RNA-Dependent Synthesis of RNA and DNA:- RNA Synthesis Offers Important Clues to Biochemical Evolution
The extraordinary complexity and order that distinguish living from inanimate systems are key manifestations of fundamental life processes. Maintaining the living state requires that selected chemical transformations occur very rapidly—especially those that use environmental energy sources and synthesize elaborate or specialized cellular macromolecules. Life depends on powerful and selective catalysts—enzymes—and on informational systems capable of both securely storing the blueprint for these enzymes and accurately reproducing the blue print for generation after generation. Chromosomes en code the blueprint not for the cell but for the enzymes that construct and maintain the cell. The parallel demands for information and catalysis present a classic conundrum: what came first, the information needed to specify structure or the enzymes needed to maintain and transmit the information? The unveiling of the structural and functional complexity of RNA led Carl Woese, Francis Crick, and Leslie Orgel to propose in the 1960s that this macromolecule might serve as both information carrier and catalyst. The discovery of catalytic RNAs took this proposal from
conjecture to hypothesis and has led to widespread speculation that an “RNA world” might have been important in the transition from prebiotic chemistry to life . The parent of all life on this planet, in the sense that it could reproduce itself across the generations from the origin of life to the present, might have been a self-replicating RNA or a polymer with equivalent chemical characteristics. How might a self-replicating polymer come to be? How might it maintain itself in an environment where the precursors for polymer synthesis are scarce? How could evolution progress from such a polymer to the modern DNA-protein world? These difficult questions can be addressed by careful experimentation, providing clues about how life on Earth began and evolved. The probable origin of purine and pyrimidine bases is suggested by experiments designed to test hypotheses about prebiotic chemistry . Beginning with simple molecules thought to be present in the early atmosphere (CH4, NH3, H2O, H2), electrical discharges such as lightning generate, first, more reactive molecules such as HCN and aldehydes, then an array of amino acids and organic acids . When molecules such as HCN become abundant, purine and pyrimidine bases are synthesized in detectable amounts. Remarkably, a concentrated solution of ammonium cyanide, refluxed for a few days, generates adenine in yields of up to 0.5% (Fig. 1). Adenine may well have been the first and most abundant nucleotide constituent to appear on Earth. Intriguingly, most enzyme cofactors contain adenosine as part of their structure, although it plays no direct role in the cofactor function . This may suggest an evolutionary relationship, based on the simple synthesis of adenine from cyanide. The RNA world hypothesis requires a nucleotide polymer to reproduce itself. Can a ribozyme bring about its own synthesis in a template-directed manner? The self-splicing rRNA intron of Tetrahymena catalyzes the reversible attack of a guanosine residue on the 5splice junction (Fig. 2). If the 5splice site and the internal guide sequence are removed from the intron, the rest of the intron can bind RNA strands paired with short oligonucleotides. Part of the remaining intact intron effectively acts as a template for the alignment and ligation of the short oligonucleotides. The reaction is in essence a reversal of the attack of guano sine on the 5splice junction, but the result is the syn thesis of long RNA polymers from short ones, with the sequence of the product defined by an RNA template. A self-replicating polymer would quickly use up available supplies of precursors provided by the relatively slow processes of prebiotic chemistry. Thus, from an early stage in evolution, metabolic pathways would be required to generate precursors efficiently, with the synthesis of precursors presumably catalyzed by ribozymes. The extant ribozymes found in nature have a limited repertoire of catalytic functions, and of the ribozymes that may once have existed, no trace is left. To explore the RNA world hypothesis more deeply, we need to know whether RNA has the potential to catalyze the many different reactions needed in a primitive system of metabolic pathways.
The search for RNAs with new catalytic functions has been aided by the development of a method that rapidly searches pools of random polymers of RNA and extracts those with particular activities: SELEX is noth ing less than accelerated evolution in a test tube . It has been used to generate RNA molecules that bind to amino acids, organic dyes, nucleotides, cyanocobalamin, and other molecules. Researchers have iso lated ribozymes that catalyze ester and amide bond formation, SN2 reactions, metallation of (addition of metal ions to) porphyrins, and carbon–carbon bond formation. The evolution of enzymatic cofactors with nucleotide “handles” that facilitate their binding to ribozymes might have further expanded the repertoire of chemical processes available to primitive metabolic systems. As we shall see in the next chapter, some natural RNA molecules catalyze the formation of peptide bonds, offering an idea of how the RNA world might have been transformed by the greater catalytic potential of proteins. The synthesis of proteins would have been a major event in the evolution of the RNA world, but would also have hastened its demise. The information carrying role of RNA may have passed to DNA because DNA is chemically more stable. RNA replicase and re verse transcriptase may be modern versions of enzymes that once played important roles in making the transition to the modern DNA-based system.
Molecular parasites may also have originated in an RNA world. With the appearance of the first inefficient self-replicators, transposition could have been a potentially important alternative to replication as a strategy for successful reproduction and survival. Early parasitic RNAs would simply hop into a self-replicating molecule via catalyzed transesterification, then passively undergo replication. Natural selection would have driven trans position to become site-specific, targeting sequences that did not interfere with the catalytic activities of the host RNA. Replicators and RNA transposons could have existed in a primitive symbiotic relationship, each con tributing to the evolution of the other. Modern introns, retroviruses, and transposons may all be vestiges of a “piggy-back” strategy pursued by early parasitic RNAs. These elements continue to make major contributions to the evolution of their hosts. Although the RNA world remains a hypothesis, with many gaps yet to be explained, experimental evidence supports a growing list of its key elements. Further experimentation should increase our understanding. Important clues to the puzzle will be found in the workings of fundamental chemistry, in living cells, and perhaps on other planets.
FIGURE 1 Possible prebiotic synthesis of adenine from ammonium cyanide. Adenine is derived from five molecules of cyanide, de noted by shading.
FIGURE 2 RNA-dependent synthesis of an RNA polymer from oligonucleotide precursors. (a) The first step in the removal of the self-splicing group I intron of the rRNA precursor of Tetrahymena is reversible attack of a guanosine residue on the 5splice site. Only P1, the region of the ribozyme that includes the internal guide sequence (boxed) and the 5splice site, is shown in detail; the rest of the ribozyme is represented as a green blob. . (b) If P1 is removed (shown as the darker green “hole”), the ribozyme retains both its three-dimensional shape and its catalytic capacity. A new RNA molecule added in vitro can bind to the ribozyme in the same manner as does the internal guide sequence of P1 in (a). This provides a template for further RNA polymerization reactions when oligonucleotides com plementary to the added RNA base-pair with it. The ribozyme can link these oligonucleotides in a process equivalent to the reversal of the reaction in (a). Although only one such reaction is shown in (b), re peated binding and catalysis can result in the RNA-dependent syn thesis of long RNA polymers.
