Retention of ribosomal ribonucleic acids in agarose gels

Biochimica et Biophysica Acta, 308 (1973) 317-323

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97650

R E T E N T I O N O F R I B O S O M A L R I B O N U C L E I C ACIDS IN A G A R O S E GELS

S. L. PETROV1C, J. S. PETROVIC* and M. B. NOVAKOVI(~ Department of Biochemistry, Boris Kidrich Institute, p.o. box 522, Beograd (Yugoslavia)

(Received December 8th, 1972)

SUMMARY Ribosomal RNAs from various organisms could be immobilized in agarose gels at high ionic strength in a thermally reversible fashion. In NaCI solutions at 23 °C, agarose capacity for retention o f liver rRNAs was about 0.4 mg/ml gel under saturating conditions. NaC1 molarities necessary for a 50 ~o retention of rat liver rRNAs increase with temperature (in the range of 23-37 °C) by 0.04-0.05 M/degree for the larger (28-S) component, and by 0.07-0.09 M/degree for the smaller (18-S) component. The retention process also displays cation selectivity; for rat liver 28-S RNA, molarities of alkali metal chlorides at 50 ~ retention and 23 °C were: LiC1 and NaCI, about 0.4 M; KC1, about 0.6 M; CsCI, about 0.9 M. The retention molarities of NaCI tend to decrease with increase in both the molecular weight and the G + C content ofrRNAs, but the relationship of these variables is clearly non-linear, and it might depend on undetermined features of r R N A structure. The retention profiles appear to be largely characteristic of respective species of rRNAs.

INTRODUCTION In a recent communication 1, we have described conditions resulting in selective retention o f the larger r R N A o f rat liver in agarose gels. This work has been further extended to include other r R N A species and experimental variables. In this paper, we present detailed evidence that ribopolynucleotide retention in agarose gels results from a thermally reversible and cation-specific association which is in many aspects similar to gelation. The retention process appears to be dependent on both the molecular size and the structure of rRNAs. MATERIALS AND METHODS Labeling and isolation o f r R N A s

In view of the need I or accurate quantitation at low polynucleotide concen* Institute for Biological Research, Beograd, Yugoslavia.

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tration, majority of retention experiments were done with uniformly labeled rRNAs. These were prepared as follows. Pea seedling rRNAs. These were isolated from 3-day-old Alaska variety seedlings. The seeds were imbibed in distilled water containing carrier-free Na2H32po 4 (10/~Ci/ml; > 1000 Ci/g P; Department of High Activity, Boris Kidrich Institute), and soaked in the same solution throughout the germination period. The harvested seedlings were promptly processed as described by Huang and Bonner 2 up to the stage of the post-chromatin supernatant. This supernatant was brought to 7 mM streptomycin sulfate3, and the precipitated ribosomal material collected by centrifugation. It was then dispersed in 2.5 Y/oosodium dodecyl sulfate-0.05 M sodium maleate, pH 5.6 (ref. 4), and 4-fold deproteinized with phenol. Escherichia coli rRNAs. These were isolated from strain B cells grown for 2.5 generations in a minimal medium containing 10 pCi [32p]orthophosphate or 0.1 #Ci [a4C]uracil per ml, and 9 g total phosphates per 1. Harvested cells were thoroughly homogenized in sodium dodecyl sulfate-maleate at room temperature. Deproteinization was carried out as above, and the separation of DNA from rRNAs by salt fractionation5. Rat liver rRNAs. Rat liver rRNAs were isolated essentially as described for microsomal RNAs 4. Labeling with 32p was done with 1-2 mCi of intraperitoneally injected precursor per animal, for at least 48 h. Ethanol-precipitated RNAs were in each case collected by centrifugation, dissolved in 0.1 M NaCI-0.1 ~ sodium dodecyl sulfate-2.5 mM EDTA, pH 7.4--0.02 M Tris-HC1, pH 7.4 (saline-detergent buffer), and freed from 5-S rRNA and transfer RNA by gel filtration on Sepharose 4B6. This treatment reduced the amount of residual protein, as measured by the method of Lowry et al. 7 and micro-biuret a method, to less than 1.2 ~o (w/w) in all RNA preparations used in this study. Sucrose gradient separations Preparative sepaiation of purified rRNAs was done in an SW 25 rotor of Spinco centrifuge, using linear 10-40 ~o sucrose gradients (made in the saline-detergent buffer) at 20 °C, and centrifuging for 24-25 h at 21 000 rev./min. Agarose gels Sepharose 2B (lot 4680) and Sepharose 4B (lot 4088) were purchased from Pharmacia, Uppsala, as hydrated gels ready for use. Agarose A 37 (batch F 9509) was obtained from L'Industrie biologique francaise, Villeneuve-La-Garenne; Miles agarose (batch 505 A) was supplied by Miles-Seravac, Maidenhead, both in dry form. Agarose powders were suspended in the saline-detergent buffer and heated in a boiling water bath until clear, to prepare 2 or 4 700solutions. After cooling, the gel cakes were blended with an excess of the saline-detergent buffer, to produce columnoperable granules. The granulated gels were packed in jacketed glass columns supported by porous teflon discs (LKB Produkter, Stockholm). Flow rates necessary for reproducible RNA retention patterns at any given temperature and salt molarity were below 1 ml solvent per h per ml gel bed. The gel columns were prior to use washed with two to three bed volumes of the corresponding salt solution buffered to pH 7.4. Temperature control in the range of 25-37 °C was effected to better than 0.5 degrees.

