolated
by
ribonuclease
H
digests
using
sets
of
deoxyoligonucleotides
complementary
to
sites
along
the
whole
length
of
the
23S
RNA
sequence
(cf.
ref.
19);
these
were
reported
in
our
earlier
studies,
but
have
now
been
more
precisely
localised
by
oligonucleotide
fingerprinting
using
the
'alternative'
chromatographic
elution
system
(cf.
Fig.
2)
and,
in
the
case
of
cross-link
2,
ribonuclease
U2
digestion
of
the
cross-linked
ribonuclease
T,
residue.
Of
the
seven
new
cross-links,
six
represent
tertiary
contacts
in
the
folding
of
the
rRNA
and
thus
can
be
used
to
impose
spatial
constraints
on
the
phylogenetically
established
secondary
structure
of
23S
RNA
in
model
building
studies.
The
most
important
topographical
data
are
provided
by
cross-
links
3,
4
and
6,
which
interconnect
specific
sites
in
the
L2
binding
domain
(17)
with
sites
in
the
vicinity
of
the
'peptidyl
transferase
ring',
at
the
junction
of
helices
73,
74,
89,
90
and
93
(see
Fig.
3).
The
analysis
of
cross-link
3
(see
Table
1)
is
similar
to
that
of
a
tertiary
cross-link
tentatively
reported
earlier
(cross-link
H,
ref.
14),
but
in
the
latter
case
the
3'-site
of
the
cross-link
was
localised
to
positions
2584-2588.
These
nucleotides
lie
adjacent
to
positions
2607
-2609
in
the
secondary
structure
at
the
base
of
helix
93
(see
Fig.
3),
and
thus
cross-link
3
is
not
incompatible
with
this
earlier
observation.
However,
a
cross-link
involving
the
previously
reported
site
was
not
observed
in
these
experiments.
Cross-link
6
connects
helices
71
and
92
of
the
secondary
structure
and
was
preliminarily
localised
through
ribonuclease
H
digests
using
sets
of
DNA
templates
(19).
Directed
digests
to
excise
this
cross-link
revealed
two
contacts
between
the
same
fragments
(cross-links
4
and
6)
which,
taken
together
with
cross-link
5,
demonstrate
a
very
close
juxtaposition
of
the
loop
ends
of
helices
70,
71
and
92.
A
three-dimensional
model
of
the
tRNA
binding
domain
of
23S
RNA
In
addition
to
the
locations
of
intra-RNA
cross-links
analysed
in
this
study,
Fig.
3
also
shows
topographical
data
obtained
from
RNA-protein
cross-linking
studies
carried
out
in
our
laboratory
(reviewed
in
ref.
11,18).
These
two
independent
sets
of
cross-
linking
data
define
points
of
topographical
neighborhood
'in
situ'
and
were
used
to
impose
constraints
upon
the
folding
of
the
secondary
structure
of
23S
RNA
in
three-dimensional
model
building
studies.
RNA-protein
cross-linking
data
provide
the
link
between
the
tertiary
structure
of
the
rRNA
and
the
spatial
distribution
of
the
proteins
within
the
ribosomal
subunit.
Both
recent
models
for
the
tertiary
structure
of
16S
RNA
'in
situ'
in
the
30S
subunit
(7,8)
relied
upon
the
known
relative
positions
of
the
ribosomal
proteins
as
defined
by
low
angle
neutron
scattering
studies
(25)
to
correlate
the
tertiary
structure
of
the
RNA
with
the
spatial
arrangement
of
the
proteins.
At
the
moment,
a
corresponding
map
is
not
available
for
the
50S
subunit.
However,
a
model
has
been
proposed
for
the
arrangement
of
the
proteins
within
the
50S
subunit
(26),
based
on
immuno
electron
microscopy
(IEM)
data
and
the
immunological
analysis
of
a
large
number
of
inter-protein
cross-links.
The
high
degree
of
compatibility
of
the
IEM
model
with
the
neutron
scattering
map
in
the
case
of
the
30S
subunit
(see
e.g.
ref.
11)
suggests
that
the
IEM-derived
protein
arrangement
of
ref.
26
for
the
50S
subunit
is
likely
to
be
reliable.
