Biochimica et Biophysica Acta, 741 (1983) 23-29 23 Elsevier
BBA 91263
THE EFFECT OF IONIC AND TEMPERATURE SHIFTS USED FOR IN VITRO RIBOSOME SUBUNIT RECONSTITUTION UPON THE LARGE MOLECULAR WEIGHT RIBOSOMAL RIBONUCLEIC ACIDS
JOHN SYKES and EMILIA METCALF
Department of Biochemistry, University of Sheffield, Sheffield SIO 2TN (U.K.)
The behaviour of purified, intact preparations of 16 S and 23 S rRNAs has been studied in their respective ribosome subunit reconstitution systems by means of sedimentation and electrophoretic analysis. Both species undergo profound conformation changes to more compact species at the temperatures and ionic conditions commonly agreed for their reconstitution into ribosome subunits. The 16 S rRNA undergoes a complete conformation change over the input range of concentration used, whereas the change for 23 S rRNA is incomplete for inputs above 1.5 mg • ml - i. Intact 23 S rRNA is also required and some preparations recommended for r-protein binding studies do not meet this requirement for reconstitution. These observa- tions are discussed in relation to the overall effectiveness of ribosome subunit reconstitution systems.
Introduction
The total reconstitution in vitro of ribosome subunits from r-proteins and r-RNA is an im- portant experimental system for the study of ribo- some structure, assembly and topology. The sim- ple one-step in vitro reconstitution Of the 30 S ribosome subunit from Escherichia coli has been widely exploited in many laboratories [1]. An equally effective and widely-adopted in vitro re- constitution system for the E. coli 50 S subunit has been more difficult to define [2,3]. The original procedure [4] for Bacillus stearthermophilus 50 S subunits did not prove to be generally applicable and the single-step reconstitutions [5,6] proposed for E. coli 50 S have not been confirmed [7]. A two-step reconstitution for this subunit appears mandatory although the efficiency of the original procedure [2] for E. coli was recently doubted by Amils et al. [8] who claimed, whilst using very similar ionic and temperature shift conditions, that 'less destructive techniques' were required for the
preparation of the rRNA and r-proteins. The dif- ferences between these two procedures have now been resolved [9] and do indeed centre on the mode of preparation of the r-proteins and rRNA rather than the minor differences in reaction con- ditions. The preparative procedures adopted by Nierhaus and Dohme [2,3,10] give reconstituted particles of higher functional activity.
The behaviour of rRNA species and r-proteins in solution and in buffers approximating those used in subunit reconstitutions has been well documented. Furthermore the systematic influence of pH, cation concentration, temperature, time etc., on the overall reconstitution process have all been examined for both 30 S [1,11] and 50 S [2,3,10]. However, the behaviour of the rRNA in the cation- and temperature-shifted circumstances now widely adopted for the one-step 30 S and two-step 50 S reconstitution has not been fully defined. This is clearly of importance since it is well known that rRNAs are extremely flexible macromolecules profoundly, and often reversibly
influenced by the ionic environment [12-14]. Fur- thermore, in both reconstitution conditions [1,2] and non-reconstitution conditions [15] the confor- mation of the rRNA (or assembling particle) and the prevailing temperature undoubtedly influence the binding of the r-proteins to the rRNA.
This paper is therefore concerned with the ef- fect on intact, purified preparations of the large molecular weight rRNA species from E. co# of the ionic conditions and temperature shifts now gener- ally agreed to be optimal for ribosome subunit reconstitution. The experiments reveal that in the appropriate reconstitution systems the 16 S and 23 S rRNA species adopt compact configurations and in the case of 23 S rRNA the change at the optimum temperature from a partially unfolded to a condensed form is incomplete at the adopted levels of RNA input and ionic conditions. In the case of 16 S rRNA in the 30 S reconstitution this change is complete.
