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Losick Mango office hours TODAY 3-5 PM Kahne’s Office October 21 Richard Losick RNA and the Origin of Life 1. Ribozymes 2. Is the ribosome a molecular fossil from an RNA world? 3. How did life evolve? Goal: To understand how life might have evolved from an RNA world. Objectives: 1. Describe self-splicing. 2. Explain why RNase P is a true ribozyme. 3. Explain why the ribosome could be a molecular fossil from an RNA world. 4. Describe how to select for ribozymes that polymerize nucleotides. 5. Explain why evolution of self-replicating ribozymes demands compartments. With RNA we throw away many of the rules! If DNA is straight-laced and uniform, RNA is freewheeling and audacious. RNA is the non-conformist. It has ceded primacy as the repository of genetic information to DNA but it has gained versatility. It is a master architect, forming complex, threedimensional structures, and it can carry out catalysis, a trick it learned long before proteins knew how to be enzymes. In short, life probably evolved from an ‘RNA world’. - Y. Truly What is an enzyme? •Enzymes are proteins that catalyze a chemical reaction. •Enzymes have an “active site” that binds substrates through ionic, H-bonds, and/or van der Waals interactions. •Enzymes facilitate the formation of a transition state, thereby lowering the activation energy. •Enzymes have a turnover number greater than one. James Sumner purified the first enzyme, urease, in 1926 and showed it to be a protein. + H 2O urease CO2 + 2 NH3 Harvard class of 1910 For over half a century, it was believed that only proteins are enzymes. But RNA molecules can be enzymes too! RNA enzymes are known as “ribozymes.” We will consider ribozymes that: • cleave phosphodiester bonds • create phosphodiester bonds • create peptide bonds Recall mRNA splicing, which occurs in two transesterification reactions Splicing is catalyzed by an enzyme complex called the splicosome Self-splicing requires no enzyme. The intron mediates its own excision! exon intron Self-splicing intron uses a free guanine nucleotide as a nucleophile. Strictly speaking, is a self-splicing RNA a ribozyme? Is it a catalyst? Why or why not? RNase P •The first true RNA enzyme (ribozyme). •It cleaves a precursor to tRNA molecules (pre-tRNAs) at a specific site. RNase P cleaves off a segment of RNA from the transcription product of tRNA genes to generate the mature tRNA RNase P RNA subunit protein subunit tRNA substrate Main points 1. Both RNA and protein can be enzymes 2. Self-splicing introns and RNase P act on phosphorous centers 2. Is the ribosome a molecular fossil from an RNA world? Self-splicing introns and RNase P act on phosphorous centers. One ribozyme is known that acts on a carbon center. It is the enzyme at the heart of the ribosome that creates peptide bonds! TheO phosphodiester bond OH O P O CH2 O O O O A OH P O CH2 O OH C O OH 3' end The peptide bond peptide bond formation is catalyzed by “peptidyl transferase” The peptidyl transferase is a ribozyme! • Peptidyl transferase is composed of RNA. • No proteins are close to the catalytic site • The ribozyme juxtaposes the 3’ ends of the tRNAs in the P and A sites. Large subunit of the ribosome Peptide bond formation is catalyzed by a proton shuttle involving the 2’ OH The 3’ oxygen captures a proton from the 2’ OH The 2’ oxygen in turn captures a proton from the nitrogen of the amino group. Main points so far 1. The most fundamental reaction in the “protein world” is catalyzed by a ribozyme. 2. The RNA component of the large subunit of the ribosome is a ribozyme that catalyzes peptide bond formation. 3. Both ribosomal RNA and tRNA participate in catalysis. 4. Is the ribosome a fossil from an earlier, “RNA world”? 3. How Did Life Evolve The discovery of ribozymes simplified the problem of the origin of life. RNA could be the information carrier and the replicase for its own duplication. Eventually, a ribozyme evolved that could synthesize proteins. Did life evolve from an “RNA world”? What is life? Life is a system that is capable of selfreplication and that is subject to Darwinian evolution. Self-replication means the system relies only on small molecules and energy. Darwinian evolution implies variation (mutation) and phenotypic expression. Can we create an RNA molecule that is capable of self-replication and variation? Evolving an RNA replicase 1. Start with RNA molecules of random sequence. 2. Incubate RNAs with ribonucleoside triphosphates and an RNA template. 3. Size select RNAs that are slightly larger (those with polymerase activity!). 4. Mutagenize size-selected RNAs. 5. Repeat the cycle of size selection and mutagenesis. Evolving RNA molecules that can polymerize ribonucleoside triphosphates. The best ribozyme with polymerizing activity to date can generate 95-nucleotide long RNAs in a template-directed manner. Structure of an evolved ribozyme capable of carrying out phosphodiester bond formation How do we get from a ribozyme replicaase to life? Three conceptual problems: 1.The self-replication problem. 2.The Darwinian selection problem 3.The prebiotic chemistry problem. The Self-replication Problem One replicase would have to make a complete copy of another. The immediate product would be a complement of the replicase. The complement in turn would be a template to make more replicases. The Darwinian Selection Problem How could this system evolve to select for better and better replicases? If a mutant ribozyme arose that worked better, it would copy non-mutant RNAs as well as mutant RNAs. The solution to this dilemma is compartmentalization. Vesicles form spontaneously from lipids Fatty acids and other lipids naturally form vesicles Chronology of Life on Earth 1. Earth and its moon arose from a collison with a giant projectile about 4.5 million years ago. 2. It took a few hundred years for Earth to cool off enough to have oceans. 3. Layered structures called stromatolites in sedimentary rocks indicate life as early as 3.6 million years ago. 4. So life likely arose between 4.2 and 3.6 million years ago. life arises from an RNA world? The prebiotic chemistry problem What was the nature of the prebiotic world that spawned self-replicating RNA molecules? In 1953, Stanley Miller and Harold Urey showed that subjecting water, methane, ammonia, and hydrogen to electrical discharges in sealed flasks generated amino acids. In 1969 the Murchison meteorite landed in Australia, bringing amino acids and bases to Earth from outer space! Francis Crick and the Theory of Panspermia