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In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex.

Abstract：

:The genomes of hyperthermophilic Archaea encode dozens of methylation guide, C/D box small RNAs that guide 2'-O-methylation of ribose to specific sites in rRNA and various tRNAs. The genes encoding the Sulfolobus homologues of eukaryotic proteins that are known to be present in C/D box small nucleolar ribonucleoprotein (snoRNP) complexes were cloned, and the proteins (aFIB, aNOP56, and aL7a) were expressed and purified. The purified proteins along with an in vitro transcript of the Sulfolobus sR1 small RNA were reconstituted in vitro, into an RNP complex. The order of assembly of the three proteins onto the RNA was aL7a, aNOP56, and aFIB. The complex was active in targeting S-adenosyl methionine (SAM)-dependent, site-specific 2'-O-methylation of ribose to a short fragment of ribosomal RNA (rRNA) that was complementary to the D box guide region of the sR1 small RNA. The presence of aFIB was essential for methylation; mutant proteins having amino acid replacements in the SAM-binding motif of aFIB were able to assemble into an RNP complex, but the resulting complexes were defective in methylation activity. These experiments define the minimal number of components and the conditions required to achieve in vitro RNA guide-directed 2'-O-methylation of ribose in a target RNA.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Omer AD,Ziesche S,Ebhardt H,Dennis PP

doi

10.1073/pnas.082101999

keywords:

subject

Has Abstract

pub_date

2002-04-16 00:00:00

pages

5289-94

issue

8

eissn

0027-8424

issn

1091-6490

pii

99/8/5289

journal_volume

99

pub_type

杂志文章

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