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Formation of hairpin helices in uridylic acid oligomers
Vol. 41, No. 6, 1970
BIOCHEMICAL
mF?MATxm
OF HAImm
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HEUCES
IN uFctmc
ACID OLIB
M. Dourlent*, J.C. Y'ihrierr, F. Run and M. Ieng CentredeBiq&ysiqueMol&xlaire, 454rl&ns-O2, France
Received November 13, 1970 Tnebehaviorof uridylicacidoligatrars underheatinghasbeen s~iedbyequil~iumandkinetic~irrrtnts,eitherin 0.5 MCsClor in presence of spermine. The absorpticplmelting curvescan be accounted far by acooperative xtoncnolecular transition. Teqeraturejmp results are consistentwith themaltingofdoublestraxIed structure. It is concluded that uridylic acid oligarrxs can form hairpin helices.
Introd~tion
F¢wxkonpolyuridylicacid (polyu) hasled ccmclusion that, during the cooperative coil-helix
us to the transi-
tion, the molecule folds on itself (1,Z). Three rrkain steps were assto e.qlain our results : well above Tm, thendecule is a single stranded chain doing several hairpin helices (the (3) ; on cooling, it folds on itself, lengthof thehelicalsecticnsdqerdingan salt concentration) and finally, welltier Tmtheaoleculetends to fcrma singlehairpin.Nuaerousprcblans arise fran such ancdel. Qle of them is todetermine polyrrerizaticn of polyuridylic acid at which fold*
the smallest degree of occurs. Also, it is
investing toknmthenutbr ofnonpaired residues in the loop. Astudy of oligouridylic acids (oligo U) might be helpful for such an insight.
Material
Oligo U were prepared by alkaline hydrolysis (3) of poly U in 0.3 M KOHat 37'C for 6 minutes. The hydrolysis was stq?pedbyneutralizatica?withpercNaricacid and theexcess KC104 removed by centrifugation. Then, the solution was made acid by addition of HCl and incubatedone hour. After neutralizaticol topH 8, themixturewas treated hyalkalinephosphatase andadsorbed into aDEAe cellulose oolunn.The olig~s were eluted sqx2ntially gradient at pH 8.5. The different
.8MammniLuncarbanate with a .OlMto fractions were twice lyqhilized to rexove
*Martre-AssistantattheFaculty
of Sciences (Or%anS). 1590
Vol. 41, No. 6, 1970
the salt.
BIOCHEMICAL
The purity
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of each fractim
was checked by chramtcgraphy.
Wry
good separation was obtained up to (Up)lOU. The higher poorly resolved fractions wxe mixed together and chrcmatqraphied on Whakmn pa3M 1 M-ammniurn acetate + 4 vol. 95 % ethanol (4)). lcmwidth,were cutup. After elution, theprcductswere (6 vol.
Strips of paper, identifiedby
alkaline hydrolysis as 0Jpj14U, WpP)17U, (QD)~~U, and @pP)30U.From LJpUto (Up),OU, each oli.gcamr was guite pure ; the last four products wcxe polydispersed. For all experiments, the solutions ware made in 0.5 M CsCl, 0.05 M Na caccdylate, O.ooOl M EDTA, pH 6.6. Contamination of the prcdwts bydivalentmtalions has notbeenchecked.
0
Fig. 1
Fesults
Meltingcurves polymerization
atxl discussicm
10
20
30
T’C
40
0.5 M CzZl as a function of the degree of (indicated on the figure).
in
lhemelting 4o”c
and
-
curves of fig. l&low thatbetwee.n 872, the absorbance of the oligomer
is nearly constant up to (Up),,V, while a decmase is clearly observed for (I@p)14U,at-xdlcnger oligawrs. TIndepends on the cl~~inlengthandcmbe readily obtained for (UpP,30U.Assming symmtricalmlting curves, Rnvalues canbe estirrated for the threeothermes. The values are listed in Table 1. Anirrportantfindingisthatthesecurvesare~~tantheconcentratiou c in the range l-10 optical densities (260 mn), yielding strmg aridenceforan~~~lecularcanformationalchange.~acooperative bimolecularprocess is involved in the formationof shcartdoublehelical sequences smh an oli.goA-oligoAatacidpH or oligo A-oligo U at neutral pH,thereacticncanbedescribedinafirstapproxima~byatwostate 1591
BIOCHEMICAL
Vol. 41, No. 6, 1970
t&e
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(AH) on the degree of polyr&zation.
equilibrimbetwem the fully unpaired andpaired states, increasing c by a factir In c (5,6). In the present case, lInbys-everaldegrees.Thisisnotobserved.
and !thva.ries as 10 wuld increase
derive the equili&immnstantK frcmther&ting curves an3 to cmpute the mlix mthalpy AH of the reaction. The van t'Hoff plots are quite linear beizwea-l- 6OC ad + 15’T (fig. 2). The values of AH are given in Table 1. As I-
I
I 3.5
Fiq.
