volume 7 Number 81979
Nucleic A c i d s Research
The 3' terminus of 16S rRNA: secondary structure and interaction with ribosomal protein SI
Raymond C.Yuan, Joan A.Steitz, Peter B.Mcore and Donald M.Crothers
Departments of Molecular Biophysics and Biochemistry and Chemistry, Yale University, New
Haven, Connecticut, USA
Received 27 September 1979
ABSTRACT
We report studies of the secondary structure and SI ribosomal protein
binding properties of the c o l i d n fragment, containing 49 residues from the
31 terminus of £. c o l l 16S rRNA. Temperature jump relaxation kinetic
measurements reveal two helices 1n the structure. One of these, melting at
81°C 1n 5 nfl Mg 2+ , 1s associated with the 9-base pair hairpin helix predicted by the nucleotide sequence. The other melting t r a n s i t i o n , at 21°C
1n 5 mM Kg , 1s assigned to a 4-base pair helix which constrains the
pyr1m1d1ne t r a c t of the c o i i d n fragment Into a bulge loop. SI protein
forms a strong 1:1 complex with the c o l i d n fragment, with an association
constant of 5 x 10° M" 1 1n 5 mM Kg . More protein molecules are bound,
but with weaker a f f i n i t y , when the SI concentration 1s Increased. SI
binding causes melting of the c o l i d n fragment secondary structure, as
Inferred from the observed absorbance Increase. The SI binding site on the
c o l i d n fragment has been localized 1n the region of the bulge loop, since
the melting t r a n s i t i o n corresponding to the 4-base pair helix 1s lost 1n
the complex. We discuss current models f o r the role of SI protein 1n
polypeptide chain i n i t i a t i o n in l i g h t of these and previous results.
INTRODUCTION
The colicin fragment, produced by the action of c o l i c i n E3 on E_. c o l l
Mbosomes, comprises the 3' terminal 49 residues of 16S rRNA. I t therefore
Includes sequences f i r s t suggested to be involved 1n the I n i t i a t i o n of
polypeptide chains by Shine and Dalgarno (1974). Direct evidence for
mRNA-rRNA base pairing came from the finding that mRNA I n i t i a t o r regions
could be recovered non-covalently bound to the c o l i d n fragment when 70S
I n i t i a t i o n complexes are disassembled by detergent (Steitz and Jakes,
1975). In such isolated mRNA-rRNA hybrids, the 16S rRNA residues that
participate 1n Intermolecular duplex formation appear to be those at the
pyr1m1d1ne-r1ch 3 1 terminus (Steitz and Steege, 1977).
Ribosomal protein SI has also been known for some time to play an
C Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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Important role 1n the binding of natural mRNAs and 1n I n i t i a t i o n of protein
synthesis by £. c o l l ribosomes (Van Du1n and Van Knippenberg, 1974; Van
D1e1jen et al_., 1975, 1976; Dahlberg, 1974; Szer et al_., 1975; Sobura et
a]_., 1977; Stel tz et H - , 1977). Chemical crosslinking studies located SI
adjacent to both the 31 terminus of 16S rRNA (Kenner, 1973; Czernilofsky et
ail-, 1975) and the messenger binding s i t e on the 30S ribosome (F1ser e_t
a l . , 1975). Moreover, upon treatment of colidn-cleaved ribosomes with low
magnesium concentration, a complex containing the c o l i d n fragment with SI
bound near Its 3' end was recovered (Dahlberg and Dahlberg, 1975). Isolated
SI protein was found to bind strongly to a variety of single-stranded
polynucleotides with a preference for pyr1m1d1ne-r1ch over purine-r1ch
sequences ( B e a r e _ t ^ l . , 1976; Draper e_t .al.., 1977; Szer et ^ i - , 1976; Tal
et a l . , 1972; Miller et aj_., 1974; M i l l e r and Wahba, 1974; Carmichael,
1975; Jay and Kaempfer, 1975; Senear and Steitz, 1976; Goelz and Steitz,
1977; Lipecky et al_., 1977). Circular dichroism data showed that SI unfolds
a variety of stacked or helical single-stranded polynucleotides but does
not alter the structure of double-stranded helices with a few exceptions
under mild 1on1c conditions (Bear ejt j j _ . , 1976).
Based on these observations, various models were proposed whereby SI
interacts directly with either the mRNA or with the 3' terminus of 16S rRNA
to f a c i l i t a t e I n i t i a t i o n . However, more recent reports indicate that the
mechanism may be more canplex. F i r s t , Laughrea and Moore (1978a) found
that SI can bind strongly to the 30S ribosome even 1n the absence of the
colicin fragment. Second, I n t r i n s i c fluorescence studies have suggested
that SI may possess two nucleic add binding sites with d i f f e r i n g specifici t y (Draper et i l - , 1977; Draper and Von Hippel, 1978a, 1978b).
