TY - JOUR
T1 - The global conformation of an active hammerhead RNA during the process of self-cleavage
AU - Amiri, Khaled M.A.
AU - Hagerman, Paul J.
N1 - Funding Information:
The authors thank Dr David McKay for providing interhelix angles for his RNA crystal structure prior to publication, and Elsi Vacano for performing the hydrodynamic computations using DIFFROT. This work was supported by a grant from the National Institutes of Health (GM 35305 to P.J.H.), and by a graduate fellowship (to K.M.A.A.) from the United Arab Emirates University.
PY - 1996/8/16
Y1 - 1996/8/16
N2 - The RNA 'hammerhead' domain is a small element of secondary structure found in the genomes of certain plant pathogens. It possesses a core of conserved sequence at the conjunction of three helix stems, and is capable of undergoing self-cleavage in the presence of divalent cations. Both crystallographic and solution studies suggest that the domain is highly structured, with the three stems assuming a Y-shaped global conformation; however, such studies have employed either RNA analogues that were catalytically inactive, or conditions of temperature and pH for which rates of self-cleavage are slow. Thus, it was unknown whether such species represented the principal conformers during the cleavage process itself. In order to address this issue, a series of time-resolved, transient electric birefringence measurements was conducted in an effort to define the global conformation of an RNA hammerhead in real time throughout the process of self-cleavage. The current study demonstrates that the angular relationship between the two helices that flank the cleavage center is essentially unchanged between the pre-cleavage and post-cleavage forms. Moreover, despite the fact that at least one kinetic intermediate is formed during the self-cleavage reaction, there is no evidence for the existence of a significant population of intermediates with altered global conformation during cleavage. Thus, any conformational isomerism that may occur is likely to be relatively localized to the active center. Finally, it was observed that sequence elements lying outside of the conserved region, at the base of stem I, influence interhelix geometry. The current results are consistent with a structural model in which the active center possesses similar conformations pre-cleavage and post-cleavage. Such a model would help to explain the significant rate of reversal of the cleavage reaction (self-ligation).
AB - The RNA 'hammerhead' domain is a small element of secondary structure found in the genomes of certain plant pathogens. It possesses a core of conserved sequence at the conjunction of three helix stems, and is capable of undergoing self-cleavage in the presence of divalent cations. Both crystallographic and solution studies suggest that the domain is highly structured, with the three stems assuming a Y-shaped global conformation; however, such studies have employed either RNA analogues that were catalytically inactive, or conditions of temperature and pH for which rates of self-cleavage are slow. Thus, it was unknown whether such species represented the principal conformers during the cleavage process itself. In order to address this issue, a series of time-resolved, transient electric birefringence measurements was conducted in an effort to define the global conformation of an RNA hammerhead in real time throughout the process of self-cleavage. The current study demonstrates that the angular relationship between the two helices that flank the cleavage center is essentially unchanged between the pre-cleavage and post-cleavage forms. Moreover, despite the fact that at least one kinetic intermediate is formed during the self-cleavage reaction, there is no evidence for the existence of a significant population of intermediates with altered global conformation during cleavage. Thus, any conformational isomerism that may occur is likely to be relatively localized to the active center. Finally, it was observed that sequence elements lying outside of the conserved region, at the base of stem I, influence interhelix geometry. The current results are consistent with a structural model in which the active center possesses similar conformations pre-cleavage and post-cleavage. Such a model would help to explain the significant rate of reversal of the cleavage reaction (self-ligation).
KW - RNA processing
KW - RNA structure
KW - Replication
KW - Ribozymes
KW - Transient electric birefringence
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U2 - 10.1006/jmbi.1996.0446
DO - 10.1006/jmbi.1996.0446
M3 - Article
C2 - 8757281
AN - SCOPUS:0030590218
SN - 0022-2836
VL - 261
SP - 125
EP - 134
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -