THIS PROJECT INVESTIGATES COFACTOR RNA CONJUGATES TO SHED LIGHT ON THE ORIGIN EARLY EVOLUTION AND EVOLUTIONARY HISTORY OF LIFE. THE BIOLOGY AND BIOCHEMISTRY OF THIS NEW CLASS OF RNAS RECENTLY OBSERVED IN BACTERIAL RNA FRACTIONS WILL EXPAND OUR UNDERSTANDING OF HOW NUCLEIC ACIDS AND THE SMALL MOLECULE METABOLOME WORK TOGETHER HOW THEY MAY SHAPE METABOLIC FLUX IN MODERN ORGANISMS ANDHOW THEY MAY HAVE CONTRIBUTED TO CATALYTIC DIVERSITY IN ANCIENT RNA ENZYMES. THE TWO AIMS BUILD UPON STRONG LITERATURE PRECEDENT AND PRELIMINARY DATA COMBINING INNOVATIVE EXPERIMENTAL AND BIOINFORMATICS APPROACHES IN AN INTEGRATED EXAMINATION OF A NOVEL CLASS OF RNA TRANSCRIPTS.AIM 1 BACTERIAL COA RNA CONJUGATES. RNA TRANSCRIPTS THAT CARRY COVALENTLY ATTACHED COENZYME A COA HAVE BEEN UNAMBIGUOUSLY DEMONSTRATED TO EXIST IN E. COLI AND S. VENEZUELAE BUT NOTHING IS KNOWN ABOUT THEIR BIOLOGICAL SIGNIFICANCE. AN INTRIGUING POSSIBILITY SUPPORTED BY IDENTIFICATION OF ACETYL AND SUCCINYL COA ESTER CONJUGATES TO RNA IS THAT THEY ARE INVOLVED IN RNA MEDIATED MANIPULATION OF SMALL MOLECULE METABOLISM. WE WILL ESTABLISH THE IDENTITIES OF COA RNA CONJUGATES IN MULTIPLE BACTERIAL SPECIES USING AN AFFINITY CAPTURE STRATEGY SIMILAR TO ONE USED RECENTLY TO IDENTIFY NAD RNA CONJUGATES IN E. COLI. WE WILL ALSO DETERMINE SEQUENCE AND SECONDARY STRUCTURE REQUIREMENTS FOR CONJUGATE FORMATION AND USE THIS INFORMATION TO ESTABLISH PHYLOGENETIC DISTRIBUTIONS OFCOA RNA AMONG SEQUENCED GENOMES. OUR RESULTS WILL PROVIDE THE FIRST CLEAR PICTURE OF THIS PREVIOUSLY UNRECOGNIZED FACET OF RNA BIOLOGY AND THEY WILL ENABLE FUTURE STUDIES EXPLORING THEIR BIOLOGICAL ROLES. SOME OF THE IDENTIFIED TRANSCRIPTS MAY REPRESENT TRUE MOLECULAR FOSSILS HELD OVER FROM THE TIME OF AN RNA WORLD OTHERS WILL BE NEWER INVENTIONS THAT ILLUSTRATE THE VARIETY OF WAYSIN WHICH ORGANISMS EXPLOIT RNA TRANSCRIPTS TO MANIPULATE SMALL MOLECULE METABOLITES. THUS THESE STUDIES ARE HIGHLY RELEVANT TO UNDERSTANDING THE EVOLUTION OF METABOLIC DIVERSITY OF EXTANT LIFE ON EARTH. AIM 2 ARTIFICIAL FAD RNA CONJUGATES. ENZYMES ENHANCE THE CHEMICAL REACTIVITY OF COFACTORS THROUGH BOTH COVALENT AND NON COVALENT INTERACTIONS. WE RECENTLY IDENTIFIED RNA APTAMERS THAT STRONGLY DIFFERENTIATE BETWEEN OXIDIZED FAD AND REDUCED FADH2 FLAVINS. WE WILL EXAMINE BOTH NON COVALENT AND COVALENT FLAVIN RNA COMPLEXES FOCUSING ON ESTABLISHING HOW THEIR MOLECULAR INTERACTIONS INFLUENCE INTRINSIC CHEMICAL REACTIVITY OF THE BOUND FLAVIN AND THE STRUCTURAL BASIS FOR THESE INFLUENCES. DIFFERENTIAL BINDING ENERGY IS USED BY FLAVOPROTEIN ENZYMES TO PERTURB INTRINSIC REACTIVITY HALF REACTION REDUCTION POTENTIAL THUS PROVIDING A STRONG CHEMICAL RATIONALE FOR THIS APPROACH. THE RESULTS FROM THIS STUDY WILL PROVIDE CHEMICAL CONSTRAINTS ON HOW TO CONNECT MODERN RNA METABOLITE CONJUGATES WITH THEIR POTENTIAL BIOCHEMICAL ROLES IN ANCIENT AND MODERN? METABOLISMS. THESE STUDIES WILL ADDRESS LONG STANDING MODELS FOR THE EVOLUTIONARY ORIGIN OF NUCLEOTIDE COFACTORS IN WHICH THESE MOLECULES ARE PROPOSED TO HAVE ENHANCED THE CATALYTICREPERTOIRE OF ANCIENT RIBOZYMES. THE PROJECT IS HIGHLY RELEVANT TO NASAS EXOBIOLOGY AND EVOLUTIONARY BIOLOGY PROGRAM AND TO THE NRAS EARLY EVOLUTION OF LIFE ANDTHE BIOSPHERE AREA OF EMPHASIS. WE ANTICIPATE THAT THE PROJECT WILL PROVIDE CRITICAL ADVANCES RELATED TO UNDERSTANDING SMALL MOLECULE METABOLISM AND RNA BIOLOGY IN MODERN ORGANISMS AND TO UNDERSTANDING RNAS ABILITY TO PROVIDE METABOLIC CATALYSIS IN AN RNA WORLD. THUS THESE STUDIES ARE HIGHLY RELEVANT TO FIVE SPECIFIC AREAS OF INTEREST TO NASA 1 EARLY EVOLUTION OF LIFE AND THE NATURE OF THE MOST PRIMITIVE ORGANISMS 2 THE TRANSITION TO MODERN BIOCHEMISTRY 3 EVOLUTION OF GENES PATHWAYS AND MICROBIAL SPECIES 4 THE REPERTOIRE OF DEMONSTRATED RNA FUNCTIONS AND 5 CONSTRUCTION OF ARTIFICIAL CHEMICAL SYSTEMS TO TEST HYPOTHESES REGARDING THE ORIGINAL NATURE OF KEY BIOLOGICAL PROCESSES.