To explore in greater detail the possibility of a functional interaction between the 3' end of 16S rRNA and protein SI during the I n i t i a t i o n
of polypeptide chains, we have undertaken direct physical measurements of
the secondary structure of the c o l i c i n fragment. Temperature jump relaxation studies were f i r s t performed to confirm that the Isolated
49-nucleot1de fragment folds Into the predicted structure, which 1s
I l l u s t r a t e d in Figure 1. Then the effect of adding equimolar amounts of
purified SI protein was studied. The results demonstrate that SI Interacts
strongly with the isolated 3' end of 16S rRNA, forming a complex of 1:1
stoichiometry. Binding perturbs the secondary structure In the region of
the polypyrinidine t r a c t , and probably causes melting of the 4-base-pair
helix which closes the bulge loop in the structure shown in Figure 1.
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'U
"it
Figure 1. Postulated secondary structures of the c o l i c f n fragment of 16S
RNft. Th~e hydrogen base pairings are predicted from the sequence of the
c o l i c i n fragment (Ehresmann et al_., 1975; Bowman, 1972). Form (A) 1s
expected to be the most stabTe conformation at low temperature. Form (B)
1s the open conformation predicted when the lower bulge helix (a) 1s d i s rupted and the Sh1ne-Dal garno nucleotide sequence 1s exposed for mRNA
binding.
MATERIALS AND METHODS
Isolation of the C o l t d n Fragment
£. c o l l MRE 600 ribosomes were obtained from the low salt wash of the
cell extract as described by Anderson e_t .al_. (1968). Incubation with
c o l i d n E3 was carried out at 37°C f o r 45 m1n 1n 2 ml of 0.01 M Tris HC1,
pH 7.4, 0.01 M MgCl,, 0.06 M NH.C1 , and 0.006 M P-mercaptoethanol con32
taining 400 A^™ units of ribosomes (Including a small amount of
P-labeled
ribosomes as marker) and 0.2 mg of purified c o l i c i n E3 (the kind g i f t of K.
Jakes). The mixture was then phenol-extracted and the total ribosomal RNA
precipitated with ethanol before fractionation 1n a 15X-3OX sucrose gradient
containing 0.5% SDS f o r 21 hrs at 22°C and 26,000 rpm In a Spinco SW27
rotor. Fractions containing the c o l i c i n fragment and small ribosoraal RNAs
were pooled and subjected to electrophoresis on a 9J Tr1s-Acetate polyacrylamide slab gel at pH 8.0 (Steitz and Jakes, 1975). The c o l i c i n
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fragment band was Identified by autoradiography, eluted electrophoretical l y ,
and further purified by several ethanol precipitations. An aliquot of the
f i n a l preparation was fingerprinted to establish Us Intactness and purity.
The control RNA sample of sequence A,U, was kindly provided by Dr. K.
Breslauer.
Ribosomal Proteins
30S ribosomal protein SI was Isolated as reported previously (Laughrea
and Hoore, 1977). I t s concentration was measured by the Lowry (1951) assay
and Its purity (> 901) confirmed by SOS gel electrophoresis (Laemmli, 1970)
and by determining I t s ^280^^260 a ' ) S O r o a n c e r a t i o to be 1.58. The RNA
binding a c t i v i t y of SI was assayed by f i l t e r binding using 32 P-labeled R17
RNA (Senear and S t e i t z , 1976); 4 ug SI were required to retain 25 ng R17
RNA on a milUpore f i l t e r .
Protein S14 was Isolated as described by Moore (1979).
Temperature-jump Relaxation Measurements
The temperature-jump Instrument has been described 1n detail (Crothers,
1971). The cell requires 1.4 ml of sample, and has an optical path of 0.7
cm. Solutions containing colicin fragment for the temperature-jump experiments were dialyzed against either Buffer I , containing 20 mM sodium
cacodylate, 50 mM sodium phosphate, pH 7.0, or Buffer I I , (Buffer I + 5 mH
MgClp)- Relaxation measurements were carried out as described previously
(Cole and Crothers, 1972; Gralla and Crothers, 1973a,b). A 0.05 nF capacitor was used, yielding a heating time of 1.5 usec. At lower temperatures a
temperature-jump size of 4°C was used, but this was reduced to 1.9°C at
higher temperature to avoid solution cavitation. From the measured relaxation signal amplitude a differential melting curve can be constructed,
yielding the transition temperatures (T ) and enthalpy change (&H) by the
analysis described e a r l i e r (Gralla and Crothers, 1973a,b). After measurement, samples were assayed for degradation by gel electrophoresis and
fingerprinting.