2
1
I
,
3.6
I 3.7
I Y+K-‘)
Vant'Hoffplots obtained frcmthemeltingcurves fig. 1. The degree of polymrizatim is irdicated line. 1592
shmn in above each
Vol. 4 1, No. 6, 1970
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
alreadyobser&l for thevalues of 'IIn, the absolutevalueof with the degree of polymerization, showing the Tative transition. Itwxld have been interesting towork order to seewhether atransiticmdoescccurwiththe
atlw
AH increases character of the
teqeratures in shorter0ligmxs.Thi.s
is inpossible in 0.5 M CsCl. Hmever, it is knmn that the stability of poly U is largely increased in presence of spemine (7). We confinred this result with poly U (M ~150,ooO) (fig. 3). The stability of oligo U is also increased butto a smaller extent than that of poly U. It may be noticed that Q)loU begins to fold at O'C (fig. 3). The molar enthalpies are given in Table 1. These results are not explained.
0
Fig.
3
Melting
10
20
.3l
T=‘C
40
-4 M Na caccdylati pH 6.6 with qermine curves in 0.5.10 cxmcentration 10e4 M, as mm, qxcmine HCl
Teqerature-jmp expfximnts have been performed on (Up)30U samples. Thewbolerangeof thetransitioncouldnotbecoveredbecause the solution froze on the colder electr& at lower texqeratures. This is due to a large ~eraturegradientintheterrpesature-jwp cell. Whenmaasuremmts ~epossible,therelaxaticlsignalcouldbeac~~~forby~y~ relaxationtims (fig.4).Theshorterrelaxa6~times~no~~~le teqesature dependenceandcorrespcsdedtotherisetimeoftheapparatus (= 3 ps).This effect is ~bablyrelated tochange instackingeguilibria wkichhasbeens~tobeveryfast(6).ThelclngerreLaxationtimeTdecreased frm (22 * 3) PS at 6.4'C to (8 * 2) us at13.5V. In this range of 1593
Vol. 41, No. 6, 1970
Fig.
4
I
I
20
40
I
Ups)
1
60
Typicaloscillc6cx+ trace of a txqerature-jwp experiment, ad its semilogsrithmic analysis. Final equilibrimbqerature 6.4"C.
relaxatimtbres, thesignal-W-noiseratio islmdaccuracyinthe analysis of the curves is vwzypcor. FCC a nmxmolecular coilhelixtransiticm, which agrees with the "all or none" nwAe1,
cdl.
.
53 L
helix
% -,L kDT
(K=
lb theratecmstant5~d~ ]k = K [~(l+Ki
canbe -'
,
=]b+k&
ccarputedfran
equilibrium
adkinetic
]b = b(l+K)]-1
AnArrheniusplotof]kand]bgives theactivatimenthalpies related to each rate constant (fig. 5). While B$ is found to be positive = 30 Kcal de-l) , ~isnegative (s*=5.5K~almAe-~).This ( s* haskeI-lZihd~abserved for the ccqexativehelix-coil transitimof oligoA-oligoUdoublestranded~lexes(6)andhdlfmoleculesoftransfer FSA (8).Thus, the teqerature-jmpresultsseemtobe inagreemzntwiththe cocperat~velneltingofs~~doublestranded~~~andsupportthehairpin mdelassd fran~libriumdata.Humxz, itmustbepoinkdoutthat thetenperaturerangecov~dintheexperkentsis -anathepocar accuracy of them u3xmntsdoes notccqdetely mltie thepc6sibility of awiderrelaxatbnspectrum.Nevertheless thispc6sibilitymuldnotbe in 1594
Vol. 41, No. 6, 1970
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
K 10 5
2 1 .5
,2
Fiq.
