RNA-catalyzed thioester synthesis and the relationship between size and activity in RNA catalysis
With the rapidly increasing numbers of catalytic or functional RNA molecules being isolated by SELEX methods, it is surprising that there is no agreement as to the optimal size for random RNA libraries. While it is suggested that longer random regions are more advantageous, it is believed from an evolutionary perspective that smaller random regions are sufficient to isolate complex RNA catalysts. In order to explore the utilization of coenzymes in an RNA world as well as to determine a relationship between size and activity in catalytic RNA molecules several ribozymes have been isolated from size heterogeneous RNA pools that either catalyze coenzyme formation themselves, or catalyze reactions directly involving these coenzymes. A novel adenosine-initiated transcription by the T7 class II promoter [straight phi]2.5 has been developed to explore the ability of RNA to utilize adenosine derivatives such as coenzyme A (CoA) in catalysis. The CoA-initiated transcription method has been used to isolate a series of thioester synthetase ribozymes. Thioesters play an important role in modern cellular metabolism and a great deal of evidence exists that suggests their involvement in the origin and early evolution of life. The isolated ribozymes can utilize a variety of acyl or aminoacyl adenylate substrates to form acyl or aminoacyl thioester derivatives of CoA respectfully. Both the coenzyme synthetase ribozymes and the thioester synthetase ribozymes were isolated from a size heterogeneous RNA pool containing RNA with random regions of 30, 60, 100, and 140 nucleotides. In addition, several ester synthesizing ribozymes have also been isolated from the same heterogeneous pools. Comprehensive kinetic analyses have been conducted on these ribozymes and there appears to be an optimal random region size around 60 nucleotides. All of the ribozymes described above are linked by a synthetic scheme where the multiple chemical steps required to synthesize peptide bonds from thioester intermediates constitute a synthetic pathway catalyzed by a series of individual metabolic ribozymes in an entirely RNA-based metabolic pathway.