Methods
Growth and isolation of ribosome subunits. Escherichia coli A19 (Hfr, rel, met, ms) was grown in aerated glucose mineral salts medium plus methionine as described by Dean and Sykes [16].
The harvesting and the breakage of the bacteria and the methods for the bulk isolation of the 30 S and 50 S ribosome subunits using cycles of dif- ferential and zonal ultracentrifugation were as de- scribed by Sykes et al. [17]. Particular care was taken to exclude other nuclease activity by using sterilized glassware, rotors, feed lines etc., at every stage during the preparative procedures. In addi- tion all reagents were made using sterilized, glass- distilled water. The buffered sucrose solutions for the zonal centrifugations were made by dissolving the quantity of sucrose required in half the final volume of sterilized, distilled water with the addi- tion of 0.2% (v /v) diethylpyrocarbonate [18]. The solution was stirred overnight at room temperature and then boiled for 10 rain to remove the dieth- ylpyrocarbonate. This solution was mixed with an equal volume of double-strength TMN (buffer 1) [17] and the final sucrose concentration checked with a refractometer.
Extraction of rRNA. The rRNA was extracted from pooled zonal ultracentrifugation samples
containing the 30 S particle peak essentially as described by Dean and Sykes [16] and Sykes et al. [17]. However all apparatus used was sterilized and where appropriate all reagents were made in sterilized, distilled water.
The rRNA was extracted from pooled zonal ultracentrifugation samples containing the 50 S particle by first making the samples 1% (w/v) with sodium dodecyl sulphate (SDS) and 3% (v/v) with respect to diethylpyrocarbona~e before extracting the rRNA as described above. It was found neces- sary to take these additional precautions and also to repeat the phenol extraction two further times in order to ensure the absence of nuclease activity in the final preparation. The rRNA preparations from 50 S subunits were much more susceptible to degradation than those from 30 S and greater care was required in their preparation to remove all traces of nuclease activity and ensure a prepara- tion which remained stable throughout the recon- stitution procedures. All the final rRNA prepara- tions were stable stored under ethanol at - 2 0 ° C until required. Certain preparations of the rRNA species were made by the method described by Hochkeppel et al. [19]. The concentration of RNA used in these experiments was determined spectro- photometrically assuming a value of EI~,, at 260 nm of 227 [20].
30 S and 50 S subunit reconstitution conditions. The buffers used, level of 16 S rRNA input and single-step incubation etc., for the 30 S subunit reconstitution were exactly as described by Traub et al. [21], except that r-proteins were not added to buffer solution V since examination of the rRNA under total reconstitution conditions was required and not total reconstitution.
Similarly the buffers used, level of rRNA input and two-stage incubation procedure for 50 S re- constitution were as described by Nierhaus and Dohme (Ref. 10, see also Ref. 9) again omitting only the r-proteins in order to study the effect of these reconstitution conditions on the rRNA alone. It should be noted that, in agreement with Nowotny et al. [9], pure 23 S rRNA from isolated 50 S subunits (see above) was used in these experi- ments.
Polyacrylamide gel electrophoresis of rRNA. Electrophoresis was performed as described by Dean and Sykes [16].
Sedimentation analyses. These were performed at 20°C in a Beckman-Spinco analytical ultra- centrifuge equipped with an R.T.I.C. unit with schlieren and ultraviolet optical systems. Parts of the analytical cells coming into contact with the preparat ions were cleaned by treating in distilled water for 10 min at 90°C and rinsing in sterilized, distilled water. Fresh, sterile, disposable syringes were used for introducing each sample into the analytical cell.