5
Arrhenius and vant'Hoffplots kinetic data for (UpP,,U.
obtained
discrepancywiththehairpinmzdelandmightbedue the solution or/am3 to a degree of ccqerativity fit with the "all or rime" mdel.
franequilibrimand
to thepolydispemity of too smll to give reasamble
C3XEluSian
Amq thedifferentresults r-ted here, sane of them clearlyshawthat~hairpinm3delisasatisfactoryrepresentaticnofthec&eredfomofoligoUsystms. The teqerature-jmpmasurermmts are in favor ofdoublehelicalstrmture, AHandandimreasewiththe concentratim of the solution rnmberofresidues but are ir&pemIentonthe ~isCpllyafewdegreesbelow?mofpolyUfcor (UpP)mU.Foldingcanoccur forachaincontaining llresidsles. Inprincipleour st~ycouldallowtheccqmtationof structural andthfzm&~al~~tessof furCWentalinterestsuchas:nu&erof residues~~elocp,eJlthalpyofanU-Upair,nucleationpar~~ (representedby u inlAeoretical.treatmzntS of thehelix-mil transition) aczomting for the coqmativityof the transition. Unfcxtunately, the polydispersity of the soluticns does not allow to give m3Aqful results fen: suchcaqxtations.E8etter fractimationof thepro3ucts,ifsmcessful, should enable us to cmpare experirrwtdl results with thearetical mdels.
1595
Vol. 41, No. 6,197O
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
we wish to thank Prof. Charles S&Ix-on for his interestinthiswork.
1. 2. 3. 4. 5. 6. 7. 8.
Thrierr, J.C. ad w, M., Bio&im. Biqr~hys. Acta. 182 (1969) 575. (in p-s). Thrierr, J.C., Dourlent, M. and Leng, M., J. Pkd. Bi= sinpkins, H. and Richards, E.G., J. Mol. Biol. 29 (1967) 349. Thach, R.E., and Doty, P., Science 148 (1965) 632. A&equist, J. and Dade, V., Jourri:f Am. Chem. Sot. 87 (1965) 1450. Pkxhke, D., lhesis, ~aunschweig, 1968. szer, w., J. ml. Biol. 16 (1966) 585. Xfm-eir, R., Riesner, D., ?-&~s, G., Win-, w., mid, R. d Zachau, H.G., E'EBSrdzers 2 (1969) 15.
1596
BIOCHEMICAL
mF?MATxm
OF HAImm
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HEUCES
IN uFctmc
ACID OLIB
M. Dourlent*, J.C. Y'ihrierr, F. Run and M. Ieng CentredeBiq&ysiqueMol&xlaire, 454rl&ns-O2, France
Received November 13, 1970 Tnebehaviorof uridylicacidoligatrars underheatinghasbeen s~iedbyequil~iumandkinetic~irrrtnts,eitherin 0.5 MCsClor in presence of spermine. The absorpticplmelting curvescan be accounted far by acooperative xtoncnolecular transition. Teqeraturejmp results are consistentwith themaltingofdoublestraxIed structure. It is concluded that uridylic acid oligarrxs can form hairpin helices.
Introd~tion
F¢wxkonpolyuridylicacid (polyu) hasled ccmclusion that, during the cooperative coil-helix
us to the transi-
tion, the molecule folds on itself (1,Z). Three rrkain steps were assto e.qlain our results : well above Tm, thendecule is a single stranded chain doing several hairpin helices (the (3) ; on cooling, it folds on itself, lengthof thehelicalsecticnsdqerdingan salt concentration) and finally, welltier Tmtheaoleculetends to fcrma singlehairpin.Nuaerousprcblans arise fran such ancdel. Qle of them is todetermine polyrrerizaticn of polyuridylic acid at which fold*
the smallest degree of occurs. Also, it is
investing toknmthenutbr ofnonpaired residues in the loop. Astudy of oligouridylic acids (oligo U) might be helpful for such an insight.
Material
Oligo U were prepared by alkaline hydrolysis (3) of poly U in 0.3 M KOHat 37'C for 6 minutes. The hydrolysis was stq?pedbyneutralizatica?withpercNaricacid and theexcess KC104 removed by centrifugation. Then, the solution was made acid by addition of HCl and incubatedone hour. After neutralizaticol topH 8, themixturewas treated hyalkalinephosphatase andadsorbed into aDEAe cellulose oolunn.The olig~s were eluted sqx2ntially gradient at pH 8.5. The different
.8MammniLuncarbanate with a .OlMto fractions were twice lyqhilized to rexove
*Martre-AssistantattheFaculty
of Sciences (Or%anS). 1590
Vol. 41, No. 6, 1970
the salt.
