Nucleic Acids Research
Volume 8 Number 19 1980
Chemical modification studies and the secondary structure of HeLa cell 5.8s rRNA
John M.Kellyt and B.Edward H.Maden
Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, UK
Received 11 August 1980
ABSTRACT
Various secondary s t r u c t u r e models have been proposed f o r 5.8 S rRNA.
In t h i s paper HeLa c e l l 5.8 S rRNA is shown t o possess several s i t e s t h a t
a r e reactive t o carbodiimide a t 25O, and other regions t h a t a r e unreactive.
Previous work has established the d i s t r i b u t i o n of reactive and unreactive
cytidine residues along the primary s t r u c t u r e (11). The secondary
s t r u c t u r e model of Nazar e t a l . (7) is f u l l y compatible with the chemical
r e a c t i v i t y data whereas other models a r e p a r t l y incompatible.
We
conclude t h a t the model of Nazar e t a l . provides the b e s t approximation so
f a r available t o the conformation of i s o l a t e d 5.8 S rm.
Findings on the
e f f e c t of temperature on the chemical r e a c t i v i t y of d i f f e r e n t p a r t s of the
s t r u c t u r e a r e summarized.
The findings described i n t h i s paper should
provide a b a s i s f o r examining the s p e c i f i c i n t e r a c t i o n of 5.8 S rRNA
with 28 S rRNA.
. .
INTRODUCTION
Eukaryotic 5.8
S rRNA
e x i s t s i n the ribosome in the form of a s p e c i f i c
complex with 28 S rRNA (1-3).
The complex survives deproteinization by
phenol, but i s dissociated by treatments which d i s r u p t hydrogen bonds (1-3).
Detailed understanding of t h e i n t e r a c t i o n of 5.8 S rRNA with 28 S rRNA
requires knowledge of possible secondary s t r u c t u r e i n t e r a c t i o n s within 5.8 S
rRNA i t s e l f .
This follows from t h e f a c t t h a t 5.8 S rRNA i s transcribed
before 28 S rRNA i n the ribosomal transcription u n i t (3-6).
Therefore
during ribosome formation there i s a short i n t e r v a l during which the 28 S
sequence i s unavailable f o r interaction.
I t i s almost c e r t a i n t h a t
i n t e r n a l secondary s t r u c t u r e forms i n the 5.8 S sequence during t h i s period.
Therefore during t h e subsequent i n t e r a c t i o n with 28 S rRNA such i n t e r n a l
s t r u c t u r e must be e i t h e r u t i l i z e d o r modified.
Nazar e t a l . (7) determined the nucleotide sequence of a mammalian
( r a t hepatoma) 5.8 S rRNA and proposed a secondary structure.
Other
secondary s t r u c t u r e s f o r 5.8 S rRNA have also been proposed (8-10).
--
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@ IRL Press Limited, 1 Falconberg Court, London W1V 5FG. U.K.
We
Nucleic Acids Research
have described the reaction of specific cytidine residues in HeLa c e l l
5.8 s rRNA with sodium bisulphite (11).
The distribution of reactive
and unreactive cytidines along the primary structure was in good agreement
with the secondary structure model of Nazar e t a l . (7).
Here we describe
the reaction of 5.8 S rRNA with l-cyclohexyl-3-(2-morphollnoethyl)-carbodlimide, hereafter called carbodiimide.
The findings provide further data
on the chemical reactivity of 5.8 S rRNA, including Information on a
number of s i t e s which were inaccessible t o analysis using bisulphite.
On
the basis of the chemical reactivity data the model of Nazar et a l . appears
to be the best approximation to the secondary structure of the molecule in
i t s isolated form ( i . e . detached from 28 S rRNA).
He also briefly describe
changes in s u s c e p t i b i l i t y of 5.8 S rRNA to chemical modification as a function
of temperature.
HETHODS
Growth and l a b e l l i n g o f
cells
HeLa c e l l s , grown i n modified E a g l e ' s medium, were l a b e l l e d with
as
Pi
d e s c r i b e d (12) e x c e p t t h a t the actinomycin ' c h a s e ' s t e p was omitted.
Cytoplamnic RNA was i s o l a t e d by phenol e x t r a c t i o n as d e s c r i b e d ( 3 ) .
rRNA was obtained as i n r e f .
5.8 S
3 and was p u r i f i e d by rerunning on a 10-25%
sucrose g r a d i e n t ( 1 1 ) .
