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Local rather than global folding enables the lead-dependent activity of the 8-17 deoxyribozyme: evidence from contact photo-crosslinking.

Abstract：

:The 8-17 deoxyribozyme is an in vitro selected enzyme capable of sequence-specific cleavage of RNA. While selected to be a magnesium and zinc-utilizing enzyme, the 8-17 DNAzyme has been shown to utilize lead for its catalysis. Fluorescence-based experiments have indicated that the magnesium- and zinc-utilizing versions of the DNAzyme-substrate complex need to form a defined tertiary structure to be active, but no such global folding is required for the lead-mediated activity. Here, we have investigated this phenomenon, including the use of contact photo-crosslinking to map the tertiary fold of the lead-dependent DNAzyme. While our results recapitulate that global folding is not required for the lead activity, they reveal strikingly distinct lead-mediated modes of activity under conditions of low versus moderate solution ionic strength. Even in very low salt buffers, where no global folding of the 8-17 DNAzyme occurs, the active site of the enzyme appears to form a distinct local fold, one that cannot be modelled easily by DNA/RNA constructs that preserve key sequence and secondary structure features of the active site.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Liu Y,Sen D

doi

10.1016/j.jmb.2009.11.028

subject

Has Abstract

pub_date

2010-01-15 00:00:00

pages

234-41

issue

2

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(09)01405-3

journal_volume

395

pub_type

杂志文章

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