Results
The conformation of "16 S' rRNA during 30 S subunit reconstitution
The sedimentation characteristics of the r R N A isolated f rom the 30 S r ibosome subunits as de- scribed in Methods in 0.01 M aceta te /1 M NaC1, p H 4.6 and in 10 m M Tris-HC1, p H 7.8/0.3 m M MgC12/30 m M N H 4 C 1 / 6 mM fl-mercaptoethanol (buffer 1) are shown in Fig. la. The extrapolated
0 sedimentat ion constants (s20,w) are 15.07 S in the ace ta te /NaC1 buffer and 16 .44 S in buffer 1.
b
2 5 " ~ '
o X ~ 20
u 15 g
101 , , • ~ ~o 1 2 3 4 5 6 1 2 - - 3 4 5
RNA mg m1-1
Fig. la. Sedimentation coefficient vs. concentration for rRNA isolated from 30S ribosome subunits as described in Methods: (O I) in 0.01 M acetate/0.1 M NaC1, pH 4.6 (s20,,,0 = 15.07 S) and (Q Q) in this buffer after incubation in 30 S reconstitution buffer and precipitation with ethanol. (11 I) in buffer 1, pH 7.5 0 (s20.w =16.44 S) and (A A) in 30 S reconstitution buffer (s2°0.w = 21.36 S). b. Sedimentation coefficient vs. concentration for large molecular weight rRNA isolated from 50 S ribosome subunits: (O e) in 0.01 M acetate/0.1 M NaCI/pH 4.6 (S2o, w ° _- 22.41 S) and (Q ®) in this buffer following incubation in 50 S reconstitution buffer and precipitation with ethanol. (11 II) in buffer 1, pH 7.5 (S°o,w = 22.0 S) and (A ~,) in 50 S reconstitution buffer.
25
These values are within the range of those reported for a similar preparat ion f rom E. coli B in the a c e t a t e / N a C l buffer (16.3 S) by Kurland [20], and an unfract ionated r R N A preparat ion from E. coli W3101 (17.5 S) in 0.05 M Tr i s -HCl /0 .1 M KC1, p H 7.0 [13]. However, unlike the preparat ion f rom E. coli B [20] the '16 S' r R N A prepared by the methods described here did not degrade when heated for 10 min at 60°C in either ace ta te /NaC1, p H 4.6 or buffer 1, p H 7.5.
It is concluded that the method of preparat ion descr ibed gives a stable, covalent ly intact, nuclease-free sample of '16 S' r R N A from the E. coli A19 30 S r ibosome sub-unit. This is confirmed by the observations made during the subsequent reconsti tution experiments.
The sedimentat ion behaviour of this r R N A in
Fig. 2a. Analytical ultracentrifuge pattern for rRNA from 30 S ribosomes in 0.01 M acetate/0.1 M NaCI, pH 4.6. Concentra- tion 3.2 mg. ml-i, 24 min at 59 780 rev. min-1. Sedimentation from right to left (see Fig. la). b. Analytical ultracentrifuge pattern for rRNA from 30 S ribosomes after incubation in 30 S reconstitution buffer. Concentration 2.6 mg.m1-1, 24 min at 59780 rev.min -t (see Fig. la). c. Analytical ultracentrifuge pattern for rRNA as in 2b above, precipitated with ethanol and then resuspended in 0.01 M acetate/0.1 M NaC1, pH 4.6. Concentration 2.4 mg.ml-l 24 min at 59780 rev.min-l (see Fig. la and c.f. Fig. 2a).
26
F 2 7 ~
2.1
c~ uJ 1 5
8 x~
to x~ 0 3
2 . 7
21
1 5
0 9
0 3
i J _ b
1 2 3 4 5 6
D i s t a n c e m i g r o t e d ( c m )
Fig, 3. Absorbance profiles at 260 nm for polyacrylamide gel electrophoretic separations for 5 h at 5 mA/gel , 20°C for: a. 15.07 S rRNA in 0.01 M acetate/0.1 M NaCI, pH 4.6 (see Figs. la and 2a). 25 tlg RNA loaded, b. 21.36 S rRNA after incubation in 30 S reconstitution buffer (see Figs. la and 2b). 27 t~g RNA loaded, c. As 3b above then precipitated with ethanol and resuspended in 0.01 M acetate/0.1 M NaCI, pH 4.6.27 beg RNA loaded.
