The topological configuration and conformational analysis of mRNA in translation

NOTESThe topological configurationgle-stranded mRNA on earth? Are such contormatonalfeatures correlalive with protein structures or not?and conformational analysisThrough study and analysis of he thcoretical model ofmRNA and through simulation unfolding of experimentalof mRNA in translationRNA hairpin structures. we found thal the conformation ofsingle-stranded mRNA was closely correlarive with that ofLIU Shuqun' & LIU Ciquan'QAits corresponding hairpin structure. which implied that1.Kunming Intitne of Zoology. Chinesc Academy of Sciences. Kun-conformation of single-stranded mRNA had 'memory' tn 65077 Chingthat of mRNA hairpin structure. These results wouldC. Moderm Biological Cenler. Yunnan University, Kunming 60091,provid valuable information for further study ing the rela-Correspondence should be addressed to L.iu Ciquan (e mail: brg@tionship between mRNA structure and (ruanslational rate aswell as nascent polypepide structurc.public .km.yn.cn)Abstract The theoretical model construction of mRNA1 Materials and methodshairpin structure and single-straoded structure as well as theRNA includes messenger RNA, transfer RNA. andsimulation studies on RNA structure determined by theribosomal RNA. Experimental and theoretical methodsX-ray erystal diftraction and nuclear magnetic resonanceprovid researchers with abundant RNA structural informa-rerealed that in translation, after mRNA being unfolded intoion. It is worth pointing out that stems and loops are thesingle-stranded structure, its topological configuration wascommon structural characteristics of all RNAs'". Stack-closely correlative with the origioal bairpin structure. Thconformational features of single-stranded mRNA appeareding and base pairing drive a single-stranded RNA mole-as helical regions alternating with curly regions to differentcule to fold the chain on itself into complex hairpin struc-extents. which might exert the infuence on the foldiag oftures including helical regions (sterms) linked by the non-oasceot polypeptide by various regulating effects includinghelical regions. The non-helical regions are composed ofdifferent translational rates.hairpin loops. internal loops. bulges. multiple junctions,Keswords: hairpin struchure of mRNA, topological confguration ofand single-stranded chain. No complete mRNA strutturehas been determined until now, but we can get hairpin orsingle-stranded mRNA.single- stranded structures of other RNAs. And these struc-All the RNA molecules in vivo have their own struc-tures possess the same features as those of mRNAtures. The structures of tRNA and rRNA can cary outstructures, so our structural analysis of RNA prest'ntsimporant biological functions; but for mRNA. the rela-universal sense.( i) Sample Collection. IUBQ (PDB:"i ID) wastionship between structure and function is more compli-cated" because its functions are implemented by a com-the strnucture of ubiquitin refined at 1.8 A resolution“. Itsplex and dynamic translation machine一-ribosomel-l. Incoding sequence (mRNA). obtained from ISSDI . wasranslaion, the interactions among ribosome, tRNA. andused for constructing mRNA structures and simulatingmRNA are dynamic and harmonious, and the biosynthesishairpin structure unfolding into single-stranded structure.Using the advanced search engine SearchFicld ofof nascent peptide depends on the recognition and combi-PDB, we got 74 experimental structures of RNAs 1hatnaion of the anticodons on tRNAs to codons on MRNA toPDB could collect up to 31 December 2000. Among them.a certain extent. so the mRNA on nibosome must be in athere were 68 hairpin structures determined by the X-raysingle-stranded state's. Namely. the termary structurer olcrystal diffraction or nuclear magnetic resonance (NMR)mRNA fomned by loop and stem regions has to be un-and 6 single-stranded structures determined by the X-rayfolded, and its hairpin structures unfasten continuouslycrystal dillraction. These structures were used for simu-Into 5'→3' single-stranded chain, which is convenientlating RNA hairpin unfolding and analying conforna-for tRNAs 10 proofread the decoding region of mRNAI5I.tional features of single-stranded RNA.Furthermnore, the decoding region conformation of sin(ii) Simulation method. Firstly. the