Equilibrium Sedimentation Measurements
Sedimentation equilibrium experiments were performed at 4°C 1n a
Spinco Model E analytical ultracentrifuge equipped with an UV optical
scanner. An aluminum-housed Kel F double-sector centerpiece with sapphire
windows was used. Cells contained approximately 100 i±l samples. Equilibrium 1n the short column was reached after 24 hours and an equilibrium
gradient curve was obtained by scanning at 266 nm. The colicin fragment
and ribosomal protein SI were dialyzed against Buffer I I I , containing 100
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nM KC1, 20 nH sodium cacodylate, 5 mM MgCl 2 , pH 7.0.
Equilibrium sedimentation titration of colicin fragment by SI was
carried out by addition of equal amounts of protein to both the RNAcontaining sample cell and to the reference cell, a procedure which leads
to some simplifications of the sedimentation equilibrium equations. The
equilibrium equation which applies to each component of a complex mixture
Is, assumming a dilute solution (Tanford, 1961),
M.
w
1n which Cj 1s the molar concentration, M^ the molecular weight, tp. the
density Increment (ap/dc) at constant chemical potential of d i f f u s i b l e
components (Casassa and Eisenberg, 1964), w is the angular velocity, r the
distance from the axis of rotation, and RT has I t s usual meaning. Equation
(1) yields for the contribution of species 1 to the gradient of absorbance A:
dA. «.dC,
dm C.
2
- T " " S 1 " *1C1
r = F T f «1C1Mitpi
1 1
dr
dr Z
d/
2 RT 1 1 1 1
<2)
where t^ is the extinction coefficient of species 1. The total absorbance
gradient of the sample sector is the sum of terms given by Equation (2)
corresponding to the concentration C., while the reference absorbance
gradient 1s calculated from the corresponding terms containing C 1 ^. Hence
the logarithmic gradient of the absorbance difference 4A » A s a m D i e A
reference
11 s
d /MA
dr 2 "
a?
2 R T
S ^ - * ^
(3)
In principle, Equation (3) allows one to calculate diMA/dr^ f o r an
associating mixture, assumming that values are known for the density increments tp., and that t r i a l values of the association constants are taken so
that the C. can be calculated. However, 1t frequently is v i r t u a l l y
impossible to obtain enough of a pure complex so that Its density Increment
can be measured. In that circumstance we take advantage of two simplifying
assumptions: (1) cp^ can be replaced by Its equivalent for a two-component
solution:
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(4)
1n which 7.| 1s the partial specific volume and p 1s the solution density.
(2) We assume that the molar volume V = 7..M. of complex 1s the sum of the
molar volume of the components of the complex. Then, defining • as we did
earlier (Gralla et a±., 1974)
2 RT d i n AA
—5
of
5~
dr
Equation (3) can be rearranged to
M x (l - V l P )
•
=
1 + a6t/ei
(6)
1n which the subscripts account for the components assumed to exist 1n the
problem at hand: Component 1 1s free c o l i d n fragment, 2 1s free S I , and
12 1s the equimolar complex. (Only component 2 1s present in the reference
c e l l . ) The quantity fit allows for an absorbance change when components 1
and 2 are mixed:
C
12 " f l
+ e
2
+ 8t
(?
)
and a » c i 2 ^ C l + C12^ 1 s t * ie f r a c t 1 o n °^ colicin fragment present as
complex. Assuming again that the equiraolar complex SIC 1s the only product
of the binding of SI and colicln fragment (C):
SI + C , i S l C
a can be calculated from the e<juat1on
+ KSJ + 1)
[(KC,. + KS,. + I ) 2 - 4K 2 C T S T ] 1/2
which can be derived for a bimolecular association by standard methods. In
Equation (8), Cj and S, are respectively the total molar (input) concentration of colicin fragment and SI.
Equations (6) and (8) are tested by their ability to predict the
experimental variation of v (see Equation 5) with the concentrations S^ and
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Cy, assuming K to be an adjustable variable. All other parameters in the
two equations are known or measureable, or can be estimated from the assumption of a d d i t i v i t y of molar volumes. I f species such as the 1:2 or 2:1
complex are Important in the mixture, the experimental values of • w i l l not
be adequately predicted by the two equations.
Solution Concentrations
The concentration of c o l i d n fragment (M, « 1.75 x 10 ) was determined
using 1.0 as the absorbance at 26Q nm of a 50 pg/ml solution In a 1 cm
path. The concentration of SI protein (M2 • 6.5 x 10 ) stock solutions was
determined using 0.76 as the absorbance at 280 nm of a 1 mg/ml solution
(Laughrea and Moore, 1977). The ratio of molar extinction coefficients at
266 nm needed 1n Equation (6) was estimated to be c 2 /c, - 0 . 1 .