the 30 S ribosome subunit reconstitution system described by Traub et al. [21] at the conclusion of the 30 min incubation period at 40°C is also shown in Fig. la. It is apparent by comparing the plots in Fig. la that the sedimentation characteris- tics have altered markedly during the incubation for reconstitution and the extrapolated sedimenta-
0 = 21.36 S. Fig. 2a and b, tion constant is now S2o, w respectively, show typical analytical ultracentri- fuge schlieren diagrams for these preparations each revealing a single, sharp sedimenting boundary. In the case of the '16 S' rRNA, incubated for the
stipulated time in the 30 S reconstitution buffer (Figs. 1 and 2b), precipitation of this with ethanol and resuspension in 0.01 M acetate/0.1 M NaCI, pH 4.6, completely restored the original sedimen- tation characteristics as shown in Fig. la and 2c.
The corresponding polyacrylamide gel electro- phoresis profiles to this sedimentation behaviour are shown in Fig. 3. In sharp contrast to the profound alteration in sedimentation properties it is evident that the preparation behave identically in gel electrophoresis. Polyacrylamide gel electro- phoresis does not distinguish between RNA con- formers [24].
From these results it is concluded that the in- tact, stable '16 S' rRNA, isolated from 30 S ribo- somes via the phenol procedure, undergoes a sharp and complete conformation change after ap- propriate incubation in the 30 S subunit recon- stitution system [21] to yield a conformer which, judged by the 41.7% increase in sedimentation constant and reduced dependence upon concentra- tion, is considerably more compact that the origi- nal preparation in acetate/NaC1 or buffer 1. This conformation change is completely reversible since precipitation of the rRNA after incubation and then resuspension in ace ta te /NaCl buffer restores the original sedimenting species (Fig. la). The identity of migration of all the preparations in gel electrophoresis (Fig. 3) confirms this conformation change in the absence of molecular size change.
The conformation of 23 S rRNA during 50 S ribo- some subunit reconstitution
The sedimentation vs. concentration depen- dence of the large molecular weight rRNA isolated from 50 S ribosome subunits using the procedures described in Methods is shown in Fig. lb. The extrapolated sedimentation constants for this
0 = 22.41 S in 0.01 M acetate/0.1 M rRNA are Szo, w NaC1, pH 4.6 and Szo, w ° = 22.0 S in buffer 1, pH 7.5. This preparation of '23 S' rRNA was stable to 10 min heating at 60°C in both buffers although following heat treatment in buffer 1 the rRNA at a concentration of 2 mg. ml - ] appeared to be poly- disperse. This was not due to degradation since precipitation with ethanol and resuspension in 0.01 M acetate/0.1 M NaC1, pH 4.6 restored the single, sharp sedimenting species as shown in Fig. 4b. Nonetheless it is to be noted that stable, covalently
Fig. 4. Analytical ultracentrifuge patterns, sedimentation is from fight to left, for rRNA prepared from 50 S ribosomes: a. in 0.01 M acetate/0.1 M NaCI, pH 4.6. Concentration 2.5 mg. ml- 1, 8 min at 59780 rev. rain- J (see Fig. lb and 5a). b. in 0.01 M acetate/0.1 M NaCI, pH 4.6 after incubation in 50 S reconstitution buffer and precipitation with ethanol (see Figs. lb and 5c). Concentration 2.5 mg.m1-1, 8 min at 59780 rev. min-1, c. in 50 S reconstitution buffer after incubation. Concentration 1.12 rag. ml-I, 8 min at 59 780 rev. min -I. d. in 50 S reconstitution buffer after incubation. Concentration 2.5 mg.ml-I, 8 min at 59780 rev-min-1 (see Fig. lb and 5b).