Chemical M o d i f i c a t i o n of 5 . 8 S rRNA
•Sty
(a) Carbodiimlde
1 u g of P labelled 5.8 S rRNA (1 x 1O c.p.m.)
with 20 u g of unlabelled "carrier" 18 S rRNA were dissolved in water and
lyophilized prior to modification.
The reaction mixture (total volume
5O ul) contained this RNA with 10 mg/ml carbodiimide dissolved In 0.02 M
tris HCl, lo mM MgCl- at pH 8.9.
incubations were carried out in sealed
capillary tubes.
In the main experiments incubation was at 25 . A 16 hr
reaction period, followed b y fingerprinting, revealed extensive reactivity
in several sequences and little o r none in others. A 16 hr incubation
period was therefore used for detailed analysis o f reactivity at 25 . At
37° and 5o° the reaction periods were reduced to 6 hrs. and 2 h r (see
results and discussion).
Reactions were halted by adding one fifth volume
of 0.85 M NaCl and 2.5 volumes of ethanol followed by storage at -2o°.
The RNA was precipitated twice, divided Into suitable aliquota and lyophllized prior to fingerprinting.
(b) Sodium bisulphite
5.8 S rRNA (1-1O ug) was reacted with 3 M
NaHSOj, pH 6.0 in the presence of 10 mM MgCl_ for 9 h r at 37° or 6 hr at
4522
Nucleic Acids Research
5O .
Removal of unreacted NaHSO, and of bisulphite adducta, and further
preparation of the treated 5.8 S rRKA for fingerprinting, were carried out
as described (11).
RNA fingerprinting analysis
The RNA samples (treated or untreated) were digested with Tj^ rlbonuclease or pancreatic rlbonuclease in 0.01 H Tris/HCl; O.Ol H EDTA,
pH 7.4 for 3O-45 minutes at a 1.10 (w/w) enzyme to substrate ratio.
Fractlonation of these digests by high voltage electrophoresls on cellulose
acetate (pB 3.5) and
DEAE paper (7% formic acid) followed the general
procedures described by Brownlee (13).
The voltages and separation times
varied depending on the particular analysis required.
Interpretation of data
(a) Reaction with carbon-in^de
Carbodiimlde reacts at approximately
equal rates with free urldine and guanosine (or UMP and GMP) at given pa
value in the pH range 8-9 (14,15).
It reacts about twice as rapidly with
pseudouridine as with uridina due to the availability for reaction of an
extra heterocyclic nitrogen atom (16).
Cytidine and adenosine are un-
reactlve in this pH range because of the considerably higher pK values of
their heterocyclic nitrogen atoms (14,15,17).
In various small RNA molecules of known sequence, carbodiimlde reacts
preferentially with unpaired D residues and only slightly with G (18-20).
Yeast tRNA
is an exception, possessing two reactive G residues (21).
The generally more frequent occurrence of reactive D than reactive G
residues may be due to the low propensity of unpaired U for stacking (22,20).
In the present analysis a number of products were recovered In
approximately the same relative molar yields In fingerprints of untreated
and carbodiimide treated 5.8 s rRNA, whereas all remaining products were
recovered in lower relative yields after carbodiimlde treatment.
It was
assumed that in products of the former category no significant reaction had
taken place.
It was also inferred that no reaction had occurred in the
immediately preceding G residue for a T. rlbonuclease product, or in the
immediately preceding 0 residue for a
pancreatic ribonuclease product,
since the reaction of these nucleotides with carbodiimlde blocks subsequent
cleavage of the adjacent phosphodlester bond by the respective enzyme (14,21).
Unreactlve products served as references for calculating the extent of
reactivity In other products.
It was generally
possible to localize
reactivities either to a single nucleotide or to a small group of nucleo-
4523
Nucleic Acids Research
tides by using data from overlapping T\ and pancreatic rlbonuclease products.
Several G residues were Inferred to be unreactlve where the previous bisulphite data til) and the structural models strongly Indicated base-pairing
with C.
(Parts of the proposed secondary structure are Invariant In the
different models, as described later).
Finally, several new, carbodlimlde
containing products appeared in the fingerprints of modified RNA.
Hovever,
a number of these streaked or were recovered In low yields and a complete
analysis was not undertaken.