BIOCHEMICAL
The purity
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of each fractim
was checked by chramtcgraphy.
Wry
good separation was obtained up to (Up)lOU. The higher poorly resolved fractions wxe mixed together and chrcmatqraphied on Whakmn pa3M 1 M-ammniurn acetate + 4 vol. 95 % ethanol (4)). lcmwidth,were cutup. After elution, theprcductswere (6 vol.
Strips of paper, identifiedby
alkaline hydrolysis as 0Jpj14U, WpP)17U, (QD)~~U, and @pP)30U.From LJpUto (Up),OU, each oli.gcamr was guite pure ; the last four products wcxe polydispersed. For all experiments, the solutions ware made in 0.5 M CsCl, 0.05 M Na caccdylate, O.ooOl M EDTA, pH 6.6. Contamination of the prcdwts bydivalentmtalions has notbeenchecked.
0
Fig. 1
Fesults
Meltingcurves polymerization
atxl discussicm
10
20
30
T’C
40
0.5 M CzZl as a function of the degree of (indicated on the figure).
in
lhemelting 4o”c
and
-
curves of fig. l&low thatbetwee.n 872, the absorbance of the oligomer
is nearly constant up to (Up),,V, while a decmase is clearly observed for (I@p)14U,at-xdlcnger oligawrs. TIndepends on the cl~~inlengthandcmbe readily obtained for (UpP,30U.Assming symmtricalmlting curves, Rnvalues canbe estirrated for the threeothermes. The values are listed in Table 1. Anirrportantfindingisthatthesecurvesare~~tantheconcentratiou c in the range l-10 optical densities (260 mn), yielding strmg aridenceforan~~~lecularcanformationalchange.~acooperative bimolecularprocess is involved in the formationof shcartdoublehelical sequences smh an oli.goA-oligoAatacidpH or oligo A-oligo U at neutral pH,thereacticncanbedescribedinafirstapproxima~byatwostate 1591
BIOCHEMICAL
Vol. 41, No. 6, 1970
t&e
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
(AH) on the degree of polyr&zation.
equilibrimbetwem the fully unpaired andpaired states, increasing c by a factir In c (5,6). In the present case, lInbys-everaldegrees.Thisisnotobserved.
and !thva.ries as 10 wuld increase
derive the equili&immnstantK frcmther&ting curves an3 to cmpute the mlix mthalpy AH of the reaction. The van t'Hoff plots are quite linear beizwea-l- 6OC ad + 15’T (fig. 2). The values of AH are given in Table 1. As I-
I
I 3.5
Fiq.
2
1
I
,
3.6
I 3.7
I Y+K-‘)
Vant'Hoffplots obtained frcmthemeltingcurves fig. 1. The degree of polymrizatim is irdicated line. 1592
shmn in above each
Vol. 4 1, No. 6, 1970
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
alreadyobser&l for thevalues of 'IIn, the absolutevalueof with the degree of polymerization, showing the Tative transition. Itwxld have been interesting towork order to seewhether atransiticmdoescccurwiththe
atlw
AH increases character of the
teqeratures in shorter0ligmxs.Thi.s
is inpossible in 0.5 M CsCl. Hmever, it is knmn that the stability of poly U is largely increased in presence of spemine (7). We confinred this result with poly U (M ~150,ooO) (fig. 3). The stability of oligo U is also increased butto a smaller extent than that of poly U. It may be noticed that Q)loU begins to fold at O'C (fig. 3). The molar enthalpies are given in Table 1. These results are not explained.
0
Fig.
3
Melting
10
20
.3l
T=‘C
40
-4 M Na caccdylati pH 6.6 with qermine curves in 0.5.10 cxmcentration 10e4 M, as mm, qxcmine HCl
Teqerature-jmp expfximnts have been performed on (Up)30U samples. Thewbolerangeof thetransitioncouldnotbecoveredbecause the solution froze on the colder electr& at lower texqeratures. This is due to a large ~eraturegradientintheterrpesature-jwp cell. Whenmaasuremmts ~epossible,therelaxaticlsignalcouldbeac~~~forby~y~ relaxationtims (fig.4).Theshorterrelaxa6~times~no~~~le teqesature dependenceandcorrespcsdedtotherisetimeoftheapparatus (= 3 ps).This effect is ~bablyrelated tochange instackingeguilibria wkichhasbeens~tobeveryfast(6).ThelclngerreLaxationtimeTdecreased frm (22 * 3) PS at 6.4'C to (8 * 2) us at13.5V. In this range of 1593
Vol. 41, No. 6, 1970
Fig.
