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Differential chemical probing of a group II self-splicing intron identifies bases involved in tertiary interactions and supports an alternative secondary structure model of domain V

Published online by Cambridge University Press:  01 September 1998

MARIA COSTA
Affiliation:
Centre de Génétique Moléculaire du C.N.R.S., 91190 Gif-sur-Yvette, France Present address: Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California at Santa Cruz, Santa Cruz, California 95064, USA.
ERIC L. CHRISTIAN
Affiliation:
Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio 44106-4960, USA
FRANÇOIS MICHEL
Affiliation:
Centre de Génétique Moléculaire du C.N.R.S., 91190 Gif-sur-Yvette, France
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Abstract

Dimethyl sulfate modification was used to probe for tertiary structural elements in the group II intron Pl.LSU/2 from the mitochondrial pre-ribosomal RNA of the brown alga Pylaiella littoralis. Modification of the lariat form of the intron under conditions that allow both native folding and conformational homogeneity is found to be generally consistent with secondary and tertiary structural features identified previously for group II ribozymes. A comparison of chemical probing at temperatures just below and above the first melting transition illustrates the cooperative unfolding of tertiary structure and identifies novel candidates for tertiary interactions in addition to defining elements of secondary structure. Substitution of the GAAA terminal loop of domain V is shown to be compatible with retention of conformational homogeneity (despite the loss of an important tertiary interaction), but produces a concise methylation footprint in domain I at the site previously shown to harbor the receptor for that loop. The analysis also identified two nucleotide positions in domain V with novel secondary and potential tertiary structural roles. The proposed refinement of domain V secondary structure is supported by an expanded comparative analysis of group II sequences and bears increased resemblance to U2:U6 snRNA pairing in the spliceosome.

Type
Research Article
Information
RNA , Volume 4 , Issue 9 , September 1998 , pp. 1055 - 1068
Copyright
© 1998 RNA Society

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Footnotes

Reprint requests to: Maria Costa; Center for Molecular Biology of RNA, Sinsheimer Laboratories, University of California at Santa Cruz, Santa Cruz, California 95064, USA.