One sequence, two ribozymes: Implications for the emergence of new ribozyme folds

Science

Volumn 289, Issue 5478, 2000, Pages 448-452

  Schultes, Erik A ^a   Bartel, David P ^a  

^a WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

RIBOZYME;

ARTICLE; EVOLUTION; GENE DUPLICATION; PRIORITY JOURNAL; PROTEIN FOLDING; RNA SEQUENCE; RNA STRUCTURE; SEQUENCE ANALYSIS;

BASE SEQUENCE; CATALYSIS; EVOLUTION, MOLECULAR; GENE DUPLICATION; HEPATITIS DELTA VIRUS; MOLECULAR SEQUENCE DATA; MUTATION; NUCLEIC ACID CONFORMATION; POINT MUTATION; RNA; RNA, CATALYTIC;

EID: 0034698298     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.289.5478.448     Document Type: Article

References (38)

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22. The U73C substitution of construct HDV1, which is predicted to favor the HDV fold without changing the core of either ribozyme, does not lead to a change in the dominant fold, as determined by Pb(II) cleavage. This explains why HDV1 retains much of the ligase activity of the intersection sequence (Fig. 26). The observation that HDV1 cleavage activity is within 20-fold of the prototype HDV ribozyme indicates that the small fraction of HDV1 molecules that assume the HDV fold are highly active. Because U73C affects the fold and not the core of the ribozyme, the intersection molecules that assume the HDV fold must also be highly active.
23. 15 variants was based on two sequences that were similar to that shown in Fig. 2A and also had detectable ligation and cleavage activity: 5′-Ggactcccat tagactgggc cGCCTCCTCG CGGCgggagt tGGGccaggg aggtaagccc tttctggGCt AAgggccca and 5′-Ggactcccat tagactgggc cGCCTCCTCG CGGCgggagt tGGGccaggg aggtaagccc tttctggGCt AAggccca (lowercase letters indicate positions mutagenized such that they each had a 10% probability of changing to one of the three other nucleotides; uppercase letters indicate positions not mutagenized because of their importance for HDV self-cleavage activity). Selection for ligation activity was performed as described (35). Pool activity reached that of the prototype ribozyme by round three, at which point the pool was cloned and sequenced. Each of the 20 isolates had a different nucleotide sequence, indicating that there are many highly active sequences in the vicinity of the intersection sequence. Sequences of active clones informed the design of LIG1, LIG2, and LIG4.
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37. -1 (14). All except seven ribozymes were transcribed from synthetic oligonucleotide templates. The others (LIG P, LIG2, LIG1, INT, HDV1, HDV2, and HDV P) were transcribed from polymerase chain reaction (PCR)-amplified DNA that had been generated from plasmid DNA template and appropriate primers. This removed the small amount of sequence heterogeneity observed with long synthetic templates, eliminating concern that sequence heterogeneity might increase the apparent activity of inefficient ribozymes.
38. We thank W. Johnston for assistance with in vitro selection (23) and helpful discussions and N. Lau for assistance with Web fig. 1. We thank P. Kim as well as M. Lawrence, P. Unrau, M. Glasner, N. Bergman, and others in the lab for comments on the manuscript. Supported by the NSF/Alfred P. Sloan Foundation (E.A.S.) and the NIH (D.P.B.).