Proc. Nat. Acad. Sci. USAVol. 73, No. 2, pp. 307-1S1, February 1976Biochemistry

Studies on secondary structure of single-stranded RNA frombacteriophage MS2 by electron microscopy

(magnesium chloride/hairpin loops/molecular maps)

ANN B. JACOBSON*Max-Planck-Institut fur Biochemie, Abteilung Virusforschung, 8033 Martinsried bei Muinchen, Germany

Communicated by R. B. Setlow, November 3,1975

ABSTRACT A method allowing the demonstration andstudy by electron microscopy of secondary structure of viralRNA has been developed. Single-stranded RNA from the bac-teriophage MS2 has been analyzed in the electron micro-scope in the presence of various concentrations of MgCI2.Depending on the salt concentration, the molecules displayone to three large open loops which range in size from 10 to20% of the total RNA lengh, and smaller closed loops whichare approximately 3-5% of the total RNA length. Within onespreading, the conformation of the molecules is variable.However, the average complexity of the molecules increaseswith increasing salt, and individual loops which are infre-quent at low salt increase in frequency with increasing salt.By analyzing the manner in which the individual loops ap-peared, it was possible to show that all molecules could bedescribed by one basic pattern of secondary structure forma-tion.

Secondary structure, that is, the presence of short, helical,base-paired hairpin loops, has been demonstrated in single-stranded RNA by a variety of physical techniques. For thesmall RNA-containing bacteriophages, whose RNA has amolecular weight of 1.1 X 106, both hydrodynamic andspectroscopic data show that 60-80% of the RNA exists inthe form of short base-paired loops (1-5). Recently, se-quence analysis has also shown that the bacteriophage RNAcontains many internal regions which are self-complementa-ry and which could produce hairpin loops by base pairing(6, 7). Calculations based on the known free energies of theindividual base-pairs suggest that the most stable moleculesare those in which the individual loops are relatively short,generally under 50 nucleotides in length. Although it is notknown whether more than one stable arrangement of loopsis possible, the specificity of the fragments obtained afternuclease digestion suggests that one arrangement is pre-ferred (8). In addition to the physical data, a variety of ge-netic and biochemical experiments suggest that specific sec-ondary structure occurs in phage RNA molecules, and isprobably responsible for the regulation of both transcriptionand translation during viral replication (9).

Recent studies have shown that specific patterns of loopscan be visualized on ribosomal RNAs in the electron micro-scope (10, 11). These studies show that secondary structuremapping can be used, in ways similar to denaturation map-ping of double-stranded DNAs (12), to obtain informationabout the biological properties of RNA molecules. To date,studies of this sort have generally been unsuccessful withRNAs of lower (G+C) content, (13), and it has thereforebeen suggested that the hairpin loops which occur on non-ribosomal RNAs may be too small to be visualized in theelectron microscope. The one exception is a recent report on

the possible occurrence of two specific loops on the RNA ofRD-114 (14).RNAs from the small RNA-containing bacteriophages

have a moderate (G+C) content and messenger function.Due to the large amount of information available abouttheir secondary structures, they are particularly useful mate-rial with which to look for new methods for the visualizationof secondary structure in the electron microscope. A methodsuccessful for the demonstration of specific loop patterns inthis RNA should be applicable to similar RNAs of animal or-igin.

I studied the RNA from the bacteriophage MS2 in theelectron microscope after spreading it in the presence ofMgCl2. Under these conditions all the molecules display avariety of both large and small loops. The number and com-plexity of the loops depend on the salt concentration used.Plots of the individual molecules clearly show that all of thestructures observed fit into one basic pattern of secondarystructure.

MATERLALS AND METHODS

MS2 phage stocks were grown and purified according tostandard procedures (15). The RNA was extracted with phe-nol and purified by sedimentation for 3 hr at 340,000 X g ona 5-20% sucrose gradient containing 50 mM NaCl, 25 mMTris-HCl, and 1 mM EDTA, pH 7.5. The leading peak wascollected and precipitated with ethanol. The RNA pellet wascollected by centrifugation, dissolved in H20 at a concentra-tion of 5-7 ug/ml, and stored frozen until use.