RNA IMMOBILIZATION IN AGAROSE

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Chemicals LiCI was prepared from reagent grade Li2COa by neutralization with HC1. NaC1, KC1 and CsC1 were all analytical grade chemicals. 2.5-M or 5.0-M solutions were made from thoroughly dried salts, and their actual molarities routinely checked by refractometry before making suitable dilutions. RESULTS

General features of rRNA retention in agarose 9els Capacity for r R N A retention was studied with commercial agarose-gel preparations, as well as with gels prepared from dry commercial agarose, using unfractiohated rat liver rRNAs. All examined gel preparations gave essentially similar r R N A trapping patterns, reaching a capacity of 0.36-0.41 mg r R N A per ml gel between 1.1 and 1.4 M NaC1 at 23 °C (Fig. 1).

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Fig. 1. Capacity of agarose gels for retention of unfractionated rat liver rRNAs. (a), Sepharose 2B; (b), Sepharose 4B; (c) Agarose A 37, 2 % gel; (d) Miles agarose, 2 % gel; (e) Miles agarose, 4 % gel (for further information concerning the above gels refer to Materials and Methods). Numbers above bars refer to NaC1 molarity employed to effect retention. 5.0-ml gel columns were used. Retention of r R N A s at suitable salt molarities was unaffected by their concentration in the range of 0.01-1 mg/ml influent, provided the flow rate was kept sufficiently low. It was thus possible to concentrate r R N A s from rather dilute solutions (e.9. those ensuing from methylated albumin kieselguhr chromatographyg). Native D N A in concentrations up to 0.4 mg/ml influent did not noticeably decrease immobilization of r R N A at 2.0 M NaC1 (Fig. 2). The native D N A itself, however, was not retained in the gels to any significant extent (Fig. 2). Liver r R N A retained in agarose gels could not be released by an extensive washing of gel columns with 1.0-1.5 M NaC1, or by resuspension of gels containing trapped R N A in such solvents. It could be completely recovered by either eluting with 0.01-0.2 M NaC1 at p H 7, or by raising the elution temperature to 37 °C (see also the next section and Fig. 3), using NaCI molarity which at 23 °C causes retention of r R N A in the gels.

Dependence of rRNA retention on temperature and species of alkali metal cation The temperature dependence of r R N A retention was further examined in some detail for the two kigh-mol, wt r R N A s from rat liver. As seen in Fig. 3 and Table I,

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Fig. 2. Independence of rRNA retention on the presence of native DNA. Sepharose 2B, 10 mlcolumn in buffered 2 M NaCI, 5.0-ml fractions. After Fraction 5, NaC1 concentration changed to 0.1 M. Samples: 0.11 mg unfractionated rat liver rRNA uniformly labeled with. [6-14C]orotic acid, with A, 0.14 rag; B, 0.28 mg, and C, 0.70 mg native rat liver DNA, in 2.0 ml 2 M NaCI each. The non-retained portion of rRNA amounted to 7-8 ~ of total radioactivity in all columns. TABLE I D E P E N D E N C E ON T E M P E R A T U R E OF NaCI MOLARITIES AT 50 ~ RAT LIVER rRNAs

RETENTION OF

Values obtained by graphical extrapolation from the data plotted in Fig. 3, which were taken with 10-ml columns of Sepharose 2B using rRNAs separated by density gradient centrifugation (see the caption to Fig. 3). Molarity o f 50 % retention 23 °C

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in the range of 23-37 °C the NaC1 molarities necessary for r R N A retention show a slightly saturating increase for both polynucleotides. In any case, the smaller r R N A is trapped at somewhat lower molarities of NaC1 than would be predicted from the behavior of the larger r R N A if the retention molarities of NaCI linearly decreased with increasing molecular weight. At 23 °C, the smaller rRNAs of two non-vertebrates displayed even more pronounced behavior of this type (Fig. 5). To learn whether the immobilization process depends on cation species employed, retention of the larger r R N A from rat liver was studied in solutions of four alkali metal chlorides. As shown in Fig. 4, the r R N A retention molarity ranges of these cations did significantly differ, with LiC1 being the most efficient, and CsC1 the least efficient effector of R N A retention. Ratios of molarities of alkaline chlorides at 50 ~ retention of this R N A (Table II) showed some correspondence with ratios of