Tables 1 and 2 give the recoveries of
Hol*r flalda
EpOt
Tl
T2
T2t
saqnapca
c
-
C-<3
tHKU
OH
T3
»-G
T4
C-*-G
T5
A-A-C
T6
o-c-c-c-a
T7
A-A-C-Q
ocntxol
(tb«or«tic»l)
11.15
(10)
cazbodllsld*
trutad
9.44
r^ctlvit,"1'
-
(.20
(6)
6.16
-
1M.159
0.91
(1)
0.22
75%
-
2.36
(3)
2.91
-
1.04
(1)
1.0O
-
1.01
(11
O.M
-
0.45
«D
0.33
-
-
1.44
(21
1.34
-
aozma
-
(c)
T8
QB-G
14
0.21
(0.2)
0.19
1O%
T9
0-0
K.30
1.99
(l.B)
2.06
0*
no
T-O
71
1.02
(1)
1.12
OV
TlOa
pC-G
0.42
(O.4-O.S)
0.42
-
TlOb
pc
0.12
(0.1-O.2J
0.17
-
Til
D-C-C
1.92
(2)
1.S0
5%
T12
C-T-C
57
1.03
(1)
1.13
0*
1.15
(1)
0.15
2i»
J
(1)
0.92
m
Id)
(11
0.33
70»
(d)
1.06
(1)
0.32
7O»
34,1H
TU
A-0-«
3J
T1U
CMJ-C -C
27
Tl*b
C-C-O-C
144
C-O-A-C
S3
J13
T16
C-O-A-C-C
0.67
HI)
0.21
7«
(c)
Tl*
O-C-U-C
14C.148
1.10
(11
0.24
sot
(•)
Tl«
C-A-C-U-O-C
112,113
0.91
(1)
O.M
25«
Tlt.l
A-O-C- A-U-C-G
91,94
0.67
(11
0.4O
55%
Tl«.2
A-O-C-A-C-U-C-C
1»,.>2
T20
A-C-A-C-U-U-C-G
T31
A-C-A-C-A-C-U-O
T2t
B-O-C-C- U-C-C-C-fl
T2J
A-C-U-C-U-U-A-C
5.7.1
T23*
A-A-U-D-QB-C-A-G
T24
A- A- D-C- A -A-O-G
75.76
65,66,69
131
(f)
;
(1)
Iol,li32
I.K!
(1)
0.31)
•5%
(I)
".a
o.r?
(1)
O.H
4O%
'g>
115.U6.1H
0.10
(11
0.03
95%
Oi)
40%
13)
)
O.M
(1)
C)
0.61)
• j %
11)
(1!
0.17
55%
[k)
yltlfla of Tl rlixgmcl—** product! trxm H«LJ C*11 f-.B S
and crttnt of r>«ctixm aftrr tree
of 5,a S rWW with carbodljnldc *t 25
Plag«xpxlat« of iWLa call S.I I rfm haw bare pc*llchad (11,25).
Tha reaction with caxbodllnld* was csxrlad out a*
davcrlbad In tha Methods •action.
ftolax ylalds aftax cartodilmid* trrstxart u*ra calculatad i i r i H o g that T4, C-A-U,
was 1. OB this a*txaf>tlon products TB-T12 al*o g«vt similar yialds as 1A control fingerprints.
Tlalda ara tba M I D I
of thxaa datarmlnatlona froa looapaodaot axparlosnts.
Uotaai(•) K»t>tn In this colissn rafar to poaltlons of
trxldloa raslduas In tha saquanca.
Cb) laactlvltlaa ara axprasaad to tha naaxast 5*.
Xn prodocts with aora than ooa u
raaldua, reactivity *-*y ba uoaqaally dlstxlbutadi in a fav casas C m*j also ba raactlT* (sac follcvlng ootao).
(c) Products
T6 and TIC wars obuioad In low yl«ld In control fInoarprints, probaJoly do« to lncoaplat* dloa«tlODi tbay axa fro» ex ovaxlap
tha atabla b«llx (*). 013* in Tit Is claaxly rvactlra, bat tha praclaa valoa u y ba soiwwhat mxallaLls.
<dj Thvsa t w
%
pxDdocts roD cloaa togatbar, bet ara aapaxatad aftax long sacocd illaarnirn m u .
TUb (0144) acoscnts fox nost of tha
raactMty.
(a) Moat of tba raactlvity Is In Ul4a - s«a oota (a) In labl* 2.
