The global conformation of an active hammerhead RNA during the process of self-cleavage

Khaled M A Amiri, Paul J Hagerman

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

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).

Original languageEnglish (US)
Pages (from-to)125-134
Number of pages10
JournalJournal of Molecular Biology
Volume261
Issue number2
DOIs
StatePublished - Aug 16 1996
Externally publishedYes

Fingerprint

Isomerism
RNA
Birefringence
Nucleic Acid Conformation
Plant Genome
Conserved Sequence
Structural Models
Divalent Cations
Ligation
Temperature
Population

Keywords

  • Replication
  • Ribozymes
  • RNA processing
  • RNA structure
  • Transient electric birefringence

ASJC Scopus subject areas

  • Virology

Cite this

The global conformation of an active hammerhead RNA during the process of self-cleavage. / Amiri, Khaled M A; Hagerman, Paul J.

In: Journal of Molecular Biology, Vol. 261, No. 2, 16.08.1996, p. 125-134.

Research output: Contribution to journalArticle

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