Intermediates and kinetic traps in the folding of a large ribozyme revealed by circular dichroism and UV absorbance spectroscopies and catalytic activity.
The folding thermodynamics and kinetics for the ribozyme from Bacillus subtilis RNase P are analyzed using circular dichroism and UV absorbance spectroscopies and catalytic activity. At 37 degrees C, the addition of Mg2+ (Kd approximately 50 microM) to the unfolded state produces an intermediate state within 1 ms which contains a comparable amount of secondary structure as the native ribozyme. The subsequent transition to the native state (Kd[Mg] approximately 0.8 mM, Hill coefficient approximately 3.5) has a half-life of hundreds of seconds as measured by circular dichroism at 278 nm and by a ribozyme activity assay. Surprisingly, the formation of the native structure is accelerated strongly by the addition of a denaturant; approximately 30-fold at 4.5 M urea. Thus, the rate-limiting step entails the disruption of a considerable number of interactions. The folding of this, and presumably other large RNAs, is slow due to the structural rearrangement of kinetically trapped species. Taken together with previous submillisecond relaxation kinetics of tRNA tertiary structure, we suggest that error-free RNA folding can be on the order of milliseconds.
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Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637, USA. taopan@midway.uchicago.edu.com
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D001412:Bacillus subtilis / Q000201:enzymology*; D002384:Catalysis; D002942:Circular Dichroism; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism; D043262:Ribonuclease P; D012965:Sodium Chloride / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet; D014508:Urea / Q000494:pharmacology
D016337:RNA, Catalytic; D012360:RNA, Transfer, Phe; D015636:Magnesium Chloride; D012965:Sodium Chloride; D014508:Urea; D004722:Endoribonucleases; D043262:Ribonuclease P