intact, nuclease-free preparations of '23 S' rRNA, as described, were generally more difficult to achieve than for the '16 S' rRNA. Preparations of 23 S rRNA incompletely resolved from nucleases or containing 'hidden breaks' rapidly degraded under reconstitution conditions. This difficulty, and its possible contribution to the relative ef- ficiencies of different 50 S reconstitution systems has been noted by Amils et al. [8]. Alternative methods of preparing rRNAs to enhance their interaction with r-proteins have been proposed [19]. However, the 23 S rRNA prepared by the latter method was found to be degraded during the incubations required for 50 S reconstitution and the 16 S rRNA prepared by this method was insoluble in the 30 S reconstitution buffers even after incubation at 40°C for 5 min. It is therefore
27
concluded that the alternative rRNA preparations described by Hochkeppel et al. [19] are not suita- ble for reconstitution experiments.
Fig. 4c and d show the sedimentation pattern at two different concentrations for the stable, cova- lently intact preparation of '23 S' rRNA, prepared according to Methods, after incubation in the 50 S reconstitution system [!0]. Fig. lb shows the sedi- mentation vs. concentration dependence of this rRNA in the reconstitution system. The sedimen- tation behaviour and the nature of the preparation is clearly altered in a complex manner during the incubation for reconstitution. The sedimentation vs. concentration plot (Fig. lb), by comparison with that in acetate/NaC1 and buffer 1, reveals a considerably lowered dependence of sedimentation upon concentration and an elevated range of sedi- mentation coefficients. These are both consistent with the rRNA adopting a more compact config- uration in the 50 S subunit reconstitution buffer compared with the conformation in buffer 1 or acetate/NaC1 buffer. This conformation change, in the absence of any molecular weight change, is confirmed by the identity of the polyacrylamide gel electrophoresis profiles (Fig. 5) of the original samples, the samples in the reconstitution system and those obtained by precipitation from the re- constitution system with ethanol and resuspended in acetate/NaC1 buffer. Precipitation by ethanol from the reconstitution mixture, followed by resus- pension in acetate/NaC1, pH 4.6, buffer also res- tores the original sedimentation profile (Fig. 4a and b) and characteristics (Fig. lb). The extent of configurational change is however dependent inter alia upon the level of rRNA input. This is revealed by the wide scatter of sedimentation coefficients over the range of concentration of '23 S' rRNA (Fig. lb) and the sedimentation patterns which at concentrations below approx. 1.5 mg. ml- ~ are apparently monodisperse and clearly polydisperse at higher levels of rRNA input (cf. Fig. 4c and d). These conformation changes in '23 S' rRNA, al- though reversible as shown above (Figs. 5 and lb), are therefore incomplete in the 50 S reconstitution system, particularly compared with those noted for '16 S' rRNA above. The extent of conformation change depends upon the concentration of rRNA, the ionic conditions (particularly the level of Mg 2 +) and the degree of equilibration of the rRNA with
28
2 7
2:1
15
o g
0 3
i ~ i ~ " ~ l ~:r -
2 7
21
~7~5
c 0 9
o ~ o3
2 7
1.5
o g / J
o~
1 2 3 4 5 6 7 Distance migrated (cm)
Fig. 5. Abso rbance prof i les at 260 nm for po lyac ry lamide gel
electrophoresis separations of rRNA from 50 S ribosomes for 5 h at 5 mA/gel, 20°C: a. rRNA in 0.01 M acetate/0.1 M NaC1, pH 4.6 (see Fig. 4a and lb). 25 t~g rRNA loaded, b. after incubation in 50 S reconstitution buffer (see Fig. lb and 4 d). 22.5 ~g rRNA loaded, c. as 5b above then reprecipitated with ethanol and resuspended in 0.01 M acetate/0.1 M NaCI, pH 4.6. 22.8/lg loaded.