mRNA sec-gle-stranded mRNA can be subtly adjusted to pair with theondary structures of IUBQ und thesc 74 samples wereanticodons of A- and P-site tRNAs. Relevant researchespredicted by RNAsructure 3.5. Comparing the pre-have revealed that the structure of mRNA is related to thedicted structures with experinental structures. we foundtranslation initiation efficiencyh', the movement rate of that the theoretical structures were consistent with ex-rbosome along mRNA", and the termination of transla-perimental structures in base pairing. and the acuracytion" . Moreover. different translation rates of ribosomerate of base pairing in predicted structures was 100% ifon he different regions of single sranded mRNA can the RNA sequence length was less than 100 nucleotiles.dfferentially code protein secondary structural types'.Secondly. he termary structures o[ RNAs werc buit on theHowever. what are the conformational features of sin-basis of their secondary structure. and then the haipin384Chine中国煤化工。March 2002MYHCNMHGNOTESstructures were unfolded into single-siranded RNAs. Anear RNA structure that comprised the random nucleotidemRNA temnary structure was a complex hairpin assembledsequence, and added a K cation onto each phosphoricby its structural element- -simple hairpin- rand the essen-acidgroup to keep the whole systcm as elctroneutrality.tial structural characteristic of the simple hairpin was stemThe initial positions of K* were located on the planeand loop. So we sclected randomly the fourth hairpin onformed by the O-P-O atoms of phosphoric acid group.the mRNA secondary structure of IUBQ， which waswith equal distances l0 the lwo oxygen atoms. Then thecomposed of 24 nucleotides, with I0 base pairs located alwhole system was soaked in one layer of aqueous solu-stem region and 4 dissociative bases at loop region. Be-tion". Finally, the energy of the system was minimized tocause this hairpin possessed the typical structural featureof RNA. and it was convenient to analyze its conforma-tion. we built its ternary structure and corresponding sin-gle-stranded structure on Silicon Graphics 02 WorkstationISGI Inc.. Silicon. CA, USA) using Biopolymer softwaremodule of Insight II 2000 (Molecular Simulations Inc.,[SA1. and stretched the unfolded single-stranded RNAsegment twice to get the fourth hairpin structure of 1BQmRNA and its three corresponding extended single-stranded segments (lig. 1). Furthermore, we built the lin-(abh)(a)b)(c鸟c)d)mawr T tvF:g. 1. The fourth huirpin structure of IUBQ mRNA. its crrespondingg. 2. The secondary and lermary structures of ICQS und .DUH国unfolded singlc- stranded smucturc. and extended single- stranded struc-NMR-determined bhairpin structure of ICQ5: (b) secondany sIructure ofture. (a) The fourth hairpin structure of IUBQ mRNA built on SGI 021CQ5 predicted by RNA strucnure 3.5: (C) the X-ray sTystal dfrac-Workstaton: tb) secondary strnucture predicted by RNAstructure 3.5: (心tiondermined sngle-stranded structure of IDUH: (d) secundary sIruc.the single stranded srucure thar was not stretched: (d) the sin-ture of IDUH predicted by RNAstrucrure 3.5: le) haumpin strucrurer ofgle-stranded structure uhat was stretched once from stnucture IC); (e) theIDUH built on SGI ()2 Worksiation on the biasis of slrulurts (as. (b).single- stranded structure that was stretched twice fom structure (C). Theand (d). The single stranded regions in 1C) along the dasheid lines weresingle- stranded regions along the dashed lines were the stem regions ofstems of IDUH haipin structure. The boxes in [b) and山Indicate thehairpin sructure. In (8)。()--c), the arow drection is from5' to3'reguons of identucal secondary structures of ICQ5 and IDUH In lah le.and (e). the aTow drection is from5" w3'1L;u. s. Q.. Yang. J. Bai, C. L er a.. Analysis of the cnergies of the tiple base miplets. J. MoL. Struc. (Thechem. in the pres.Chinese Science Bulletin Vol. 47 No. 5 March 2002中国煤化工385MHCNMHGNOTESopimize linear RNA molecular structure by Discovermodule of Insight II . Speically the Amber force-fieldluas adopted. and the Derivative was set as 0.1. Weyadopled steepest descents method to minimize RNAstructure for 400 steps. and then conjugated the gradientmethod to minimize 1000 steps. The optimized linearaRNA structure was used for conformational analysis ofsingle- stranded