4
I
I
20
40
I
Ups)
1
60
Typicaloscillc6cx+ trace of a txqerature-jwp experiment, ad its semilogsrithmic analysis. Final equilibrimbqerature 6.4"C.
relaxatimtbres, thesignal-W-noiseratio islmdaccuracyinthe analysis of the curves is vwzypcor. FCC a nmxmolecular coilhelixtransiticm, which agrees with the "all or none" nwAe1,
cdl.
.
53 L
helix
% -,L kDT
(K=
lb theratecmstant5~d~ ]k = K [~(l+Ki
canbe -'
,
=]b+k&
ccarputedfran
equilibrium
adkinetic
]b = b(l+K)]-1
AnArrheniusplotof]kand]bgives theactivatimenthalpies related to each rate constant (fig. 5). While B$ is found to be positive = 30 Kcal de-l) , ~isnegative (s*=5.5K~almAe-~).This ( s* haskeI-lZihd~abserved for the ccqexativehelix-coil transitimof oligoA-oligoUdoublestranded~lexes(6)andhdlfmoleculesoftransfer FSA (8).Thus, the teqerature-jmpresultsseemtobe inagreemzntwiththe cocperat~velneltingofs~~doublestranded~~~andsupportthehairpin mdelassd fran~libriumdata.Humxz, itmustbepoinkdoutthat thetenperaturerangecov~dintheexperkentsis -anathepocar accuracy of them u3xmntsdoes notccqdetely mltie thepc6sibility of awiderrelaxatbnspectrum.Nevertheless thispc6sibilitymuldnotbe in 1594
Vol. 41, No. 6, 1970
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
K 10 5
2 1 .5
,2
Fiq.
5
Arrhenius and vant'Hoffplots kinetic data for (UpP,,U.
obtained
discrepancywiththehairpinmzdelandmightbedue the solution or/am3 to a degree of ccqerativity fit with the "all or rime" mdel.
franequilibrimand
to thepolydispemity of too smll to give reasamble
C3XEluSian
Amq thedifferentresults r-ted here, sane of them clearlyshawthat~hairpinm3delisasatisfactoryrepresentaticnofthec&eredfomofoligoUsystms. The teqerature-jmpmasurermmts are in favor ofdoublehelicalstrmture, AHandandimreasewiththe concentratim of the solution rnmberofresidues but are ir&pemIentonthe ~isCpllyafewdegreesbelow?mofpolyUfcor (UpP)mU.Foldingcanoccur forachaincontaining llresidsles. Inprincipleour st~ycouldallowtheccqmtationof structural andthfzm&~al~~tessof furCWentalinterestsuchas:nu&erof residues~~elocp,eJlthalpyofanU-Upair,nucleationpar~~ (representedby u inlAeoretical.treatmzntS of thehelix-mil transition) aczomting for the coqmativityof the transition. Unfcxtunately, the polydispersity of the soluticns does not allow to give m3Aqful results fen: suchcaqxtations.E8etter fractimationof thepro3ucts,ifsmcessful, should enable us to cmpare experirrwtdl results with thearetical mdels.
1595
Vol. 41, No. 6,197O
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
we wish to thank Prof. Charles S&Ix-on for his interestinthiswork.
1. 2. 3. 4. 5. 6. 7. 8.
Thrierr, J.C. ad w, M., Bio&im. Biqr~hys. Acta. 182 (1969) 575. (in p-s). Thrierr, J.C., Dourlent, M. and Leng, M., J. Pkd. Bi= sinpkins, H. and Richards, E.G., J. Mol. Biol. 29 (1967) 349. Thach, R.E., and Doty, P., Science 148 (1965) 632. A&equist, J. and Dade, V., Jourri:f Am. Chem. Sot. 87 (1965) 1450. Pkxhke, D., lhesis, ~aunschweig, 1968. szer, w., J. ml. Biol. 16 (1966) 585. Xfm-eir, R., Riesner, D., ?-&~s, G., Win-, w., mid, R. d Zachau, H.G., E'EBSrdzers 2 (1969) 15.
1596