Hyperchromidty Measurements
The hyperchromism of the colicin fragment Interacting with SI protein
was measured on the Cary 14 spectrophotometer. A sensitive slide wire with
an absorbance range from 0-0.20 O.D. was used. A constant c e l l temperature
of 4°C was maintained throughout the experiment by circulating water at
constant temperature through both the thermally-jacketed sample and the
reference c e l l s . Both the colicin fragment and SI protein samples used
were dialyzed against Buffer I I I . During the experiment, Increments of
5-100 ^1 of SI protein was added to sample and reference cells and the
absorbance changes at 266 nm were measured.
RESULTS
T-jump measurements confirm the existence of two double helices 1n
the c o i i c i n fragment. T-jump studies of the melting of RNA helices
t y p i c a l l y show a very fast (a few ^sec or less) relaxation component,
characteristic of the unstacking of bases in single stranded regions, and a
slower (10 \isec or more) component which corresponds to melting of a double
helix. The amplitude of the resolvable component, plotted as a function of
temperature, yields a d i f f e r e n t i a l melting curve for the h e l i x . In simple
systems there is one such transition for each double helix contained 1n the
RNA molecule (Gralla et al_., 1974; Crothers et al_., 1974). We found two
resolvable melting transitions for the c o l i d n fragment, confirming the
existence of two helices 1n the secondary structure.
Figures 2 and 3 show the d i f f e r e n t i a l amplitude and relaxation time
data for the two resolvable relaxation effects observed 1n the melting of
the c o l i d n fragment. The Tra and AH values corresponding to the two effects
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I
I
I
I
I
I
V
I
I
15
b)
10
A A/AT
—I
4^H
1
^—I
1
1
h
I
I
i
I
I
I
10
AA/AT
5
I
0
10 20 30 40 50 60 70 80 90 100
TAVE C O
Figure 2. Differential thermal transition profiles for the coiicin fragment
relaxation effects as determined directly by temperature jump. The amplitudes (6A) for the effect (a) and (b) were obtained from the zero-time
intercept 1n mV on the semi-logarithemic plot and divided by the temperature-jump size. The resultant A A/AT 1s then plotted versus the temperature
corresponding to the m1d-po1nt of the temperature-jump size (4.0 deg-c for
(b) and 1.88 deg-c for (a)). The peaks in the transitions were identified
as the melting+ temperature (Tm). Plot (I) 1s the melting profile in buffer
I (0.05 M Na phosphate, 20 nM sodium cacodylate, pH 7.0); plot I I 1s the
melting profile in buffer I I (0.05 M Na+ phosphate, 20 mM sodium cacodylate
and 5 mM magnesium chloride, pH 7.0).
a and b are reported in Table 1.
The relaxation time constants T shown in Figure 3 support the assignment of each transition to melting of a single helix. Time constants in
the range from 50 to 200 usec are characteristic of RNA helices which close
small hairpin loops (Grail a and Crothers, 1973b; Grail a et_ a±., 1974;
Crothers .et al_., 1974). More complex melting phenomena, including involvement of tertiary structure, usually occur on a slower time scale (Crothers
et^ a K , 1974). Hence the relaxation kinetic data strongly support the
existence of two helical sections in the low-temperature secondary structure
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o.oi
3.60
3.50
3.40
3J0
3.10
3JX> 2-90
2.80
4 * I0 3 CK)
Figure 3. Variation with reciprocal temperature of the relaxation times
for the two relaxation effects exhibited by the colicin fragment. The
relaxation times for each effect were measured at 266 nm and were calculated from the slope of the sem1-logar1them1c plot (Cole and Crothers,
1972).
of the c o l i d n fragment of 16S rRNA.
The assignment of these to the helices
shown 1n the secondary structure diagram 1n Figure 1 1s considered l a t e r .
SI protein and the colicin fragment form a strong 1:1 complex. We
examined the Interaction between SI and the c o l i d n fragment using the
technique of sedimentation equilibrium, with the results summarized 1n
Figure 4.
The quantity ijr, defined by Equation 5, is proportional to the
apparent molecular weight in the solution.
The increase 1n ij> as SI protein
is added to a constant amount of RNA reflects the increasing molecular
weight as complex formation occurs.
The dotted line 1n the figure shows
the expected variation 1n </ i f formation of a 1:1 complex were quantitative,
corresponding to an i n f i n i t e l y large association constant.
The solid line
shows the f i t we obtained by adjusting the association constant K f o r the
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Table 1.
Parameters for melting of the two helices 1n the colicin
fragment of 16S rRKA.