RNA IMMOBILIZATION IN AGAROSE

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Fig. 3. Retention of rat liver rRNAs on Sepharose 2B as dependent on NaCI molarity and temperature. 10-ml gel columns with 0.1-0.3 mg [U-a2PIRNAs isolated by density gradient centrifugation as described under Materials and Methods. 0 - 0 , 28-S RNA, 23 °C; O - O , 28-S RNA, 30 °C; O - - - O , 28-S RNA, 37°C; x - x , 18-S RNA, 23°C; A - A , 18-S RNA, 30°C; x - - - x , 18-S RNA, 37 °C. Fig. 4. Retention of rat liver 28-S rRNA in Sepharose 2B as dependent on molarity of various alkali chlorides (RCI). 10-ml gel columns with 0.2-0.3 mg 28-S r[U-32PlRNA, isolated by density gradient centrifugation as described under Materials and Methods. O - O , LiCl; 0 - 0 , NaCI; O - - - O , KCI; O - - -O, CsCI. TABLE II MOLARITIES OF ALKALI METAL CHLORIDES AT 50 % RETENTION OF RAT LIVER 28-S RNA AT 23 °C

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Molecular properties o f r R N A s and their retention in agarose gels W h e n highly purified, gradient-separated r R N A s of several organisms were i m m o b i l i z e d f r o m NaCI solutions at 23 °C, the observed r e t e n t i o n ranges (Fig. 5) showed certain dependence o n molecular weight. This dependence was neither linear, n o r strict. Thus, 18-S r R N A from rat liver, h a v i n g considerably lower molecular weight t h a n either E. coli 23-S R N A or pea 25-S R N A 11, reached either 50 % or s a t u r a t i n g r e t e n t i o n at lower NaCI molarities. A possible dependence o f retention molarity o n G + C c o n t e n t could be inferred from the fact that the two rat liver r R N A s , which differ very considerably in G + C

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Fig. 5. Retention of rRNAs from various organisms on Sepharose 2B at 23 °C, as dependent on NaC1 molarity. 10-ml gel columns with 0.1-0.3 mg r[U-32P]RNAs isolated by density gradient centrifugation as described under Materials and Methods. Q-O, 28-S RNA from rat liver; O-O, 18-S RNA from rat liver; x - x , 25-S RNA of pea seedlings; 0 - - -0, 23-S RNA from E. coli; × . . . . x, 17-S RNA of pea seedlings; 0 - - - O , 16-S RNA from E. coli. content 1J2, show the largest difference in retention molarities. Members of the two invertebrate R N A pairs, with much less pronounced differences in G + C content ~3,14 showed much closer spacing of retention molarity ranges (Fig. 5). DISCUSSION

Gel-like aggregation of rRNAs at high ionic strength has been used for many years to effect their separation from D N A and tRNA, or to attempt their fractionation s, is, 16. It is possibly linked to both the high molecular weight of rRNAs and alternation of helical and nonhelical parts in their molecules 17,18. The RNA-agarose association probably shares certain features with such an aggregation. However, the capacity for r R N A retention is limited, arguing strongly against nonspecific aggregation or random precipitation as mechanisms of immobilization. Some 1014 r R N A molecules are retained per ml of agarose gel in saturating conditions, meaning that approximately one nucleotidyl residue is immobilized per 100 or more galactosyl units. The non-retention of native D N A in conditions which allow an essentially complete immobilization of rRNA might be explained in terms of their different overall secondary structures, and also of their respective hydration parameters 19' 2 o, once that adequate hydration data become available for all classes of ribopolynucleotides. The observed cation selectivity, thermal reversibility, and dependence on temperature also underline the specific of the retention process. The retention profile appears to be characteristic for the given species of rRNA. RNA-agarose association could perhaps be compared with formation of "quaternary associates" between anionic polysaccharides and agarose 21, i.e. with a co-gelation. Sequences rich in guanylic acid could be important in RNA-agarose association, in view of poor water solubility, high self-association 22, and ionic strengthdependent gelation 23 of guanosine derivatives. However, as might be inferred from the retention profile of rat 18-S R N A compared to those of E. coli 23-S or pea 25-S RNAs, the "zeroth-neighbor ''24 guanylate content probably does not directly control RNA-agarose association.

RNA IMMOBILIZATION IN AGAROSE

323

The relation of molecular weight and the retention molarity was non-linear even with liver rRNAs, which display the largest difference in both molecular weight 11 and G ÷ C content12 14-among rRNAs compared here. With E. coli and pea rRNAs, where the nucleotide compositions of the pair members are rather similar, the spacing of NaC1 molarities needed to effect the same extent of retention was much smaller than with rat rRNAs, but anyway characteristic of rRNA species examined. This suggests that RNA-agarose association reflects overall molecular properties and structural differences between individual polynucleotides, and could perhaps be exploited as a general method for characterization of these macromolecules. -

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A C K N O W L E D G MENT

We acknowledge excellent technical assistance by Mrs Rada Tepavac. REFERENCES 1 2 3 4 5 6 7 8 9 l0 11 12 13 14 15 16

17 18 19 20 21 22 23 24

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