the environment [14]. The two-phase 50 S recon- stitution system generally used [9] clearly does not permit this equilibration since on initial mixing it achieves an ionic environment of 20 mM Tris-HC1, pH 7 .6 /4 -7 .5 mM magnesium aceta te /400 mM ammonium chloride/0.02 mM EDTA disodium s a l t / 2 mM fl-mercaptoethanol and after 20 min incubation at 44°C the magnesium ion is raised to 20 mM followed by further incubation at 50°C. The observations here show that although this results in considerable configurational change in the rRNA it is incomplete and the incubation
conditions do not permit equilibration of the '23 S' rRNA with the environment, particularly at higher rRNA inputs. The rRNA therefore exhibits a variety of complex sedimentation behaviour (Fig. l b) reflecting this and not amenable to detailed analysis since there are slow components sedi- menting unresolved in the presence of faster com- ponents and the further possibility of some aggre- gation of the rRNA in the reconstitution buffer with the elevated levels of Mg 24 cannot be eliminated by the data.
Discussion
The 16 S rRNA prepared by the phenol proce- dure used in these experiments is intact, nuclease- free and during the course of the one-step incuba- tion under 30 S sub-unit reconstitution conditions undergoes rapid, pronounced and complete con- formational change to a considerably more com-
0 = 21.36 S. Not pact species sedimentating at Szo.w unexpectedly this process is completely reversible on reprecipitation and taking the rRNA up at low temperature in 0.01 M acetate/0.1 M NaCI, pH 4.6 buffer. The complete adoption of this compact configuration by all the input 16 S rRNA in the ionic and temperature conditions of the reconstitu- tion mixture must be related to the known depen- dence of the association of the r-proteins with the rRNA upon temperature and conformational change in this assembly process [1]. Similar ob- servations regarding this conformational change to
0 = 21.36 S species have been made [22,23]. a s20,w
In contrast, 16 S rRNA prepared by the acetic acid method of Hochkeppel et al. [19] is ap- parently less stable than the phenol preparations and although suitable for r-protein binding studies is not suitable for the prolonged incubations of reconstitution experiments.
In the two-step procedure for the reconstitution of 50 S, where the Mg 2+ is raised to 20 mM and the temperature from 40 to 50°C in the progress of the reaction, the 23 S rRNA undergoes a similar conformation change to a more compact species, although at levels of rRNA input above 1.5 mg- ml-~ this change is incomplete. This incomplete conversion of the rRNA is clearly dependent inter alia on the the input of rRNA vis fi vis the effective Mg 2+ level in the reconstitution system
and this factor may be re levant to the overal l eff iciency of the reconst i tu t ion. This would be a fur ther factor in the d iscrepancies no ted [9] be- tween the Ami l s et al. [8] and Nie rhaus and D o h m e [10] recons t i tu t ion systems, since the former group used r R N A from 70 S r ibosomes (i.e., 23 S plus 16 S) as total input R N A , whereas the la t ter used 23 S r R N A only as in the present exper iments .
The efficiency of r ibosome subuni t reconst i tu- t ions is usual ly j u d g e d by the bas ic res tora t ion of the physical , chemical and funct ional p roper t ies of subuni t s bu t rare ly by the quant i ta t ive yield of recons t i tu ted par t ic les f rom the input levels of r R N A and r-proteins . By compar i son with the 30 S recons t i tu t ion a factor in the more complex 50 S reconst i tu t ion , as present ly adopted , may be the need to ensure that the total react ion condi t ions pe rmi t a comple te confo rma t iona l change of the 23 S r R N A and this is clearly dependen t upon the inpu t level of r R N A in add i t ion to the prevai l ing ionic env i ronment and temperature . The presence of r -pro te ins in the comple te recons t i tu t ion system m a y also assist in pul l ing the equi l ib r ium over to a more un i fo rm con fo rma t ion a l though this is not the case in the 30 S system. Schulte et al. [15], a l though not using a full recons t i tu t ion system, observed that a cr i t ical and marked b ind ing de- pendence is shown between r -pro te in L24 in the M g 2+ range 1 0 - 3 - 1 0 -2 M.
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29
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