If) Tbaaa two products also n o cloa«
togathar, but axa ^ust sapaxmtad aftar long aamnri Haafulnn ruas.
Both spots axa highly raactlva, aspaclally T19.2.
fluta tba raactlrlty In T19.2 la graatar than In tha oraxlappiao, product P21 ;tablt 2), tbaxa pust also ba xaacti*ity In Ol2
and/or powlbly 024.
(g] • • activity avldaocad by thla product stay ba wbolly or partly dua to tha pracc-dlro, C81 (product P14,
tatla 2).
Ch) 0139 was IS% raacUva (product P24, tat la 2).
Slnca pxodoct T23 la 95* xaactlw tbaxe M t also lm raactivlty
In U12« and/ox Ol2f.
(J) PIUJULL T3J* *l«oat cosplataly dlsapp*ara aftar raactloc.
This U doe to raactivlty In 075 (product
P10) and C7t and/or 0a77 la— product P5J.
T33 l a partly raactlva, probably at US.
d ) «*• also prodocta P22 and P l l .
4524
Nucleic Acids Research
Holax Yields
nrliln.'* 1
Spot
MqiMBC*
control
(tlMoratlcal)
19.0
19. S
(2O)
(19)
PI
0 + T
H
C
-
P3
A-C
-
s.&s
(5)
-
0.46
0.92
7.O6
(0.4-0.5)
r*
PC
P5
Oa-C
re
o-c
-
F7
O-A-C
-
PS
A-O-C
-
P9
O-A-A-C
-
no
A-D
Pll
A-A-U
(WI.69
>u
G-O-C
-
H3
O-A-O-C
ru
A-OG-A-C
PIS
0-A-A-O-A-A-C
pie
O-U plus
0-T
(76)
CM)
30,34,146
154.71
38,91
351
1M
(1)
11.53
6.27
0.54
0.15
7.33
1.78
2.32
1.00
2.15
0.66
1.50
0.90
0.61
0.47
3.43S
2.23
(S)
4.7J
(1)
(I)
(2)
4.7JS
0.9»
2.47
1.06
1.91
O.97
(3)
O.M
(1)
0.77
(1)
(2)
(1)
(2)
(1)
I
P17
O-A-U
PIS
G-A-A-U
75
l.U
(1)
P2O
0-0-0
14
?21
O-O-A-0
16
O.95
0.S3
(0.6)
(O.B)
P22
G-A-G-A-A-O
65
O.M
(1)
P23
0-C-O-C-C
P24
O-O-G-D
P25
0-O-tJfc-C-C-A-U
-
US
14,18
reactivity
U.M
I
87,94
ctxbodllalda
treated
0.45
0.76
O. 20
(2)
(ill
(1)
(0.2)
1.71
0.43
0.93
0.2»
0.55
0.34
O.12
0.05
Hots*
Q>)
651
(c)
15%
40%
(<U
2O%
-
(•)
40*
(f)
4M
(g)
15%
25%
6O%
O%
no
7O%
00
4O%
o>
-
(k)
15%
(1)
75%
Mqnanccs and polar ylalda of pmcriitlc rlbonoclaaM product-! from L* c»ll 5.8 1 rtott and w U n t of r—ctlon
tr— tnant of 5.6 I rUJft with CAib H *<*6m *t 25°
••action cortditlona war* th« UMEM AJ in t*bl« 1.
Hol*r yields aft«r c«xbodiiaid* traatnant w*r* calculattd
that p9, O-K-h-C, was 1.0.
(On this assusptlon s«v«ral other products including P2o also gav« approalsmtaly the
•asM nolar yields *m in control fingerprints).
The values shown are the asens froo three independent experiment*.
Reactivities axe given to Dearest i\,
Hotsst(a) mnfeers refer to positions of uridine residue* in the eequerce.
IttKtMrs in parenUwses refer to utidine residues imedlately preceding the indicated products.
Q>) Inactivity o a t
be due to precediog U residues.
(c) Reactivity could be due to 076, OB77 OX both.
(d) Reactivity could be at U66
and/or UG9. (e) The fact that this product is alcost undlnlnlshed after the caxbodiimid* reaction shows that the
preceding UHB i s practically unxeactive.
Therefore ao%t reactivity In U-C-U-G (product T17) aost be due to 01*6.
(f) Inactivity in this product en»t be In C8O and/or G51.