Accordingly,
we
have
incorporated
our
complete
intra-RNA
and
RNA-protein
cross-link
data
into
a
model
for
the
three-
dimensional
structure
of
23S
RNA
in
the
large
ribosomal
subunit,
using
the
protein
map
(26)
to
align
the
arrangement
of
the
RNA
with
the
spatial
distribution
of
the
proteins.
The
tRNA
binding
domain
of
the
23S
RNA
involves
sites
in
the
region
comprising
helices
64
to
93
(Fig.
3),
and
the
RNA-protein
cross-linking
data
in
this
portion
of
the
molecule
define
sites
in
close
topographical
neighborhood
with
proteins
Li,
L2,
L5,
L6,
L14,
L18,
L27
and
L33
(see
Fig.
3).
With
the
exception
of
L33,
epitopes
to
all
of
these
proteins
have
been
located
on
the
ribosomal
subunit
by
IEM
studies.
However,
cross-link
data
pairing
L33
with
both
LI
and
L27
allows
it
to
be
placed
between
the
latter
two
proteins,
close
to the
base
of
the
'LI
protuberance'
(26).
The
computer
version
of
the
23S
RNA
model
is
presented
in
Fig.
4.
The
view
given
is
of
the
interface
side
of
the
50S
subunit.
The
overall
shape
of
the
large
subunit
observed
in
electron
micrographs
(including
the
'stalk',
'central
protuberance'
and
'LI
protuberance')
is
clearly
recognisable
in
the
model
(cf.
ref.
26).
The
tertiary
structure
of
the
23S
RNA
is
presented
as
series
of
cylindrical
elements
corresponding
to
the
individual
helices
of
the
secondary
structure
(cf.
ref.
7).
No
information
is
available
for
the
detailed
arrangement
of
single
stranded
regions
of
the
molecule,
or
for
the
effects
of
loops
or
bulges
upon
individual
helices,
and
therefore
they
are
not
indicated
in
the
computer
model,
although
present
in
the
'wire
and
tube'
model
(cf.
ref.
7).
This
preliminary
model
provides
a
framework
within
which
to
incorporate
steadily
incoming
new
cross-link
data.
Although
the
general
folding
pattern
of
the
RNA
in
the
large
subunit
has
become
clear,
sufficient
data
are
not
available
at
this
stage
to
present
a
detailed
model
of
the
complete
23S
RNA.
In
contrast,
the
high
degree
of
spatial
constraints
imposed
on
the
organisation
of
helices
64-93
of
the
secondary
structure
define
a
distinct
self-
contained
domain
in
the
three-dimensional
model
of
the
23S
RNA,
positioned
in
the
upper
portion
of
the
large
subunit
on
the
interface
side.
Helices
constituting
this
detailed
region
of
the

4330
Nucleic
Acids
Research,
Vol.
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No.
15
.!
I
~
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
I-
I
o
IIzD
9
V
O
/
Do
3
o
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Q\
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/
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Nucleic
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No.
15
4331
model
are
denoted
by
heavier
shading
in
Fig.
4
and
numbered
with
respect
to Fig.
3.
The
other
regions
of
the
structure
are
also
included
in
Fig.
4
to
illustrate
the
orientation
of
the
tRNA
binding
domain
of
23S
RNA
within
the
complete
molecule:
this
is
established
through
a
number
of
long
range
connections
(see
ref.
18
for
discussion),
e.g.
intra-RNA
cross-links
1
and
2
and
a
tertiary
interaction
between
positions
413
-416
and
2407-2410
in
the
23S
RNA
sequence
(27)
(see
Fig.
3).
These
remaining
helices
are
however
left
unnumbered
to
emphasize
the
preliminary
nature
of
these
regions
of
the
model.
Figure
4a
shows
the
locations
of
RNA-protein
cross-link
sites
within
the
tRNA
binding
domain
in
the
three-dimensional
model.
We
have
previously
reported
cross-links
to
LI
between
helices
77
and
78
(11)
within
the
known
binding
region
of
the
protein
(16),
and
have
since
been
able
to
precisely
assign
an
additional
LI
cross-link
site
in
helix
68
to
positions
1876-1878
(M.
Osswald
and
R.
Brimacombe,
unpublished
results),
providing
evidence
that
the
orientation
of
this
latter
helix
is
directed
towards
the
'LI
protuberance'
in
the
ribosomal
subunit.
