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P-B3-04 Ribosome facilitates refolding of rhodanese and lysozyme by suppressing aggregation
B3 Structures and new functions of RNA P-B3-02 RADIO?BK&OGICAL IMPLICATIONS OF RNA TARGET ANALYSIS E. Kempner’ and S. Bernstein2 ‘NIAMS and‘NE&NIH (USA) The reported target sizesfor ribozymes irradiated at -135°C (PNAS, 1996) are Ribozyme structural Activity
P-B3-01 TkIE STRUCTURE AND MELTING BEHAVIOR OF THE LOOP I FRAGMENT OF COLE1 RNAI. HUANG, ‘II-I, LIN’, TH, LM’, I-ID, YANG, JL’, KABERDIN, VR’ and LM-CHAO, S*. ‘Inst. Biomed. Sci., and ‘Inst. Mol. Biol., Academia
Sinica (Taiwan)
We have employed NMR techniques to probe the structure of several RNA fragments related to the loop I region of ColE 1 RNA I. The secondarystructure deduced from NMR spectra suggeststhat the loop I fragment consistsof a stem of 11 base-pairs which contains a U-U base-pairand a bulged C base, a 7 nucleotide loop, and a single-stranded S’end of 12 nucleotides.The UV-melting study of a 42-mer further revealed a multi-step melting behavior with transition temperatures of 32’C’ correspondingto the opening of the loop region and 71°C due to the overall melting of the stem region. The structural distortion induced by the U-U mismatch was fiuther studied with a synthesized 9-mer RNA containing the U-U mismatch at the center. The results showed that the stem region assumes an A-form structure with distortion restricted only at the base pairs adjacent to the U-U mismatch. Molecular dynamic simulation further reveal that the U-U pair forms a pair of hydrogen bonds through a mixed 2-3 and 3-2 bonding of the U-U bases.
79 kDa 80 14.8k1.8 368 319 15.9f4.2 The structural target size agrees with the mass of the complete RNA molecule; therefore a radiation hit anywhere cleaves the polynucleotide backbone. The activity target size, however, is much smaller and agrees with the minimal active size of the ribozymal unit (15.5 kDa). Radiation hits outside this unit do not damage that part of the ribozyme because the ribose sugar moiety apparently blocks energy transfer along the polymer. Shortdistance transfers of energy would increase the predicted target size beyond the minimal unit. Our data suggest that there is a maximum energy spread of 15 nucleotides, and probably much less. Alternatively, this “additional mass” may be due to associated water, and would predict not more than l-2 waters per nucleotide. Both of these values are much lower than previously suggestedestimates.
P-B3-04
P-B3-03 X-RAY “&OOTPRINTING” ANALYSIS OF THE MG DEPENDENT FOLDING OF THE TETHYMENA THERMOPHILA
RIBOSOME FACILITATES REFOLDING OF RHODASESE AND LYSOZYME BY SUPPRESSING AGGREGATION Ghnsh N’. Haara K2. P Swkar S. N3. Department of Biochemistry and Biophysics University of Kalyani. Kaiyani-741235. W.B.. India Parpar: The it~\alvemetu of ribosome ta protein folding has recently beea recognized. Ribosomc facilitated recoastitutron al acttvte of a cntplc of detmtwed proteins. In th present nork tbc mode of acttoa of ribxome aas investigated b! usmg two monomctic proteins rhadanese atd Iysozyme. Methods: Rhcdatmse and Iysvzyme was deaaturcd in buff& contains 6Y Guaaidme-HCI. Deaaturcd etqme aas diluted tata buffer Aggrcgatioa (Turbidtty) was measured as absorbance at 320 am in presetue or absence of different concentrations of E. Co/i 70s ribosome along wtth enzyme actnlty Results: 111vitro dcnntumtwn of these two proteins uerc trrcvcrslblc. siacc the refolded pol!peptidc clams aggrcgatcd rapidly. as fotmd directI\ b a stmng. cotxeattation dependcat increase itt turbidity Ribosome inhthted aggregation as indicated by specific supptession of turbidtty in an energy independent way. Hcnvevcr. mere suppression of aggregation reactions tbat competed nith correcl protein folding. was not enough to reactivalc lbe protein as revealed b! enzyme acti\@ Conclusions: We threfore coaclt& tltat aggregation suppression alone can not reactivate the protein otkr structural coadttions like keeping thiol groups in reduced condition for rlnxlanese aad mainletmnce of S-S bonds for Iwxymc were aecessa~ for the reconstitution of the acme &e