(g) Product T5, A-A-C, was unre active but T13, A-O-O was
partly reactive.
This implies partial reactivity in U3fl 1 m i l lately preceding P15. It i s possible that the structure
i s eoswwhat disordered in the region of intersection Y, with loosening of the helix at UM but protection of C39 and
042 by stacking.
(h) P20 and P21 represent the unoethylated fore of >25. Inactivity in thlo region i s probably in
Oil with possibly SODS contribution from Cl*. Unl4 i s mreactlva (Tfi, table 1).
(j) inactivity Is probably in
065 with s o n contribution froa G*2.
(k) This product was recovered in LOW yield in control fingerprints, probably
doe to incomplete digestioni i t i s froa th# very stable holix (e).
(li JeacUvity cust be at 0125) c residues are
in stable helix (e).
standaxd products from untreated and carbodiimide treated 5.8 S rRNA.
Footnotes to the tables Indicate how reactivities were localized to
particular sites.
Further details are given in ref. 23.
(b) Reaction with bisulphite
Reactivities of C residues to bisulphite
were analyzed by the same general procedures as described previously (11) .
RESULTS AND DISCUSSIGN
Considerable simplification is achieved by first discussing the
4525
Nucleic Acids Research
Intoudkan Y
loop I
H.IU (o)
»
V (30)
H.IU(b)
H.II. (c)
LoopH
O-C' U " C "G-U-G-C-G-U-C-G-A-U-G' A / * " A G-C-G.^-C-AC-G q-AG-C-il-A-C. A ,*0-A-OC-(^VC
-C
Q A .U
K»y
mort than on* of th«M
(b) Ctrbedlimldc, 25
or both
omor mor« of H N N
A
#
G-A-
A
U
I
u
u
(c) Bisulphllc,
4526
A
A
U/
h
A-lS
u
V
G
6
A
U
Nucleic Acids Research
reactivity data In relation to the secondary structure model of Nazar et al.
(7), with which there Is good agreement.
Other models, with which there Is
less good agreement, are discussed later.
Reaction with carbodiimide at 25°: the Nasar model
Figure la shows the Nazar model, with the main structural features
named as In our previous work (11).
Figure lb showB the sites in 5.8 S
rRNA that are reactive or unreactive to carbodiimide.
The reactivities
were Inferred from the data in tables 1 and 2 as outlined in the methods
section, tha footnotes to the tables and the following text.
summarizes the bisulphite reactivity data.
Figure lc
Particular aspects of the
carbodiimide data are now discussed.
Dnreactlve regions
In the proposed helical regions (a) , (b) and (c) ,
and in the distal parts of helix (d), several nucleotides show little or no
evidence of reactivity:- the respective oligonucleotldes (T4, 8, 9, 10, 11,
12, P13 and 20) were recovered in unchanged relative yields after carbodiimide treatment of 5.8 S zRNA, whereas most other products were recovered in
lower yields relative to these products.
The unreactive nucleotides are
depicted by open circles In figure lb.
Secondary modification sltea In helical regions are unreactive
of
particular Interest is the lack of reactivity at secondary modification
sites in the proposed helices.
¥57 (product T12), ?71 (product Tlo) and
Figure 1.
HeLa cell 5.8 S rRKA, arranged according to the secondary
structure model of Nazar et al. (7).
(a) Helical regions and loops are
named according to ref. 11.
potential meeting points between helices are
n
denoted "Intersections X and Y .
Conformation at these Intersections is
unknown.
The two nucleotides between square parenthesis in loop III are
almost certainly absent in Xenopua laevis 5.8 S rRNA, as Inferred from
sequence analysis of three independent, cloned genes (ref. 5, and L. Ball
and B.E.H.M., manuscript in preparation).
Experiments are in progress
to determine whether other 5.8 S rRNA sequences are in need of revision
at this point.
Until this matter is clarified we have retained the
existing numbering system for all nucleotides.
For either possible
sequence, loop III contains the T, product T15, C-O-A-G.
Location of
other nucleotides in the various 1^ and pancreatic ribonuclease digestion
products are given in tables 1 and 2.
(b) Reactivities of nucleotides
to carbodiimide at 25°. Open circles denote no significant reactivity.
Shaded circles or ellipses denote slight to moderate reactivity (10-45%).
Dark circles denote extensive reactivity ( 5O%).