As
noted
above,
the
most
important
intra-RNA
cross-link
data
for
defining
this
region
of
the
model
are
provided
by
the
three
long
range
intra-RNA
cross-links
3,
4
and
6.
Cross-link
3
connects
the
loop-end
of
helix
65
(extending
from
the
right
hand
side
of
helix
65
in
Fig.
4)
to
the
single
stranded
region
at
the
base
of
helix
93
(helix
93
is
barely
visible
in
Fig.4a,
being
concealed
by
helix
92).
Cross-links
4
and
6
connect
helices
70,
71
and
92,
and
are
located
at
the
front
of
the
model
(as
viewed
in
Fig.
4),
at
the
base
of
the
'central
protuberance'.
Additional
data
are
provided
for
the
arrangement
of
all
the
remaining
independent
helical
systems
within
this
region
of
the
23S
RNA.
Thus,
cross-link
8
folds
the
helix
system
76-79
tightly
back
upon
itself
to
form
a
compact
structure
which,
in
addition
to
helix
68
as
already
noted,
constitutes
the
RNA
moiety
of
the
'Li
protuberance'
(helix
79
is
hidden
from
view
in
Fig.
4,
lying
behind
helix
76).
The
sub-domain
consisting
of
helices
80-88
is
located
in
the
'central
protuberance'
of
the
50S
subunit.
This
region
is
constrained
by
the
three
cross-link
sites
to
L27,
the
cross-links
to
L5
and
L18,
the
phylogenetically
established
tertiary
interaction
(27)
between
positions
2328
-2330
and
2385
-2387
(labelled
T4
in
Figs.
3,4),
and
cross-link
8,
which
spans
the
'ring'
connecting
helices
74,
75,
80,
81,
82
and
88
(see
Fig.
3).
The
orientations
of
helices
74
and
89
are
governed
by
defined
RNA-
protein
cross-link
sites
to
L33
and
L6
respectively
(cf.
ref.
26).
Similarly,
helices
61
and
66
contain
cross-links
to
proteins
L14
and
L2
respectively.
Functional
significance
of
the
model
The
site
of
peptide
bond
formation
has
been
localised
to
the
highly
conserved
'peptidyl
transferase
ring'
connecting
helices
74,
75,
89,
90
and
93
in
the
secondary
structure
of
23S
RNA
(see
Fig.
3),
largely
on
the
basis
of
the
genetic
analysis
of
organisms
resistant
to
chloramphenicol
and
anisomycin
(both
of
which
are
antibiotic
inhibitors
of
peptidyl
transfer)
(see
ref.
12).
Furthermore,
a
photoreactive
analogue
of
puromycin
was
specifically
cross-linked
to
23S
RNA
at
positions
G2502
and
U2504
within
the
'peptidyl
transferase
ring'
(28).
Chemical
footprinting
studies
(29)
have
shown
that
chloramphenicol,
erythromycin,
carbomycin
and
vernamycin
B
(which
compete
for
the
same
binding
site
on
the
ribosome
(30)
although
they
inhibit
protein
synthesis
by
different
mechanisms)
have
overlapping
binding
sites
in
this
region
of
the
RNA
secondary
structure.
Vernamycin
B
also
gave
a
strong
footprint
at
position
A752,
thus
providing
functional
data
which
correlates
well
with
the
intra-RNA
cross-link
between
positions
748
and
2613
-2614
(number
2,
Table
1)
observed
by
us.
This
cross-link
serves
to
bring
the
loop
end
of
helix
35
(11)
into
the
vicinity
of
the
'peptidyl
transferase
ring'
in
our
model.
More
directly
pertaining
to
the
binding
site
of
tRNA,
nucleotides
2451/2452
and
2584/2585
within
the
'peptidyl
transferase
ring'
were
the
sites
of
cross-linking
to
a
photoreactive
aminoacyl
affinty
analogue
of
charged
tRNAPhe,
bound
in
the
'P'
and
'A'
sites
respectively
(3)
(see
Fig.
3).
In
other
studies
(4),
an
azidoadenosine
nucleotide
incorporated
at
position
A76
of
tRNAPhe
was
cross-linked
to
position
G1945
in
the
23S
RNA,
located
at
the
junction
between
helices
70
and
71
(Fig.
3).