Theoretical ( 4 )
Experimental
Buffer
concentration
(M)
Helix (a)
T 1)
Helix (b)
i
AH/kcal
0.025 (Na+)
13.1
-
64.2
0.050 (Na+)
16.2
30 ( 3 )
69.1
0.075 (Na+)
17.9
-
0.10 (Na+)
19.3
0.05 (Na + ),
0.005 Mg2+
20.8
1.0 (Na+)
30.1 (2)
TmX)
AH/kcal
Helix (a)
m
*H
_
_
_
_
_
_
_
71.6
_
_
_
_
-
73.5
_
_
_
_
-
80.5
_
_
30
84(3)
88.4(2)
84
T
m
*H
_
Helix (b)
T
_
(5)
54(6)
28
56(5)
36(6)
84
87
(1)
(2)
Defined as the maximum of the d i f f e r e n t i a l melting curve, Figure 1.
Extrapolated from the linear variation of T with log Na , as measured
at lower salt concentration.
(3)
(4)
Determined from the width at half height of the differential melting
curve (Graila and Crothers, 1973a,b).
Estimated from the parameters given by Gralla and Crothers (1973a).
(5)
(6)
For structure A,.
For structure A ? .
Sl-col1c1n fragment canplex; the best value was found to be about 5 x 10
A
The plateau value of </ * 2.6 x 10 Indicated 1n Figure 4 corresponds
to H = 8.25 x 10 4 , the expected result for a 1:1 complex. Other s t o i chiometries for the strong complex are not consistent with the data, since </
does not come near the plateau values of 3.4 x 10 calculated for a canplex
A
with 2 c o l i d n fragments per S I , or 4.3 x 10 expected for 2 SI molecules
per c o l t c i n fragment. Hence our data are consistent with a strong 1:1
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s T /c T
0
0.25 O50 a75 1.00
L50
2.30
2.3 -
2.0 l/'/IO
M« 1.73 x 10
J . . . .
I . . . .
I . . . .
I
Figure 4. Sedimentation equilibrium t i t r a t i o n of colicin fragment by
addition of Increasing amounts of SI protein. | , defined by Equation 5, 1s
proportional to the apparent molecular weight 1n the mixture, but lacks the
buoyancy correction term (1 - vp). ST and CT are respectively the total or
Input concentrations of SI and c o l i d n fragment; the upper scale gives the
molar ratio of the two. The values of • expected for molecular weights of
1.75 x 104 (colicin fragment) and 8.25 x 1<T (1:1 complex with SI) are
shown by horizontal lines. The line for K = » shows the expected variation
of f for quantitative complex formation. The solid curve shows the results
predicted by Equations 6 and 8, using v = 0.5 for c o l i d n fragment, as
determined from the measured values of <r 1n absence of S I , and V - 0.738
for SI (Laughrea and Moore, 1977). The partial specific volume of the
complex was estimated from add1t1v1ty of molar volumes of the components.
The hyperchromism of the 1:1 complex was determined to be Jc/c, - 0.035, as
described below. Buffer I I I , 4°C.
complex of s t a b i l i t y constant 5 x 10 M , without significant contribution
from strong complexes of other sto1ch1ometr1es.
We also explored the sensitivity of the complex s t a b i l i t y to added
Mg2+ and varying temperature, with the results shown 1n Figure 5. Reducing
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2.5
2.0
4.0
6.0
8.0
[MgZ+]/mM
Figure 5. Variation of * with T and Mg2+ concentration: 2+ O, 100 mM KC1, 20
mM sodium cacodylate, pH 7.0, with varying amounts of Mg , T ° 4°C;
a, Buffer I I I at varying temperatures. The input ratio of SI to c o l i d n
fragment was 0.5 for the Mg2+ dependence measurements, and 1.0 for the
temperature dependence studies.
2+
the Mg concentration Increases the complex s t a b i l i t y above the value
2+
2+
found in 5 mM Mg (Figure 4). In the absence of Mg the measured value
of </ is equal within experimental error to the result expected for quantitative formation of a 1:1 Sl-col1cin fragment complex. Temperature change
had l i t t l e effect on the apparent molecular weight of the complex.
As a control on the s p e c i f i c i t y of Sl-col1c1n fragment Interaction, we
measured the binding of the c o l i d n fragment to ribosomal protein S14,
wtiich 1s among the farthest removed proteins from the 16S rRNA in the
ribosome assembly map (Nomura, 1973). </ was unchanged upon addition of
S14, indicating no association under the conditions of Figure 4. A similar
negative result was found for the interaction between the control RNA A,U7
and protein S I .