Purified RNA was prepared for electron microscopy by amodification of the basic protein film technique (16). Fortymicroliters of a solution containing 0.5-0.7 gg of RNA perml, 50% (vol/vol) formamide, 10 mM triethanolamine-HCl,pH 8.5, 0.1 mg/ml of cytochrome c, and MgCl2 at the ap-propriate concentration was applied to a distilled water sur-face measuring 10.9 X 10.9 cm. The film which formed waspicked up on collodion-coated grids, contrasted with alco-holic uranyl acetate (16), and shadowed with platinum at anangle of 8°. All solutions and utensils which came in contactwith the RNA were autoclaved prior to use to inactivate nu-cleases.

RESULTS

Spreadings were performed at 15 different MgCl2 concen-trations ranging from 0.1 to 5 mM. In the electron micro-scope two different types of loops were seen. Depending onthe salt concentrations, the molecules contained one or morelarge open loops closed only at their base. These loops com-prise from 10 to 20% of the total length of the RNA. Withinthese large loops and on linear regions of the RNA, smallerloops, approximately 100 to 200 nucleotides long, were ob-

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* Current address: Departement de Biologie Moleculaire, Universi-ty of Geneva, Geneva, Switzerland.

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Proc. Nat. Acad. Sci. USA 73 (1976)

FIG. 1. Electron micrograph of single-stranded RNA from the bacteriophage MS2 showing the four basic conformations that can be ob-served in spreadings performed in the presence of MgCl2. (A+B) Linear molecules; (C+D) molecules with one large loop; (E+F) moleculeswith two large loops; (G+H) molecules with three large loops. Magnification X52,200.

served. These are closed along their entire length, and corre-spond in appearance to the type of loop which has been de-scribed for ribosomal RNA.

It has been possible to classify all molecules in a spreadingaccording to the number of large open loops that they con-tain. Four conformations have been observed. These are il-lustrated in Fig. 1. Molecules may be linear with variousnumbers of small closed loops (Fig. 1A and B). They mayhave one large open loop in the center (Fig. 1C and D). Thisloop is approximately 15% of the total length of the RNAand is always associated with one small closed loop at its base(Fig. 1C). The large loop will be referred to as OL and theloop at its base as L1. Additional small loops can form withinthe large loop, making it appear smaller, as in Fig. 1D. Thecircumference of the loop now corresponds to approximate-ly 10% of the total length of the molecule. An additionalclass of molecules has two large open loops. The second loopsometimes contains a number of small internal loops (Fig.IE) or may be completely open (Fig. IF). Finally, structuresoccur which contain three large loops, where both arms ofthe molecule appear to fold back on themselves (Fig. 1G andH).The relative frequency of molecules containing one, two,

or three large open loops varies with the salt concentration atwhich the RNA is spread. These results are seen in Fig. 2.The spreadings at each magnesium concentration were pho-tographed and approximately 200 molecules on several pho-tographs were scored for the number of molecules that wereeither linear, or contained one, two, or three large openloops. The results are shown as the percent of the total mole-cules scored exhibiting a particular conformation at eachMgCl2 cncentration. It can be seen that at 0.1 mM MgCl2most of the molecules are linear and the rest have only onelarge loop. At 0.6 mM MgCl2, 24% of the molecules are lin-ear, 58% have one loop, and 17%, two loops. At 1.5 mMMgCl2 45% of the molecules have two loops while 13% aremore complex and contain at least three large loops. (The

complex molecules containing more than one large loop areobserved only at high salt and are sometimes difficult to in-terpret due to the poor quality of the spreadings which areobtained under these conditions.) Fig. 2 shows that mole-cules with increasingly complex loop structures are found

100 ,I* linearo 1 major loop

2 major loopso = 3 loops

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060-

40-

20

0 2 3 4 5MgCI2 (mM)

FIG. 2. The effect of increasing concentrations of MgCl2 onthe conformation of single-stranded RNA. The percent of mole-cules in each spreading with zero, one, two, or three large openloops is plotted as a function of increasing salt.

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Biochemistry: Jacobson

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FIG. 3. Model showing the procedure by which secondarystructure maps are constructed. The baseline of the map repre-sents the normalized length of the molecule. Closed loops are plot-ted as black rectangles above the baseline; large open loops areplotted as open rectangles twice as high as the closed loops. Rec-tangles beneath the baseline indicate the entire length of a com-plex loop which contains more than one component.

more frequently at higher salt concentrations and that struc-tures with less complexity are converted to structures withgreater complexity by the addition of loops. This suggeststhat all of the structures observed are variations of one basicpattern. If the different conformations observed were not re-lated, one might expect that two or more curves would in-crease in parallel with increasing salt.

