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J Mol Biol. 2015 Jul 17;427(14):2340-7. doi: 10.1016/j.jmb.2015.05.005. Epub 2015 May 14.

Minimal Hammerhead Ribozymes with Uncompromised Catalytic Activity.

Author information

1
Department of Chemistry and Biochemistry and The Center for the Molecular Biology of RNA, University of California at Santa Cruz, Santa Cruz, CA 95064, USA.
2
Department of Chemistry and Biochemistry and The Center for the Molecular Biology of RNA, University of California at Santa Cruz, Santa Cruz, CA 95064, USA. Electronic address: wgscott@ucsc.edu.

Abstract

We report here that a single additional trans-Hoogsteen base-pairing interaction in the minimal hammerhead ribozyme transforms an RNA sequence possessing typically modest catalytic activity into one possessing greatly enhanced catalytic activity that is instead typical of full-length natural hammerhead RNAs that have additional extensive tertiary contact interactions. Formation of this additional base-pairing interaction requires only that the substrate RNA sequence contains a U at a position seven nucleotides 3' to the cleavage site. No additions or changes are required in the minimal hammerhead ribozyme enzyme strand sequence (providing that the naturally occurring GUGA tetraloop of Stem II is maintained). This finding unambiguously demonstrates that a single Hoogsteen base-pairing interaction, in full-length hammerheads possessing this interaction, is sufficient for stabilizing the ribozyme active site, including alignment of the attacking nucleophile for the required inline hammerhead ribozyme reaction mechanism. This finding also implies that the idiosyncratic arrays of additional tertiary contacts observed in all naturally occurring full-length hammerhead sequences have evolved to prevent deleterious alternative pairing interactions within the context of the variety of natural sequences arising in vivo. Finally, this finding greatly simplifies and rationalizes the design of fast-cleaving engineered synthetic ribozymes as RNA nucleolytic reagents and as subjects for enzyme kinetics and mechanistic investigations.

KEYWORDS:

RNA catalysis; RNA structure; RNA tertiary contacts; hammerhead ribozyme; ribozyme kinetics

PMID:
25981451
PMCID:
PMC4464905
DOI:
10.1016/j.jmb.2015.05.005
[Indexed for MEDLINE]
Free PMC Article

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