Reactivities are
attributable to individual nucleotides except as follows:Ellipses
signify that one or both of two adjacent nucleotides are reactive.
Branched arrows signify that one or more of the indicated nucleotides are
reactive.
In the region 091-0102, localization of reactivities to carbodiimide ie tentative.
(c) The reactivities of cytidine residues to bisulphite, redrawn from data in ref. 11 with conventions as In (b).
4527
Nucleic Acids Research
U14, both In Its unmethylated and Its methylated form (products P2O and T8),
are all unreactlve.
As mentioned 1A the methods section, free pseudourl-
dlne is about twice as reactive as urldine to carbodiimide (16).
There-
fore the lack of reactivity of the pseudouridines in 5,8 S rRNA must
signify that they are in protected locations.
their proposed positions in figure 2:-
This is consistent with
each is shown paired with an A
residue, and each A? pair is sandwiched between GC pairs on either side.
Similarly, the partly 2•-O-mothylated U(m)14 is shown paired with G141,
and this pair is also sandwiched between GC pairs.
HffHit(d) is partly reactive
In contrast to the above findings, the
proximal part of the AD rich helix(d), near 'intersection Y 1 , shows
evidence of partial reactivity.
The decreased yield of Pll, (U)-A-A-U
clearly signifies reactivity of U66 and/or U69.
At the base of the helix
two products show considerable reactivity, P22 and T19.1.
It is likely
that a major part of the reactivity in product T19.1 is in U91.
In
product P22, reactivity may possibly be shared between 065 in the base of
the helix and G62 in the adjacent 'intersection Y'.
Two other uridines
are contained within the partly reactive product T21, but in this instance
reactivity could also be due to G81 in loop IV, or (less probably) to G89,
opposite U66.
Thus, the collective data on the proximal part of helix(d)
indicate some reactivity at least as far into the helix as D66.
The
distal part of the helix, containing ?71, is unreactlve, as discussed
above.
Helix Imperfections show varied reactivities
are depicted in the proposed helices (a) and (b).
Various imperfections
The two looped out
uridines, U144 and U146 in helix (a), both appear to be highly reactive
on the basis of detailed criteria summarized in footnotes (d) and (e) of
table 1 and (e) of table 2.
Another imperfection in helix (a) contains
the mismatched U5, opposite C152.
Product T23, containing U5, is partly
reactive, and it seems probable that this reactivity is mainly due to U5
itself.
C152, opposite US, was previously found to be partly reactive to
bisulphite (figure lc, ref. 11).
In helix Cb) the symmetrical mismatch
at the left hand end, near 'intersection X', is only slightly reactive:U27 (product T14a) showed little or no reactivity, and the 0 doublet, 112
and 113 (product T18) showed slight reactivity.
In summary, the most
reactive of these helix imperfections is the site with the two looped out
uridines in helix (a), whereas the least reactive (to carbodiimide) is
the symmetrical mismatch at the left end of helix (b).
4528
(This mismatch
Nucleic Acids Research
showed appreciable reactivity of its cytidine residues to bisulphite)
figure lc and ref. 11).
The major loops are highly reactive
Each of the major loops, I, III,
IV, V and VI, is highly reactive to carbodiimide.
There is almost certain-
ly more than one reactive nucleotide in each of loops I, IV and VI, as
outlined in the notes to products T19.2, T23a and T22 (table 1).
These
loops also contain one or more reactive C residues (figure lc; ref. 11).
The sequence in the vicinity of Qn77 in loop IV is clearly reactive:product P5, and C78 in figure lc).
Loop V is reactive in one or both of
two adjacent U residues, and also possesses one or two reactive C residues
(figure lc; ref. 11).
diimide and bisulphite.
Loop III is also highly reactive to both carbo(See legend to figure 1 concerning a possible
sequence uncertainty in this loop).
Loop II shows little or no reactivity,
but this is the least clearly defined of the designated loops, and also
consists largely of A residues.
The 3' end Is reactive
Product T2a, C-O-U
, shows evidence of
extensive reactivity towards carbodiimide, and the C residue in this
product is reactive to bisulphite (figure lc, ref. 11).
Chemical reactivity data and alternative structural models
It is clear from the above discussion that the chemical reactivity
data can be accommodated by the Kazar model.
We now comment on other
models.