Molecular maps, showing both the position and length ofthe individual loops, were constructed for 29 molecules atfour different salt concentrations. The symbolic representa-tion used in these maps is shown in Fig 3. The plots areshown in Fig. 4. The left and right end of the molecules

A_ _ ^

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FIG. 4. Molecular plots of single-stranded MS2 RNA. (A) 0.1mM MgCl2; (B) 0.2 mM MgCl2; (C) 0.4 mM MgCl2; (D) 0.6 mMMgCl2. The molecules were normalized as described in Fig. 3.However, the absolute lengths of the molecules decreased slightlywith increasing salt. The mean lengths obtained were: (A) 1.46 40.09 Mm; (B) 1.53 4 0.09 gm; (C) 1.38 4 0.08 Mm; (D) 1.35 + 0.06JAM.

Proc. Nat. Acad. Sci. USA 73 (1976)

were obtained by assigning the small loop (LI) which ispresent at the base of the large open loop (OL) to the lefthalf of the molecule. There is some ambiguity in this assign-ment due to the fact that at low salt some of the moleculesdo not contain this loop, and at high salt some of the mole-cules become difficult to interpret. Despite this limitation,the results in Fig. 4 show that the molecules scored can beordered into one basic pattern of secondary structure. At 0.1mM MgCl2, the most frequent loop occurs between 10 and15% from the left end. This loop is retained at all salt con-centrations. At higher salt, two and-sometimes three loops ofsimilar size appear in this region. A very similar loop occursat approximately the same position on the right end of themolecules, but with lower frequency. At higher salt, thisloop increases in frequency, and also appears in the form ofa double loop. Thus at higher salt, there is striking symmetrybetween both ends of the molecules.

Another loop which occurs frequently on the molecules at0.1 mM salt is located approximately 40% from the left end.As mentioned above, this loop has been used to define theleft side of the molecules. The loop appears with increasingfrequency at higher salt concentrations and at 0.6 mMMgCl2 it appears on all of the molecules. It is always presentin molecules which have one large open loop, but may occuralone as well. Several additional loops occur between 20 and40% of the left end of the molecule; they are rarer and havenot been characterized in detail.

PER CENT OF TOTAL LENGTH

FIG. 5. Histograms showing the percent of the molecules witha loop in a given interval. The original data are seen in Fig. 4. Thestep size corresponds to 70 nucleotides. closed loops; -----open loops.

B

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Proc. Nat. Acad. Sci. USA 73 (1976)

FIG. 6. An electron micrograph of single-stranded MS2 RNA spread with 0.5 mM MgCl2 Magnification X35,100.

Two small loops have been observed regularly within thefirst large open loop (OL). The first of these occurs directlyadjacent to the small loop (Li), and when present reducesthe apparent length of the open loop from 15 to 10% of thetotal length. The second small loop occurs at the right end ofthe open loop. Molecules 17, 25, and 28 in Fig. 4D show thisstructure.The data of Fig. 4 have been summarized in the form of

histograms in Fig. 5. The percent of molecules which have aloop in a given interval is plotted. In this form, the resultscan be compared directly with those seen in Fig. 2. Theydemonstrate that the complexity of the molecules increaseswith increasing salt, and is expressed by the addition of newloops. Moreover, it can be seen that the loops which are moststable at low salt are clustered at the left half of the mole-cules. The first loop at the left is particularly stable, and itsrelative frequency increases only slightly with increasingsalt. On the other hand, the similar loop at the right end ofthe molecules is unstable at low salt, and increases in fre-quency with increasing salt. The loops which occur between20 and 40% from the left end are more frequent at low saltthan their counterparts on the right end which only becomeprominent at 0.6 mM MgCl2. A striking feature of the mole-cules at high salt is the great similarity between the loop pat-terns on the left and right arms. These give the molecule asymmetrical appearance; this type of structure might havebiological significance.The electron micrograph of Fig. 6 illustrates some of the

preceding observations. It is a low magnification field of aspreading performed at 0.5 mM MgCl2. Careful examina-tion shows that while no two molecules have a completelyidentical pattern of loops, they are all basically similar.