Optical measurements Imply RNA melting and weaker secondary binding
of additional SI protein. We studied the UV absorbance change Induced by
SI protein when 1t Is mixed with c o l i d n fragment, and obtained the results
shown in Figure 6. At the 1:1 molar equivalence point in the t i t r a t i o n ,
Nucleic Acids Research
0
I.I
3.5
0.1
5.6
0.2
8.9
0.3
14.6
0.4
0.5
ml S| added
Figure 6. Absorbance change at 260 nm, AA260. a s a Unction of the volume
of a stock solution of SI protein (13.5 ^M 1n Buffer I I I ) added to both
sample and reference cells. The starting absorbance of c o l i d n fragment
was 0.157 in 1 ml of buffer I I I . The absorbance of the c o l i d n fragment
was corrected for d i l u t i o n by the SI protein stock solution. The molar
ratio of total SI (ST) to total c o l i c i n fragment (Cj) is given on the upper
scale. T = 4°C. The vertical arrow Indicates the 1:1 equivalence point.
the absorbance, corrected for volume Increase, Increased by 3.5%. Continued
addition of SI up to a 15-fold molar excess over c o l i d n fragment produced
a total absorbance increase of 30%, about the value expected for complete
melting of the secondary structure and base stacking in the RNA fragment.
We conclude that the I n i t i a l strong 1:1 complex 1s modified during such a
t i t r a t i o n by weaker binding of additional SI molecules, which further
disrupt the RNA structure. Since SI concentrations of several uM s t i l l do
not saturate this weaker binding, we conclude that the corresponding association constant 1s less than 10 M . This value 1s consistent with the
absence of detectable secondary binding 1n the sedimentation equilibrium
experiment, which focused on small prote1n/RNA Input ratios. The RNA
unfolding observed with the c o l i d n fragment 1s analogous to the melting
generally seen for synthetic and natural RNAs (Szer e£ al_., 1976; Bear £ t
al_., 1976; Kol b et al_., 1977).
SI binding disrupts the weaker colicin fragment double helix. We used
the T-jump method to localize the RNA melting Induced by SI binding 1n the
strong complex. The d i f f e r e n t i a l melting curve for the colicin fragment
helix (a) was measured in the presence of varying amounts of SI protein
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(Figure 7). The T value was unchanged, but the relaxation amplitude
decreased 1n proportion to the molar ratio Sy/C-r of SI to c o l i c i n fragment.
The dotted line in Figure 7 shows the result expected i f quantitative
formation of a 1:1 complex disrupted the less stable helix (a), and caused
I t s melting signal to disappear. Clearly the results are in close agreement
with expectation based on this simple model.
Figure 7 also shows a control experiment 1n which another RNA molecule,
A 7 U 7 , with a T (24°) near that of helix (a), was mixed with Increasing
amounts of SI protein. Only a very small reduction of the peak relaxation
amplitude was observed 1n this case. This finding 1s consistent with much
weaker SI binding to A,U 7 than to the colicin fragment, as we also concluded
from sedimentation equilibrium experiments.
DISCUSSION
Support for the Low-Temperature Secondary Structures Shown in Figure 1A
Our observation of two d i s t i n c t melting transitions for the c o l i c i n
fragment constitutes strong evidence for the existence of two helices 1n
the RNA secondary structure. (Figure 1) As shown 1n Table 1 , both the
enthalpy of melting (84 kcal/ntol) and the Tm (88°C, extrapolated to 1 H
70
0.2
0.4
0.6
O8
1.0
1.2
Figure 7. Peak amplitude of d i f f e r e n t i a l melting curves when SI protein
1s added. O, helix a (see Figures 1 and 2) in the c o l i c i n fragment; Q
A7U7. Buffer I I . The dotted line shows the expected result I f quantitative binding occurs and disrupts helix a.
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NaCl) of the more stable helix (b) are 1n close agreement with the
ties calculated for that helix (&H = 87 kcal/mol, Tm - 84°C) using
parameters collected by Gralla and Crothers (1973a). This r e s u l t ,
addition to the NMR data reported by Baan et al_. (1977), makes the
of helix (b) highly l i k e l y .
properthe
In
existence
Based on I t s enthalpy of melting (30 kcal/mol), the less stable helix
(a) should contain 3-4 base pairs. The most stable predicted structures of
this type are shown as Al and A2. According to the parameters of Gralla
and Crothers (1973a), the two structures are not s u f f i c i e n t l y d i f f e r e n t 1n
s t a b i l i t y to be distinguished theoretically. The relative s t a b i l i t y of
structure A 1s increased because I t contains 2 AU-GC and 1 GC-GC stacks,
whereas structure A2 has the less stable AU-AU stack to accompany the
remaining AU-GC and GC-GC interactions. However, structure Al 1s r e l a t i v e l y destabilized because melting to structure B allows formation of a
new GU pair at the end of the helix (b). These two effects approximately
cancel, leaving the predicted s t a b i l i t i e s for Al and A2 nearly equal.
Our results do not eliminate the p o s s i b i l i t y that the low temperature
structure 1s a mixture of forms such as Al and A2. In p r i n c i p l e , there
should then be a new relaxation e f f e c t , corresponding roughly to e q u i l i b r a tion of Al and A2. However, the absorbance and enthalpy difference between
these two forms would probably be too small to allow detection of the
additional (slower) relaxation signal.