In
the
same
experiment,
the
tRNA
was
also
cross-linked
to
L27,
one
of
the
most
frequently
labelled
proteins
in
studies
using
probes
directed
at
the
peptidyl
transferase
center.
Thus,
G1945,
the
sites
in
23S
RNA
cross-linked
to
L27
and
the
'peptidyl
transferase
ring'
would
be
expected
to
be
adjacent
in
the
three-dimensional
structure.
Indeed,
these
regions
of
the
23S
RNA
are
in
very
close
proximity
in
our
model,
lying
at
the
base
of
the
'central
protuberance'
of
the
50S
subunit
(see
Fig.
4).
In
quite
different
studies
from
the
photo-affinity
labelling
experiments
described
above,
proteins
L2,
L3,
LA
and
L16
(in
addition
to
the
23S
RNA)
have
been
shown
to
be
essential
for
peptidyl
transferase
activity
by
partial
reconstitution
experiments
'in
vitro'
(e.g.
31,32).
Together
with
L27,
these
proteins
are
clustered
in
the
IEM
protein
map
of
Walleczek
et
al.
(26)
towards
the
geometric
center
of
the
50S
subunit.
This
supports
the
location
of
the
peptidyl
transferase
region
directly
beneath
the
'central
protuberance'
of
the large
subunit,
in
accordance
with
our
model
(Fig.
4),
although
the
position
of
the
peptidyl
transferase
center
is
often
interpreted
as
being
more
towards
the
'LI
protuberance'
(e.g.
ref.
33).
The
peptidyl
transferase
center
defines
the
location
of
the
CCA
end
of
tRNA
in
the
'A'
or
'P'
sites
on
the
large
subunit.
Using
chemical
footprinting
techniques,
Moazed
and
Noller
(6)
have
recently
identified
a
comprehensive
catalogue
of
sites
in
the
23S
RNA
which
are
responsive
to
tRNA
binding
in
the
ribosomal
'A',
'P'
and
'E'
sites.
As
noted
above,
several
of
these
sites
implicate
additional
regions
of
the
23S
RNA
secondary
structure
in
the
binding
of
ribosomal
bound
tRNA.
The
locations
of
these
tRNA
footprinting
data
within
the
secondary
structure
of
23S
RNA
are
given
in
Fig.
3,
and
their
distribution
in
the
three-
dimensional
model
is
shown
in
Fig.
4b.
The
'P'
site
footprints
are
clearly
brought
together
into
a
tight
group
in
the
three-dimensional
structure
of
23S
RNA,
lying
at
the
base
of
the
'central
protuberance'
on
the
interface
side
of
the
large
subunit.
Those
sites
protected
explicitly
by
tRNA
bound
Figure
3.
The
secondary
structure
of
positions
1271-2904
of
the
E.
coli
23S
RNA
(regions
C
and
D,
ref.
11),
showing
the
locations
of
intra-RNA
and
RNA-protein
cross-link
data
used
to
define
the
model
for
the
tRNA
binding
domain.
Helices
are
numbered
according
to
reference
11.
Intra-RNA
cross-links
are
denoted
by
double
headed
arrows
and
numbered
as
in
Table
1.
RNA-protein
cross-link
sites
are
denoted
by
arrows
and
labelled
with
the
respective
ribosomal
protein.
Where
more
than
one
site
is
given
(proteins
LI
and
L27)
these
are
distinguished
by
the
suffixes
a,
b
and
c.
The
location
of
tRNA
cross-links
or
affinity
labelling
sites
(2-4)
and
chemical
footprints
(6)
within
the
23S
RNA
are
also
given:
sites
involved
in
cross-linking
(4)
or
affinity
labelling
with
analogues
of
aminoacyl
tRNA
(2,3)
are
indicated
by
arrows
and
labelled
tRNA,
whereas
the
chemical
footprinting
data
(6)
are
defined
according
to
'A',
'P'
and
'E'
sites
(V,
0
and
0,
respectively).
T4
indicates
the
phylogenetically
established
tertiary
interaction
between
positions
2328-2330
and
2385-2387
(27).
See
text
for
further
description.

4332
Nucleic
Acids
Research,
Vol.
18,
No.
15
U
L
!:,:
L.6
.7
a.
PE.;
.,
A2
E!
eX
4.