RNA.1CQ5 was the NMR -determined hairpin structure ofRNA composed of 43 nucleotides (fig. 2()). IDUH wasthe X-ray crystal diffraction-determined structure com-(bposed of 45 nucleotides (fig. 2()). There were 37 sequen-tial identical nucleotides between 1CQ5 and IDUH, andtheir predicted secondary stuctures were approximatelyuniform (figs. 2(b), (d)). So we built the ternary hairpinstructure of 1DUH on Silicon Graphics 02 Workstationbased on the secondary strucure of IDUH and the hairpinstructure of 1CQ5 (fig. 2(e)). The 1DUH hairpin structurewas used for analysis of the conformational correlation-Fig. 3. The structure of sungle stranded RNA. [a) The single stranJedship between hairpin structure and is corresponding sin-RNA structure of 283D determincd hy the X-ray crystal ditriction: thigle-stranded structure. We deposited the hairpin structurelinear exlended RNA structure including random nuclevnde scquenceof IDUH in PDB with ID of IILI.built by therertical method. wrth encrgy of 465849251 kJimol; (C! theconformnation of RNA structure that vas energy upumletu fum linear2 Results and discussionextended RNA, with energy of -47769.13 kJ/mol.1 i ) The topological configuration features of sin-Oligodeoxynuclcotide-directed immunoelectron mi-gle-stranded mRNA. The ribosome small subunit andcroscopy experimental technique for ribosome deducedlarge subunit combine with mRNA at the beginning ofthere was a 6 bp U-urn conformation at the mRNA de-translation, then mRNA enters the ribosome by passingcoding regions! Such U-turm conformation was conven-through the conduit between large subunit and small sub-ient l0 accommodate the simultaneous condon-anticodonunit. At this time, the 16S RNA located on the neck ofinteraction of the tRNAs before and after their transloca-small subunit unfolded the hairpin structure of mRNAtion. However, what is the conformational feature of theso the conformation of translating regions of mRNA is incomplete single-stranded mRNA or of the regions out ofa single-stranded state while read by the ribosome.decoding region? The computer simulation unfolding forWe analyzed 6 X-ray crystal diffraction-determinedthe theoretical and experimental RNA hairpin structuresingle stranded fragments (1OSU, IRXA, 255D, 283D，revealed that after the hairpin structures were unfolded.333D and 373D) of RNA structures from PDB, and foundthe single-stranded regions corresponding to hairpin stemthat their structures all appeared as different helicalregions often appeared as regular helical contformation.conformations no matter whether the fragments were longbut hose corresponding to hairpin loop regions often a4p-or shor. The structure of 283D is shown in fig. 3(a). Wepeared as extended helical conformation or curly confor-can observe that there is an obvious bend where base G ismation, and to a certain extent the single-stranded mRNAlocated. and the two regions located at ight and left sidesstructure remained the conformational features that itsof the bend appear as different helical conformations, re-corresponding hairpin structure had.spectively. In addition, the energy minimization for the(1) The topological configuration of singestrandedlinear RNA structure (fig. 3(b)) indicated that the linearIUBQ mRNA. The fourth hairpin o[ IUBQ mRNA. itsRNA structure was not stable in energy. After the structurecorresponding single-stranded structure. and the two ex-u as optimized. it became an iregular helical structure (ig.tended singe-stranded structures are shown in fig I. We31C)), which was more similar to its actual structure undercan see from fig. I(c) that the single-stranded regionsthe physiological condition because water and K* wereCCAGCAGAGG and UCUUUGCUGG corresponding toadded into the sysiem before energy minimization. Thestems (lined out by dashed lines) were periodic helix. butenergy of optimized RNA structure was lower than before.the region UUGA corresponding to loop was compara-In conclusion, the single-stranded mRNA was a kind oftively extended helix. Furthermore, the single-strandedmacromolecule: it was impossible for its structure to pre-structure shown in fig. l(C) was stretched lwice. and wesent linear conformation. However, such single-strandedgot two exlended structures shown in figs. ld1. (cI. WemRNA usually appeared as helical conformation that wasfound that they still remaincd the conformational fealuredriven by base stacking.of the periodic helical region altermating with the cxtcnded中国煤化工。