related to degree

• Dai, Xiaochang, Ph.D. in Chemistry, Scripps Research 1991 - 1998

authors

• Dai, Xiaochang
• Joyce, Gerald

publication date

• 2000

journal

• Helvetica Chimica Acta  Journal

abstract

• The Tetrahymena group I ribozyme was modified by replacing all 99 component uridine residues with 5-bromouridine. This resulted in a 13-fold reduction in catalytic efficiency in the RNA-catalyzed phosphoester-transfer reaction compared to the behavior of the unmodified ribozyme. A population of 10(13) variant ribozymes was constructed, each containing 5-bromouridine in place of uridine. Five successive 'generations' of in vitro evolution were carried out, selecting for improved phosphoester transferase activity. The evolved molecules exhibited a 27-fold increase in catalytic efficiency compared to the wild-type bromouridine-containing ribozyme, even exceeding that of the wild-type ribozyme in the non-brominated form. Three specific mutations were found to be responsible for this altered behavior. These mutations enhanced activity in the context of 5-bromouridine, but were detrimental in the context of unmodified uridine. The evolved RNAs not only tolerated but came to exploit the presence of the nucleotide analogue in carrying out their catalytic function.

subject areas

• Animals
• Evolution, Molecular
• Mutagenesis, Site-Directed
• Mutation
• Phosphotransferases
• RNA, Catalytic
• RNA, Protozoan
• Tetrahymena
• Uridine

International Standard Serial Number (ISSN)

• 0018-019X

Digital Object Identifier (DOI)

• 10.1002/1522-2675(20000809)83:8<1701::aid-hlca1701>3.0.co;2-1

PubMed ID

• 11543569

start page

• 1701

end page

• 1710

volume

• 83

issue

• 8