.:
V.
......
A
A
..
A
$r
-
-
A
:...
A
b.
Figure
4.
Computer
graphics
model
of
the
tertiary
structure
of
23S
RNA.
The
view
given
is
of
the
interface
side
of
the
large subunit.
Helices
constituting
the
tRNA
binding
domain
are
denoted
by
heavier
shading
and
numbered
as
in
Fig.
3.
T4
denotes
a
phylogenetically
established
tertiary
interaction,
shown
in
Fig.
3.
Panie!
a
shows
the
locations
of
RNA-protein
cross-link
sites
in
the
tertiary
model:
these
are
indicated
by
squares
and
labelled
with
the
respective
ribosomal
protein,
as
in
Fig.
3
(the
site
involved
in
a
cross-link
to
L14
in
helix
61
is
not
visible,
being
obscured
in
this
view
of
the
model
by
helix
91).
Panel
b
shows
the
distribution
of
topographical
data
relating
to
tRNA
in
the
model.
The
tRNA
footprint
sites
(6)
ale
indicated
using
the
same
symbols
as
in
Fig.
3,
and
labelled
according
to
the
corresponding
ribosomal
binding
site
('A',
'P'
or
'E'
sites,
respectively).
Their
positions
in
the
23S
RNA
sequence
are
given,
numbered
from
the
5'-end.
Sites
involved
in
cross-linking
or
affinity
labelling
with
tRNA
are
denoted
by
A.
These
sites
are
labelled
X
and
their
positions
in
the
23S
RNA
sequence
are
likewise
indicated.
in
the
'A'
site
largely
overlap
the
'P'
site
footprints,
with
the
exceptions
of
positions
G1041,
G1068
and
G1071,
which
were
shown
to
be
dependent
upon
the
presence
of
ribosomal
bound
EF-Tu.
Nucleotides
protected
by
tRNA
bound
in
the
'E'
site
are
less
numerous
and
are
located
towards
the
'LI
protuberance'.
The
distribution
pattern
of
the
footprint
data
on
the
model
is
in
agreement
with
the
proposed
direction
of
movement
of
the
tRNA
molecule
(34)
from
'A'
to
'P'
to
'E'
sites
across
the
interface
of
the
large
subunit,
from
right
to
left
with
respect
to
the
view
shown
in
Fig.
4.
The
locations
of
'E'
site
protections
on
the
upper
side
of
the
LI
protuberance
may
indicate
that
the
path
of
the
tRNA
leaving
the
ribosome
proceeds
over
the
top
of
this
structure.

Nucleic
Acids
Research,
Vol.
18,
No.
15
4333
The
cluster
of
sites
beneath
the
'central
protuberance'
of
the
50S
subunit
would
approximately
accommodate
a
tRNA
molecule
(bound
in
either
the
'A'
or
'P'
sites)
positioned
with
its
3'-end
at
the
'peptidyl
transferase
ring'
and
the
anticodon
loop
directed
towards
the
'LI
protuberance'.
This
orientation
is
in
agreement
with
the
observed
cross-link
between
the
anticodon
loop
of
a
photoreactive
derivative
of
yeast
tRNAMet
and
LI
for
'P'
site
bound
tRNA
(35).
In
this
position,
the
tRNA
anticodon
loop
could
come
into
contact
with
the
decoding
site
in
the
cleft
of
the
30S
subunit
and
is
suitably
positioned
to
satisfy
the
cross-links
observed
to
S19,
L5
and
L27
with
the
'elbow'
of
tRNA
(36).
Further
support
for
such
an
alignment
is
given
by
cross-links
to
L33
and
a
region
of
the
23S
RNA
between
helices
81-86
using
an
affinity
label
positioned
on
the
'D'
loop
of
lupin
tRNAMet
bound
in
the
'P'
site
(P.
Gornicki,
unpublished
results).
As
noted
above,
L33
is
located
between
LI
and
L27,
the
latter
protein
(together
with
L5)
being
at
the
base
of
the
'central
protuberance'
of
the
large
subunit
in
the
50S
protein
map
(see
Fig.
1,
Ref.
26).
Moazed
and
Noller
(6)
reported
the
majority
of
footprint
sites
to
be
dependent
on
the
structural
integrity
of
the
3'-end
of
the
tRNA.