386ChineMarch 2002MHCNMHGNOTES ,helical region alhough they were stretched, but alongsuructures including symmetric internal loop. After theywith the hairpin unfolding and single sranded structurewere unfolded, the conformations of the two sungle-stra-successive stretching, their energies gradually increased.nded regions corresponding l0 intermal loop were similarThis revealed mRNA hairpin unfolding and stetchingto each other. and had a unisonous relation with the con-need geting over energy barrier in vivo, which might beformation of the contextual stems. However. the confor-completed by many factors including GTP.mations of the two strands located at asymmetric loops,(2) The topological configuration of single-strandedbulges, or other bigger loops were dissimilar to each other.IDUH. We got the single-stranded crystal structure ofAfter the hairpin structures were unfolded, the conforma-1DUH from PDB. and built its temary hairpin structuretions of the single -stranded regions corresponding to loops(fig. 2(e)) on Wiorkstation (see sec. 1). We found that thewere different from those corresponding to stems, the for-single-stranded regions corresponding to hairpin stemsmer usually presented iregular coil or bend. and the ltterwere the regular helical conformation (lined out by dashedusually presented regular helical conformation. Moreover,lines). but those cortesponding t0 loops were iregularwe found a palpable tendency-- -a bend occurred at thecoils or different bends.single-stranded regions corresponding 10 asymmetric(3) The lopological configuration of the single-loops or intemal loops where the A (sometimes the G) wasstranded RNA unfolded from the hairpin structure includ-located. However, it is worth emphasizing that ouring symmetric intemal loop. The symmetric intermalsimulation unfolding of all the samples indicated that theloop in the hairpin structure usually appeared as compara-conformations of the single- stranded RNAs were closelytive "smooth" conformain. Fig. 4 shows the hairpincorrelative with those of their corresponding hairpinRNAs no matter what the structural features of loopswere， and the conformational feature of the sin-gle-stranded RNA all showed itself as periodic helical日conformation altemating with relative cxtended helix orcurly conformation.(i) The conformational analysis of single-strandedmRNA. The computer simulation of unfolding RNAhairpin indicated that the unfolded structure of sin-gle-stranded RNA was stretched, and the pitch increased.Table I lists the distance changes between adjacent P at-oms before and after 1EOR hairpin being unfolded. Thedistances between the adjacent P atoms of the sin-1A51IEOR28SPgle-stranded RNA structures increased averagely by 1.07小A compared to those of the corresponding hairpin struc-tures. For AI3-GI4 and G18-CI9, the distances were 8.12A and 8.16 A. respectively. The torsion angle changes of1EOR before and after unfolding are listed in table 2.IASIwhere we observe that x angles change slightly. whichindicates that the bases orientation of the whole chainchanges lt; β angles of the single-stranded RNA areusually more than those of the corTesponding hairpinstructure, and they augment clearly especially in the loop.which indicates the conformation of single-stranded RNAcorresponding 1to loop region changes obvlously; more.over, the changes of other torsion angles. such asa,y. δ.and E. show at various levels. but they are unconspicuous,which implies that the conformation of the unfolded令心single-stranded RNA structure is closely correlauive withthat of the corresponding hairpin structure. These resultswere identical with the observed results of IDUH andtig. 