Rubin's model (8)
This was proposed in conjunction with the yeast
(S. cerevisiae) 5.8 S rRNA sequence, which was the first 5.8 S sequence to
be determined.
Host of the proposed secondary Interactions In this model
are concentrated into the central region of the molecule.
more recent models, the 5' and 3' regions do not Interact.
In contrast to
However,
results from optical studies on 5.8 S rRNA from yeast (9,24) and rat (24)
demand a higher degree of base-pairing than in the Rubin structurb.
Moreover the chemical reactivity data clearly establish that several
nucleotides in the 5' and 3' regions of the HeLa sequence are unreactive.
Therefore the Rubin structure is unlikely to be a sufficient description
of the secondary structure of isolated 5.8 S rRNA in general.
Luoma and Marshall's cloverleaf model (9)
This model was derived to fit data from Raman spectroscopy on yeast
5.8 S rRNA, and was then adapted to the mammalian sequence (figure 2a).
Between nucleotides approximately 25-138 the general features of the model
4529
Nucleic Acids Research
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4530
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Nucleic Acids Research
resemble those of the Rubin model. Including a loop encompassing nucleotldes 37-49, a long arm from nucleotides 66-114, and the GC rich arm from
nucleotides 116-138, which Is a feature of all 5.8 S models. In addition
there is interaction between the 5' and 3' ends.
The loop from nucleotides
37-49 was designated the "antl T loop" (9) because of the presence of the
sequence G-A-A-C, which could potentially Interact with G-T-V-C in rRNA.
A number of nucleotides which might be expected to be reactive on the basis
of the model are in fact unreactive, including two C residues In the "anti T
loop".
other nucleotides which should be unreactive on the basis of the
model are reactive, particularly In the long central arm.
Moreover the
helical location of Gm77 and adjacent nucleotides in the model conflicts
with the high sensitivity of this region to Sj^ nudease (25). Thus the
available data on the accessibility of specific sites In 5.8 s rRKA are
not in good agreement with this model.
Ford and Mathleson's models (10) These authors derived three
energetically feasible models for X. laevls 5.8 S rRNA.
Model 1 is the
Nazar model adapted to the X. laevis sequence (which differs from the
mammalian sequence at only a few nucleotides).
Models 2 and 3 are shown
in figure 2.
Model 3 differs considerably from the Nazar model In the
arrangement of nucleotides 15 to 115, though retaining the AU rich helix (d)
and its associated loop.
The available chemical reactivity data appear
to be Inconsistent with this model at a number of points (figure 2b).
Model 2 (figure 2c) differs less extensively from the Nazar model than does
model 3. The major differences are that the nucleotides constituting
loop V in the Nazar model are assimilated Into a modified helix (b-) and
a bulge appears between this helix and the base of helix (d). There are
also changes in the constitution of loop I and in the arrangement of
nucleotides at the base of helix (e). Since these changes are inter-
Figure 2. Alternative structural models for HeLa cell 5.8 S rRNA.
(a) Luoma and Marshall (ref. 9 ) . (b) Ford and Mathieson, model 3.
(c) Ford and Mathieson, model 2. The latter models were proposed for
X. laevis 5.8 S rRNA (ref. 1O) as possible alternatives to the Nazar
structure.
They are shown here adapted to the HeLa sequence, which differs
from the published X. laevis sequence at only a few points, none of which
is crucial to the models.
(However, note that the two nucleotides between
square parentheses now appear to be absent from the X. laevis sequence;
see Legend to figure 1 ) . Interrupted arrows denote nucleotides which are
unreactive but which are unpaired In the models.
Heavy arrows denote
nucleotides which are reactive, but which are paired in the models.
Only
nucleotides showing fairly large discrepancies between observed and predicted reactivities are shown.
4531
Nucleic Acids Research
dependent. Information bearing on any of them Is relevant to the structure
as a whole.
Our previous bisulphite data (11) reveal one clearcut discrepancy
with this model. Product T20 contains three C residues and Is highly
reactive to bisulphite ( 80* diminuitian In recovery).
One of these C
residues, C98, Is also recovered In the pancreatic product P7, C-A-G,
which is unreactlve.
(In fact, this product occurs twice per molecule,
also encompassing C4- Since the product showed <5% conversion to G-A-U
after reaction of 5.8 S rRNA with bisulphite (11) it can be concluded
that C4 and C98 are both unreactlve).