Clearly,
the
number
of
sites
implicated
in
tRNA
interaction
and
their
distribution
in
the
model
suggest
they
could
not
be
all
in
direct
contact
with
the
CCA
end
of
the
tRNA.
Unfortunately,
data
from
chemical
footprinting
studies
do
not
allow
one
to
distinguish
between
'true'
protections
and
alterations
in
reactivity
due
to
allosteric
effects.
However,
it
is
of
note
that
of
those
sites
reported
which
were
not
affected
by
removal
of
the
3'-terminus
and
which
are
thus
possibly
in
direct
contact
with
other
portions
of
the
tRNA
molecule,
only
C2254
(which
is
afforded
protection
by
'A'
site
bound
tRNA)
is
outside
the
regions
that
are
implicitly
associated
with
the
'peptidyl
transferase
ring'
by
cross-links
4
and
6
and
the
tRNA-23S
RNA
cross-link
at
position
1945
(see
Fig.
3).
C2254
is
at
the
loop
end
of
helix
80
and
positioned
near
to
the
'LI
ridge'
in
the
three-dimensional
model
(Fig.
4)
through
the
locations
of
cross-link
8
and
the
cross-
link
site
to
L33
(see
Fig.
3),
in
agreement
with
the
suggested
orientation
of
the
tRNA
on
the
50S
subunit.
The
striking
phylogenetic
conservation
of
certain
helical
elements
and
specific
nucleotides
within
the
rRNA
secondary
structure
(27)
probably
reflects
the
molecular
basis
of
translation
at
the
level
of
a
series
of
RNA-RNA
interactions.
The
location
of
conserved
helices
in
the
23S
RNA
on
the
interface
surface
of
the
50S
subunit
in
the
model
(18)
is
thus
in
keeping
with
the
direct
involvement
of
rRNA
in
the
processes
of
protein
synthesis.
Indeed,
many
of
the
conserved
helices
are
interposed
in
the
secondary
and
tertiary
structure,
forming
a
group
constituting
the
core
of
the
tRNA
binding
domain.
This
group
is
composed
of
helices
61,
64,
66,
67,
69-75,
80,
81,
89,
90,
92
and
93
(see
Figs.
3
and
4).
The
model
of
the
tRNA
binding
domain
of
23S
RNA
presented
here
provides
a
first
insight
into
the
three-dimensional
structure
of
a
functionally
significant
region
of
the
RNA
of
the
large
subunit.
In
conjunction
with
models
for
the
organisation
of
16S
RNA
in
the
small
subunit
(7,8),
this
affords
a
basis
for
beginning
to
understand
molecular
interactions
between
rRNA
and
ribosomal
ligands
during
the
translational
process.
Proc.
Natl.
Acad.
Sci.
USA,
81,
3607-3611.
3.
Steiner,
G.,
Kuechler,
E.
and
Barta,
A.
(1988)
EMBO
J.,
7,
3949-3955.
4.
Wower,
J.,
Hixson,
S.S.
and
Zimmermann,
R.A.
(1989)
Proc.
Natl.
Acad.
Sci.
USA,
86,
5232-5236.
5.
Moazed,
D.
and
Noller,
H.F.
(1990)
J.
Mol.
Biol.,
211,
135-145.
6.
Moazed,
D.
and
Noller,
H.F.
(1989)
Cell,
57,
585-597.
7.
Brimacombe,
R.,
Atmadja,
J.,
Stiege,
W.
and
Schueler,
D.
(1988)
J.
Mol.
Biol.,
199,
115-136.
8.
Stem,
S.,
Weiser,
B.
and
Noller,
H.F.
(1988)
J.
Mol.
Biol.,
204,
447-481.
9.
Stiege,
W.,
Stade,
K.,
Schueler,
D.
and
Brimacombe,
R.
(1988)
Nucleic
Acids
Res.,
16,
2369-2388.
10.
Noller,
H.F.
(1984)
Ann.
Rev.
Biochem.,
53,
119-162.
11.
Brimacombe,
R.,
Greuer,
B.,
Mitchell,
P.,
Osswald,
M.,
Rinke-Appel,
J.,
Schueler,
D.
and
Stade,
K.
(1990)
In
Hill,
W.
et
al.
(ed.),
The
Structure,
Function
and
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