DISCUSSION

The results demonstrate that secondary structure mappingin the electron microscope is possible with an RNA of mod-

erate (G+C) content and with messenger function. Themaps obtained are different from those which were obtainedwith ribosomal RNA in that two different types of loops areobserved and the individual molecules are more variable inappearance. The increasing average complexity of the mole-cules which was observed with increasing salt is consistentwith biochemical studies which show that helical, base-paired stretches in single-stranded RNA increase with in-creasing salt (17). The mapping procedure described seemsto have general applicability: I have examined the single-stranded RNA from Sendai virus by the same method andfound that a specific pattern of loops, which is very differentfrom that seen with MS2, can be observed along a consider-able distance of the RNA.The spreading procedure used in these studies differs

from others primarily in the presence of MgCl2 in thespreading mixture. The presence of Mg+2 may enhance thestability of particular loops or simply facilitate the visualiza-tion of specific patterns in the electron microscope. Bio-chemical studies with tobacco mosaic virus RNA and withsome of the tRNAs show that Mg+2 is important in the stabi-lization of secondary and tertiary structure, but that the ef-fect is no different from that obtained with very high con-centrations of Na+ (17-19). Thus Mg+2 does not appear tohave a unique role in the stabilization of particular kinds ofloops. However, caution is necessary in extrapolating frombiochemical data to the electron microscope studies, sinceevidence that the loops seen with the microscope are corre-lated with base composition of the RNA is lacking.An accurate understanding of the secondary structure of

coliphage RNA is of interest, since a number of experimentssuggest that the conformation of the RNA is important inthe regulation of gene expression (9). In vitro studies on thetranslation of fragmented or formaldehyde-treated RNA,and on RNA from polar mutants, clearly show that secon-dary structure influences the relative amounts of coat pro-tein, synthetase, and maturation protein synthesized. The

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Proc. Nat. Acad. Sci. USA 73 (1976) 311

synthesis of Q(3 RNA may also require a particular confor-mation of the RNA, and a model has been proposed to ac-count for the strong internal binding site of RNA synthetasein which the 3' arm is folded back on itself (20). This modelis strikingly similar in appearance to some of the structureswhich were observed in the electron microscope with MS2RNA. However, the known nucleotide sequences of the twoRNAs are different and their secondary structures may dif-fer as well.

It is of interest to compare the secondary structure of MS2RNA as seen in the electron microscope with that derivedfrom nucleotide sequence data. Sequence studies haveshown that many self-complementary regions occur alongthe RNA. These are generally under 50 nucleotides in lengthand can be arranged into a series of hairpin loops (6, 7). Acomplex cluster of loops, known as the "flower model," oc-curs in the region of the coat protein gene and contains ap-proximately 350 nucleotides (8). In the electron microscope,the smallest hairpin loops are 100 to 200 nucleotides inlength. The open loop (OL) is located in the region known tocode for the coat protein cistron (21). However, it is approxi-mately 500 nucleotides in length and probably contains thecoat protein gene and some adjacent regions as well. A simi-lar loop has been reported to occur in R17 RNA (22). Thereason for the discrepancy between the structures proposedon the basis of sequence data and those seen in the electronmicroscope is not known and further study is necessary be-fore a direct comparison between these structures can bemade. Better electron microscope mapping data are neededto position the individual loops more precisely, and the 33'and 5' ends of the molecules must be identified. In addition,it may be useful to examine the secondary structure of theRNA by spreading procedures which do not require cyto-chrome c (23), since it is possible that the presence of cyto-chrome c exaggerates the apparent length of the small loops.On the other hand, the current data available on the freeenergies involved in stacking base-pairs onto an existinghelix may not give a reliable estimate of the degree of mis-matching which can be tolerated in a long loop. In addition,the association of distant regions on the RNA is difficult topredict. For example, when examined at high magnifica-tion, the open loop (OL) appears to involve a triple strandassociation such as that diagrammed in Fig. 3. Thus the elec-tron microscope studies may help achieve a better under-standing of the rules governing the formation of secondarystructure of large single-stranded RNA molecules.

I wish to express my gratitude to Dr. H. Gassman and H. S. Hoff-man of the Rechenzentrum of the Max-Planck-Institut fur Bio-chemie for their extreme helpfulness during the course of thesestudies. H. S. Hoffman wrote the program used for plotting themolecules. I also wish to thank Prof. P. H. Hofschneider for finan-cial support and encouragement. The manuscript has been pre-pared under the tenure of an European Molecular Biology Organi-zation long-term fellowship. I thank Profs. L. G. Caro, P. H. Hof-schneider, and P.-F. Spahr for reviewing the manuscript.