4. "The NMR-detrmined harpin stuctures including synumetricIUBQ mRNA.intemal loops and their corresponding theoretical single-stranded struc-ture. The PDB IDs were IASI. IEOR. and 28SP, respecively. A. U, C.3 Conclusionand C indicated nucleotides constituting the symmetric intemal loops.The single- stranded regoos lined out corresponded with the regions ofDuring the translation process in vivo. there were》mmetric inlermal lops in haipin strucrures.complex and dynamic interactions among ribosome,Chinese Science Buletin Vol.47 No. 5 March 2002中国煤化工387MYHCNMHGNOTESTable L The distances between adjacent P atoms of IEOR betore and after beng unfolded (unt: A,1EORG2C3 C3-04G4-A5A5-A6A6-G7 (17-U08 T'8-C9 C9-G10 (Gi0AT ANI:AI:Dh'6816.446.226.476.14 5.736.285.707957.516.896.857.637.776.727.591EOR A12-A13 A13-G14 G14-A15 A15-016 L16-G17 (17-G18 G18C19 C19-G20 (20-C2! C2-C2DI6567.066.606.536.195.5476.305.50_D7838.127.667.617.298 166.67a1 Dh indicates hairpin state: b) Ds indicates single- stranded state.Tabie2 Noclotide torsions of 1EOR before and afher beng untolded(" )IEORa(P- 0<1β{O、C, )_y(Cs-C)8(C. Ce(C-031ξO;-Pi广 xG1h"56.1095.68176.21-91.02-- -11551GIs152.83-65.84-1182G2h-81.41166.8675 6845.26-171.26G2:-71.23163 6483.03-166 15C3h-103.33- 160.08-901.96-154.11-80.99169.8063.42-161.01-85.66.155.34 .G4h-69.65-179.3461.12-156.811.09-156.33(i4<-66.94-177.4355.48-155.17-3.35-|5% 59As5h-135.9063.36173.08114-12791-89.12-174.1%AS<-135.2279.40-171.50104.39-128.69-172.316h103.72152.07- 160.9992.04-124.61-174.96Abs115.34178.87-167.9284.20- 145.30-17109G7h-67.60.155.2688.88_5R 7017251-158.68-58.79-93.29-167 2078.72~167.48L'8h-78.75-155.2832.6193.46178.1↓-146.4537.84170.34-59.93-149.18('9h-89.19172.0880.4990.61-139.13-67.4-152 91)C9s-99.25-165.7775.567843-159.02-8.31-152.9%GI0h- 1005-174.3063.5191.84-134.79-89.60-1-1.24G10s-103.79-164.9263.2483.47-131.04-55.58一144.66148.62-179.49-48.9695.90-151 30A1ls132.76169.7961.09175.1413.60142.4259.51-174.4593.81-176.29AlEh161 40-159.5193.01A12s178.45-169.33-145.48-:0.73Al3h .109.25-171.02-152.51-24.04 .170.17Al3s129.88 .-179.15-173.05-156.28-168.941GI4h-100.43-179.5278.111 .92.84-168.50.-70.43-i5s.16GI4s179.83167.3463.1079.77175.47-15.20AI5h-67.38169.2663.4587.18-175.84151.04A155-68.41176.8060.0079.75177.85-14X.84-170.30148.01156.3496.95-99.15129.80171.65C165166.74168.90174.7887.79-98.77-130.84-169,916i17h148.2239.13y.07_ 156.221910-155.93-105.78-174.2554.63-165.72(S4.61GI8h .132.15-164.11-74.0794.86-160.86-176.15(i15s135.60- 173.84-175.91-166.97-58.50.71.56CI9h-68.28-166.0549.7888.21-149.31-47.16. i64.01CI9s-61.35-172.3250.S780.56-162.66-39.22-568.69G20h-85.09153.8186.2286.01 .-12724-81.43:7<.14G20s-74.22160.8477.4179.71-142.04 .-i 76.11-86.34-170.25422490.93- 152.63159.63(C21s-86.04-173.1046.41- 162.63-99.55-!5:87-51.45150.1158.3088.22-14516474167.80al h indicates the hairpin state; b) S indicates single stranded statetRNA. and mRNA. For example. the mRNA hairpin.gion and anticodon, etc. Based on the theoretical structurestructure was unfolded into the single-stranded structureof mRNA hairpin and the experimental RNA structuresby the small subunit of ibosome; the conformations ofdetermined by NMR or the X-ray crystal difraction. w比mRNA decoding region and tRNA anticodon were ad-simulated and analyzed the conformational fearures ofjusted subly t0 satisfy the pairing between decoding re-single-stranded mRNAs in large scale scope within 90中国煤化工。388ChineMarch 2002YHCNMHGNOTES，nucleoides". and found that the lopological configurationof single-stranded mRNA was closely related to uhat of thecorresponding mRNA hairpin, which appeared as regularTwo-state kinetics character-helical conformation altermating with extended helix oriregular coil. Such conformation of single strandedized by image analysis ofmRNA could cause diferent translational rates while ri-bosome reading different regions of mRNA. The resultsnuclear magnetic resonancehere provde valuable information for further study on thespectrasecondary structure formation of nascent polypeptide andQAprotein folding while mRNA is translated by ribosome.Acknowledgements This work was supprted by the National NaturalYAN Yongbin' , LUO Xuechun , ZHOU Haimeng'Sctence Foundation of China (Grant No.397704181 and the Knowledge& ZHANG Riqing^Innonation Program of the Chinese Academy of Sciences (Grant No.1. National Laboratory of Biomembrane and Membrane Botechnvlogy.KJCX 1.081Department of Biulogical Scences and Biocchnolngy. Tsinghua LniReferencesversity, Beijing 10080 China;2. Protcin Science Laboratory of the Ministry of Education. LDepartment1 Shaper. E G. The secondary structure of mRNA from Escherichuof Biologica! Sciences and Biotechnology, Tsinghua l.niversity. Bel-rol: its possible role in increasing the accuracy of ranslation.jing 10084. ChinaNucle Acids Res . 1985. 