The NMR data of Baan e_t aj_. (1977) did not reveal the existence of the
helix (a). Nonetheless, our results are consistent with theirs because we
measured Tffl values below the temperatures used for their measurements. One
curious anomaly of t h e i r results may result from transient formation of the
bulge loop. They found (at 25°C 1n 5 mM Mg2+) that a terminal AU hydrogenbonded proton resonance from the (b)-hel1x was lost compared to the spectrum
without Mg2+. Since 25°C 1s close to the T (21°C) of helix (a) 1n 5 mM
m
2+
Hg , a significant fraction of molecules would be expected to contain the
bulge loop, and to be in fast NMR exchange with the remainder of the molecules. The stress contributed by the bulge loop to the terminus of helix
(b) 1s l i k e l y to enhance the "fraying" process which i s responsible for
local helix opening and rapid proton exchange. The consequence of t h i s
exchange with solvent protons is broadening and loss of the corresponding
H-bonded proton resonance (Baan e_t a±.. 1977).
Revision of the Bulge Loop Entropy Parameters
The experimental enthalpy of the f i r s t melting t r a n s i t i o n (30 kcal/
2413
Nucleic Acids Research
mol) 1s in good agreement with the theoretical values expected from the
parameters of Gralla and Crothers (1973a) (28-36 kcal/mol). However, the
predicted Tm (54-56°) 1s substantially higher than observed (30°). He
believe that this reflects inaccuracy 1n the estimate of the entropy of
bulge loops. The numbers given by Gralla and Crothers are taken from a
theoretical extrapolation (DeUsi and Crothers, 1971) to large loop sizes
of experimental data for loops of size 1 (Fink and Crothers, 1972), and are
therefore not based on appropriate model compounds.
Our results provide an opportunity to revise the bulge loop parameters
to bring the predicted T results 1n line with experiments. We suggest
upward adjustment of the bulge loop free energies by 2.2 kcal for loops of
2 or more in Table 2 of Gralla and Crothers (1973a). For example, a loop
of size 6 would then have a free energy contribution at 298 K of 7.5
kcal/mol instead of 5.3 kcal/mol. After this adjustment, the predicted T
values for structures Al (29°C) and A2 (34°C) are both 1n reasonable agreement with the value (30°C) resulting from extrapolation of the experimental
data to 1 M s a l t .
Is the Strong SI Protein-RNA Complex Significant?
Our results show that purified SI protein binds strongly to the isolated colicin fragment under conditions which give no detectable binding
for control systems such as S14 protein with c o l i d n fragment, or A7U7 with
SI protein. The loss of the melting signal for helix (a) in Figure 1
indicates that SI protein binds in the region of the bulge loop, and the
observed absorbances Increase upon complex formation at 4°C Indicates that
binding melts helix (a). Thus, the c r i t e r i a of a strong and structurally
localized interaction are satisfied, supporting the conclusion from chemical
crosslinking studies on theribosome (Kenner, 1973; Czernilofsky e.t a l . ,
1975) that there is an interaction between SI and the 3' terminus of 16S
RNA.
On the other hand, physical (Bear et aj_., 1976; Szer ^ t al_., 1976;
Draper e_t a±., 1977; L1 pecky e_t ! ] . . , 1977) and other studies (Tal e_t . a h ,
1972; M i l l e r and Wahba, 1974; Miller et al_., 1974; Carmichael, 1975; Jay
and Kaempfer, 1975; Senear and Steitz, 1976; 6oelz and Steitz, 1977) have
shown that SI protein exhibits strong a f f i n i t y for pyr1midine-r1ch singlestranded regions in RNA. The association constant we measure for the
Sl-col1cin fragment interaction (5 x 106 H" 1 at 4°C, 5 mM Mg 2+ , 100 mM KC1)
Is comparable to the value obtained by Lipecky ejt a h (1977) for the Sl-U 12
complex (9 x 106 M"1 at 4°C, 10raMMg 2+ , 200raMNaCl). Thus we cannot rule
2414
Nucleic Acids Research
out the possibility that the strong Sl-colic1n fragment Interaction 1s an
accidental consequence of the presence 1n the RNA of a pyr1m1d1ne-r1ch
region.
One difference between our results and those of Lipecky et a l . (1977)
pi
deserves comment. We found weaker SI binding as the Mg concentration
was Increased, whereas they found the contrary. Possibly this reflects
Increased stabilization of the bulge h e l i x , whose melting 1s required for
2+
binding as the Mg concentration 1s Increased in our system. The oligo U
molecules studied by Lipecky e_t a l . (1977) are expected to have l i t t l e
secondary structure, and hence Mg should not Interfere with binding
because RNA melting 1s not required. The results reported by Szer et a l .