Results: Studiesof the folding kinetics of the Tet-
ruhymena group I intron have been initiated using
x-ray “footprintmg” in the minutes timescale. The
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P-B3-01 TkIE STRUCTURE AND MELTING BEHAVIOR OF THE LOOP I FRAGMENT OF COLE1 RNAI. HUANG, ‘II-I, LIN’, TH, LM’, I-ID, YANG, JL’, KABERDIN, VR’ and LM-CHAO, S*. ‘Inst. Biomed. Sci., and ‘Inst. Mol. Biol., Academia
Sinica (Taiwan)
We have employed NMR techniques to probe the structure of several RNA fragments related to the loop I region of ColE 1 RNA I. The secondarystructure deduced from NMR spectra suggeststhat the loop I fragment consistsof a stem of 11 base-pairs which contains a U-U base-pairand a bulged C base, a 7 nucleotide loop, and a single-stranded S’end of 12 nucleotides.The UV-melting study of a 42-mer further revealed a multi-step melting behavior with transition temperatures of 32’C’ correspondingto the opening of the loop region and 71°C due to the overall melting of the stem region. The structural distortion induced by the U-U mismatch was fiuther studied with a synthesized 9-mer RNA containing the U-U mismatch at the center. The results showed that the stem region assumes an A-form structure with distortion restricted only at the base pairs adjacent to the U-U mismatch. Molecular dynamic simulation further reveal that the U-U pair forms a pair of hydrogen bonds through a mixed 2-3 and 3-2 bonding of the U-U bases.
79 kDa 80 14.8k1.8 368 319 15.9f4.2 The structural target size agrees with the mass of the complete RNA molecule; therefore a radiation hit anywhere cleaves the polynucleotide backbone. The activity target size, however, is much smaller and agrees with the minimal active size of the ribozymal unit (15.5 kDa). Radiation hits outside this unit do not damage that part of the ribozyme because the ribose sugar moiety apparently blocks energy transfer along the polymer. Shortdistance transfers of energy would increase the predicted target size beyond the minimal unit. Our data suggest that there is a maximum energy spread of 15 nucleotides, and probably much less. Alternatively, this “additional mass” may be due to associated water, and would predict not more than l-2 waters per nucleotide. Both of these values are much lower than previously suggestedestimates.
P-B3-04
P-B3-03 X-RAY “&OOTPRINTING” ANALYSIS OF THE MG DEPENDENT FOLDING OF THE TETHYMENA THERMOPHILA
RIBOSOME FACILITATES REFOLDING OF RHODASESE AND LYSOZYME BY SUPPRESSING AGGREGATION Ghnsh N’. Haara K2. P Swkar S. N3. Department of Biochemistry and Biophysics University of Kalyani. Kaiyani-741235. W.B.. India Parpar: The it~\alvemetu of ribosome ta protein folding has recently beea recognized. Ribosomc facilitated recoastitutron al acttvte of a cntplc of detmtwed proteins. In th present nork tbc mode of acttoa of ribxome aas investigated b! usmg two monomctic proteins rhadanese atd Iysozyme. Methods: Rhcdatmse and Iysvzyme was deaaturcd in buff& contains 6Y Guaaidme-HCI. Deaaturcd etqme aas diluted tata buffer Aggrcgatioa (Turbidtty) was measured as absorbance at 320 am in presetue or absence of different concentrations of E. Co/i 70s ribosome along wtth enzyme actnlty Results: 111vitro dcnntumtwn of these two proteins uerc trrcvcrslblc. siacc the refolded pol!peptidc clams aggrcgatcd rapidly. as fotmd directI\ b a stmng. cotxeattation dependcat increase itt turbidity Ribosome inhthted aggregation as indicated by specific supptession of turbidtty in an energy independent way. Hcnvevcr. mere suppression of aggregation reactions tbat competed nith correcl protein folding. was not enough to reactivalc lbe protein as revealed b! enzyme acti\@ Conclusions: We threfore coaclt& tltat aggregation suppression alone can not reactivate the protein otkr structural coadttions like keeping thiol groups in reduced condition for rlnxlanese aad mainletmnce of S-S bonds for Iwxymc were aecessa~ for the reconstitution of the acme &e
Results: Studiesof the folding kinetics of the Tet-
ruhymena group I intron have been initiated using
x-ray “footprintmg” in the minutes timescale. The
85