13: 275.Corespondence should br udunessed to Yan Yongbin le rail: ybyan@Garrelt. R. A.. Mechanics of the ribosome. Nature, 199. 40X:lsinghua .cdu.cn3. Adam>. J, M.. Jeppesen. P, Sanger. F el al. Nucletide sequcnceAbstract Nuclear magnetic resonance (NMR) spectros-from the coal prolein cistron of R17 bacteriophage RNA. Narure,copy has become an important tool in modern biologicalresearch. NMR spectra image analysis can be used to analyze1. Fesco. J. R.. Aiberts. B. M. Doty. P. Molecular derails of thethe kinetics of biomacromolecular conformational changes.The relationship betweea the image parameters and the pro-scondary structure of rihonucleie acid, Nature, 1960. 188: 4y8.5. Limn. v Venclovas. C. Spirin, A. et al.. How are IRNA andtein dynamics was investigated by using a small globularmRNA arranged in the ribosome? An atempt to cortelate theprotein o-conotoxin S03 (0-CTX SO3), The physical mean-sitrechemistry of the tRNA. mRNA inleraction with constraintsjogs of the image parameters were characterized from the:mposed by the ribaosomal topography, Nucleic Acids Res. 1992，results. Comparison of the data from the traditional integral20 2627.area of specific resomance peaks method and the NMR image5. Iserentan. D.. Fies. w.. Secondary strucrure of mRNA and efi-analysis method showed the advantages of using NMR spec-ciency of tanslation initiation. Gene. 1980, 9: 1tra image analysis for kinetic analysis of two-state processes7 Zhang. S.. Gioldman. E, Zubay, G, Clustring of low usagemonitored by 1D proton NMR.codons and ribosome movement, J. Theor. Biol, 1994, 170: 339.Key words: image analysis, proton nuclear magnetic resonance, re-s. Plat. T.. Termunation of transription and is regulanvon in theductive unfolding, unfolding dynamics.mnptophan operon of F. coli. Cell, 1981. 24: 10.9. Thanaraj. T. A. Argos. P. Ribosome -medratcd tanslatioad pauseNuclear magnetic resonance (NMR) spectroscopyand prutein domain organization, Protein Science, 1996. 5: 1594.has become an important tool for modem biological re-10. Conn. G L.. Draper, D. E.. RNA structure, Current Opinion insearch with the ability to provide atomic resolution struc-Stnucrural Biology. 1998. 8: 278.11. Berman. H. M. Westrook. J. Feng. Z. ct a.. The Prolcin Datatural information of biological macromolccules insemi-physiological conditions'". Various types of NMRBank. Nuclec Acids Res.20012. Vijay. Kumar. s.. Bugg. C. E, Cook. w. J. Structure of ubquinexperiments have been carried out on a broad range ofmacromolecules to determine the structure. dynamics,refined at 1.8 A resolurion.J, Mol. Biol. 1987. 194: 531.1 Adzhubei, I. A.. Adzhubei, A. A.. Nridle. S.. An integrated se-mobility, and interactions. Recently, NMR has also playedquence. smncure database incorporating matching mRNA se-an important role in protein dynamics studies related toquence. amino acid sequencc and protein three-dimensionalprotein functions-. Though being conceptually helpful torstructure data. Nucleic Acids Res.. 1998. 26: 327.the analysis of the complex 'H NMR spectra of proteins,i4. .Mlatheus. D. H. Sabina. J. Zuker. M. el al, Hxpanded sequencecomplete assignment is usually dificult for the analysis ofdependence of thermodynamic parameters mproves prediction oflarge protein structures, protein-protein interactions, en-RNA secondary structure. J Mol. Biol.. 1999. 288: 911.zyme functions, and protein-ligand and protein-nucleic5. Frank. J Verchoor, A.. L. Y. el al. A model of the taslatonalacid complex structures. In contrast to the typical com-apparatus bused on a three-dimensional reconstmuction of the Es-plete assignment. a new approach to image analysis ofchenchiu colt ribosome. Biochem. Cell Biol.. 1995. 73: 757.NMR spectra has recently been introduced, for kinetic16. Malhotta. A. Haney. S. (C. A quanitive model of the E. roli 16Sanalysis in protein reductive unfolding studies'RNAin the 305 nbusumal subunit.1. Mol. Bivl, 19949. 240: 308.Previous work- showed how the wwo-phase proteinWater, P. Keenan. R.. Schmit7. U. SPR- -Where the RNA andreductive unfolding process could be analyzed by usingmenbrane wurlds meet, Science. 2000, 287: 1212.image analysis of H NMR spectra, though the meanings(Received October 31. 2001of the image parameters were not fully understood due toChinese Science Bulletin Vol. 47 No.5 March 2002中国煤化工389MYHCNMHG