(1976) on the binding of SI to MS2 RNA showed decreased a f f i n i t y as Hg 2+
was Increased, probably because RNA melting 1s required, j u s t as 1n our
system.
Does the 3' terminus of 16S rRNA Bind SI Protein to the Ribosome?
Draper and von Hippel (1979) have reported that the association constant between SI protein and 30S ribosomal subunits is 2 x 108 M"1 (1n 5 mM
Hg 2 + , 10 mM NH4C1). Not only 1s this value 40 times stronger than we
measure for coiicin fragment-Si interaction, but Laughrea and Moore (1978b)
find that removal of the c o l i c i n fragment from 30S ribosomes does not
appreciably affect the SI binding a f f i n i t y . Hence, 1t seems unlikely that
Interaction with the 3' terminus of 16S rRNA 1s primarily responsible for
holding SI protein on the ribosome.
How many RNA Binding Sites Does SI Have?
Von Hippel and his colleagues have presented evidence favoring the
existence of two polynucleotide binding sites 1n SI protein (Draper et a l . ,
1977; Draper and von Hippel, 1978a; Draper and von Hippel, 1978b). Our
results do not require such a hypothesis, since the strong complex of SI
and colicin fragment has 1:1 stoichiometry. Similarly, the results of
Lipecky et ^1_. (1977) imply a 1:1 complex for binding of Un> 7 < n < 14, to
SI protein. Direct investigation of this question w i l l require experiments
capable of measuring Independently the simultaneous SI binding of two
d i s t i n c t oligonucleotide species.
How Does SI Facilitate I n i t i a t i o n ?
Two general models have been proposed whereby SI protein aids the
binding of natural mRNAs to the E^. c o l i ribosomes during polypeptide chain
i n i t i a t i o n . In both, SI binds strongly to the 30S subunit, either via
protein-protein interactions (Laughrea and Moore, 1978b) or by u t i l i z i n g
2415
Nucleic Acids Research
one of the proposed SI polynucleotide binding sites to recognize some
portion of the 16S rRNA molecule (Draper and von Hippel, 1979). Then,
according to one model, SI uses I t s RNA unwinding a c t i v i t y to disrupt RNA
secondary structures 1n the Incoming messenger RNA (van D1e1jen et a l . ,
1976), a mechanism which might also explain the a b i l i t y of SI to stimulate
chain elongation (L1nde e_t _al_., 1979). In the second model, the RNA
unwinding a c t i v i t y 1s used to bind the polypyrimidine tract at the 3 1 end
of 16S rRNA and f a c i l i t a t e or s t a b i l i z e U s Interaction with the message
(Dahlberg and Dahlberg, 1975).
Since we argue above that SI does not depend on attachment to the 3'
end of 16S rRNA to hold 1t on the ribosome, both models are s t i l l tenable.
Moreover, the finding that NEM-treated SI retains Its a f f i n i t y for 30S
ribosomes but has lost both Us RNA unwinding a c t i v i t y and I t s a b i l i t y to
stimulate I n i t i a t i o n with natural mRNAs (Kolb et al_., 1977) 1s compatible
with either model. Only the fact that SI Is located adjacent to the 16S 3'
end (Kenner. 1973; Czernilofsky et al_., 1975) and the observation that the
protein Interacts 1n v i t r o specifically with the polypyr1tn1d1ne tract within the c o l i d n fragment (Dahlberg and Dahlberg, 1975, and the results
reported 1n this paper) lend some preference to the second model. However,
since helix (a) of Figure 1 melts below physiological temperatures, SI 1s
probably not required simply t o disrupt a secondary structure feature 1n
16S rRNA that Involves the very residues predicted to pair with mRNA.
Rather, the observation that the SI requirement for messenger binding
varies Inversely with the degree of complementarity to 16S exhibited by a
particular I n i t i a t o r region (Steitz e_t .aj_., 1977) suggests that SI somehow
acts to favor the formation of Inter- as opposed to Intramolecular base
pairs by the 16S 31 end. In addition, 1t seems unlikely that SI directly
stabilizes the mRNA-rRNA duplex once formed, since SI has no apparent
preference for double-stranded regions 1n RNA (Szer e_t &±., 1976; Bear e_t
a l . , 1976). In conclusion, while a l l data argue that the RKA unwinding
a c t i v i t y 1s both available and central to SI function on the ribosome, the
exact molecular mechanism whereby this a c t i v i t y serves to f a c i l i t a t e mRNA
binding remains to be elucidated.
ACKNOWLEDGEMENTS
This research was supported by grants Al 10243 t o J.A.S. and GM 21966
t o D.M.C. R.C.Y. was supported by a Rudolph Anderson Postdoctoral Fellows h i p . We thank C. Stocking ar.d J . Wei i k y f o r technical assistance.
2416
Nucleic Acids Research
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