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2. Gesteland, R. F. & Boedtker, H. (1964) "Some physical prop-erties of bacteriophage R17 and its ribonucleic acid," J. Mol.Biol. 8,496-507.

3. Mitra, S., Enger, M. D. & Kaesberg, P. (1963) "Physical andchemical properties of RNA from the bacterial virus R17,"Proc. Nat. Acad. Sci. USA 50,68-75.

4. Strauss, J. H., Jr. & Sinsheimer, R. L. (1963) "Purification andproperties of bacteriophage MS2 and of its ribonucleic acid,"J. Mol. Blol. 7,43-54.

5. Gralla, J., Steitz, J. & Crothers, D. M. (1974) "Direct physicalevidence for secondary structure in an isolated fragment ofR17 bacteriophage mRNA," Nature 248,204-208.

6. Vanderberge, A., Min Jou, W. & Fiers, W. (1975) "3'-Termi-nal nucleotide sequence (n = 361) of bacteriophage MS2RNA," Proc. Nat. Acad. Sci. USA 72,2559-2562.

7. Weissman, C., Billeter, M. A., Goodman, H. M., Hindley, J. &Weber, H. (1973) "Structure and function of phage RNA,"Annu. Rev. Biochem. 42,303-328.

8. Min Jou, W., Haegeman, G., Ysebaert, M. & Fiers, W. (1972)"Nucleotide sequence of the gene coding for the bacterio-phage MS2 coat protein," Nature 237, 82-88.

9. Kozak, M. & Nathans, D. (1972) "Translation of the genomeof a ribonucleic acid bacteriophage," Bacteriol. Rev. 36,109-134.

10. Wellauer, P. K. & Dawid, I. B. (1974) "Secondary structuremaps of ribosomal RNA and DNA. I. Processing of Xenopuslaevis ribosomal RNA and structure of single-stranded ribo-somal DNA," J. Mol. Biol. 89,379-395.

11. Wellauer, P. K. & Dawid, I. B. (1974) "Secondary structuremaps of ribosomal RNA II. Processing of mouse L-cell ribo-somal RNA and variations in the processing pathway," J. Mol.Biol. 89,397-407.

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13. Hsu, M. T., Kung, H. J. & Davidson, N. (1973) "An electronmicroscope study of Sindbis virus RNA," Cold Spring HarborSymp. Quant. Biol. XXXVIII, 943-950.

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15. Kolakofsky, D. (1971) "Preparation of coliphage RNA" inMethods in Molecular Biology, eds. Last, J. A. & Laskin, A. I.(Marcel Dekker, Inc., New York), Vol. 1, pp. 267-277.

16. Davis, R. W., Simon, M. & Davidson, N. (1971) "Electron mi-croscope heteroduplex methods for mapping regions of basesequence homology in nucleic acids" in Methods in Enzymol-ogy, eds. Grossman, L. & Moldave, K. (Academic Press, NewYork), Vol XXI, pp. 413-428.

17. Boedtker, H. (1960) "Configurational properties of tobaccomosaic virus ribonucleic acid," J. Mol. Biol. 2, 171-188.

18. Fresco, J. R., Adams, A., Ascione, R., Henley, D. & Lindahl,T. (1966) "Tertiary structure in transfer ribonucleic acids,"Cold Spring Harbor Symp. Quant. Biol. XXI, 527-537.

19. Cole, P. E., Yang, S. K. & Crothers, D. M. (1972) "Conforma-tional changes of transfer ribonucleic acid. Equilibrium phasediagrams," Biochemistry 11, 4358-4367.

20. Kolakofsky, D. & Weissman, C. (1971) "Possible mechanismfor transition of viral RNA from polysome to replication com-plex," Nature New Biol. 231, 42-46.

21. Jeppesen, P. G. N., Steitz, J. A., Gesteland, R. F. & Spahr,P.-F. (1970) "Gene order in the bacteriophage R17 RNA: 5'-Aprotein-coat protein-synthetase-3'," Nature 226, 23-237.

22. Delius, H. Westphal, H. & Axelrod, N. (1973) "Length mea-surements of RNA synthesized in vitro by Escherichia coliRNA polymerase," J. Mol. Biol. 74,677-687.

23. Vollenweider, H. J., Sogo, J. M. & Koller, Th. (1975) "A rou-tine method for protein-free spreading of double- and single-stranded nucleic acid molecules," Proc. Nat. Acad. Sci. USA72,83-87.

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