Since C98 is unreactive, the high
reactivity in T20 must be due to C1OO and/or O.03. This fits well with
the Narar model
(figure lc), but la inconsistent with model 2 (figure 2c).
In addition, the high reactivity of product T20 to carbodilmlde is most
readily interpreted as reactivity in OlOl and 102, as inferred above.
Finally, In the altered loop I of model 2, U27 might be expected to be
reactive to carbodilmide (figure 2c) whereas in fact it shows little reactivity (table 1, figure lb).
In summary, optical data and energetic considerations have led to the
proposal of a number of secondary structure models for 5.8 S rRNA.
Chemical reactivity data provide a means for distinguishing between the
different models. The available data fit the Nazar model well but are
Inconsistent at certain sites with the other models. We conclude that the
Nazar model is the best approximation so far available to the conformation
of isolated 5.8 S rRNA.
Possibilities for further folding, and unfolding
The above conclusion does not exhaust the conformations1 description
of 5.8 s rRNA, nor does it rule out possible mobilities in the structure,
particularly during its interaction with 28 S rRNA and in protein synthesis.
In isolated 5.8 S rRNA the intersections may be flexible, allowing further
folding.
It may be significant that, whereas all five major loops possess
chemically reactive bases, in only two of these loops, IV and VI, were the
phosphodiester backbone sensitive to Si nuclease at 0.3 H Na concentration
(25). in tRNA, discrepancies between chemical reactivity data and sensitivity to SI nuclease (26) can be explained on the basis of tertiary structure
(27). Moreover, it is likely that different parts of the structure in
figure 1 possess different stabilities. Byperchrooicity measurements as
a function of temperature have provided evidence for sequential melting
of different structural regions (24). Characterization of structurally
4632
Nucleic Acids Research
labile regions would be of Interest since such regions would be candidates
for possible rearrangements during the interaction with 28 S rRNA.
To attempt to distinguish between strongly and weakly interacting
regions we repeated both the carbodiimide and the bisulphite modification
experiments at 37 and 5O°. The incubation times were adjusted in order
to detect the specific increase of reactivity of a few nucleotides due to
localized denaturation, rather than a general, non-specific Increase in
reaction with temperature.
The findings, which have been described (23),
may be summarized as follows.
At 37° all the main structural features remained at least partly
intact. However there was increased reactivity in nucleotides associated
with the proximal part of helix (d), which, as already mentioned, were
partly reactive at 25°. The distal part of helix (d), containing f71,
remained unreactive.
In helix (a) the data also suggested some loosening
of the structure at 37°, but not complete denaturation.
Several C
residues which were unreactive or slightly reactive at 25 showed increased
reactivity at 37°, for example product T14b containing C142 and 143.
The partly methylated U(m)-G sequence also became slightly reactive.
However, U154 and its flanking G residues showed no evidence of reactivity
to carbodiimide at 37°. By contrast, at 5O most of the 5.8 S structure
reacted extensively to carbodiimide or bisulphite. Nevertheless the GC
rich helices (c) and especially (e) remained unreactive even at this
temperature, suggesting that they are sufficiently stable to remain intact
during any physiological interactions in which 5.8 s rRNA participates.
The 31 limb of helix (a) has been implicated in interaction with 28 S
rRNA (28). One may infer that a rather delicate balance of equilibria
must determine whether this sequence interacts with the 5' part of the
5.8 S sequence, as in the isolated molecule, or with 28 S rRNA in the
complex.
It is possible that the looped out and mismatched bases
in helix (a) of the isolated molecule contribute to this balance by
generating potential instabilities in this helix.
It is not yet clear whether other parts of 5.8 S rRNA are involved
in, or alter during, the interaction with 28 S rRNA.
Chemical reactivity
studies on the complex might illuminate further details of the interaction.
ACKNOWI£DGEMENTS
He thank John Goddard and Tom Hathieson for discussion and advice.
This work was supported by the Medical Research Council with a studentship
to J.M.K. and a grant to B.E.H.M.
4533
Nucleic Acids Research
* John K e l l y ' s p r e s e n t address i s : - Department o f Biochemistry, National
I n s t i t u t e f o r Medical Research, The Ridgevay, M i l l H i l l , London NW7 1AA.
Correspondence on t h i s paper should be addressed t o B.E.E. Maden,
Department o f B i o c h e m i s t r y , u n i v e r s i t y o f Glasgow, Glasgow G12 8QQ.
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