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THE RNA WORLD

  Does RNA display the ability to serve as both a genetic code and catalyze reactions?  Yes.

     Although modern life depends on DNA encoding the amino acid order of proteins, such a chemical basis is too complex for the first living things.  Modern cells use RNA for so many of the stages of this process, it has been asked whether the first cells might have survived without DNA or protein, using RNA alone.  Could RNA molecules have performed the functions of proteins while simultaneously serving as the first genetic code?  In modern living things, RNA is still the only molecule which functions both as a genotype (a genetic code) and phenotype (determining outward appearance—many RNAs are functional by themselves and are never converted to protein).  There are a number of observations which suggest that RNA was the primary functional molecule in the earliest cells.

a) Nucleotides (primarily RNA nucleotides) have very diverse roles in cells

      Nucleotides (primarily RNA nucleotides which are more easily formed) are essential molecules for cells.  The molecule which cells use for virtually all of their energy transactions (ATP) is an RNA nucleotide with additional phosphate groups added.  In other words, the energy which you are using at this moment to power muscle contractions, pump ions, move cilia, cause cells to divide, etc.  is being derived from ATP, an RNA nucleotide with additional phosphate groups.   During DNA replication, DNA nucleotides are added to an existing primer of RNA nucleotides. 

     Most “enzyme helpers” or coenzymes are either modified nucleotides or can be synthesized from nucleotides.  The coenzymes NAD, NADPH, FAD, coenzyme A, Coenzyme B12, ATP, and S-adenosylmethionine are modified RNA nucleotides (Bartel, 2000).  Cyclic AMP, a modified nucleotide, is perhaps the most common signaling molecule inside cells.  The amino acid histidine is made from a nucleotide.  DNA nucelotides are made from RNA nucleotides (the DNA base thymine is derived from uracil) (Jeffares, 1998; Joyce, 1989).  The fact that majority of metabolism relies on modified RNA cofactors and the existence of these cofactors in all living organisms studied to date indicate that RNA was central to metabolism in early life (Landweber, 1999).  The Ribonucleotide AMP is a common component of many coenzymes (typically acting as the site which binds the enzyme rather than a catalytic function).  Early proteins seem to have adopted older coenzymes from RNA world (Jaday, 2002).

 

b) RNA molecules can act as enzymes (both those found in living cells and sequences generated in the absence of life)!

      Although proteins are indispensable in modern cells (for example, proteins called enzymes catalyze chemical reactions), RNA molecules can be found which perform many of the tasks done by modern proteins.  The modern genetic code is much to complex to have been functional in the first cells since it requires 3 components: DNA sequences which code for RNA sequences which code for proteins; the proteins perform the vital functions of the cell.  RNA is simpler than DNA and was probably the original genetic code which performed the vital functions of those first pre-cells.   It had been thought that RNA could not perform the functions of proteins because the 4 RNA bases could not replace the important reacting parts (functional groups) of amino acids.  Now it is known that RNA bases can be modified (both inside cells and in the absence of life); modified RNA bases have been found which possess most of the functional groups found in amino acids (Robertson, 1995).  The fact that the modified bases of tRNA are found in all living things indicates that RNA nucleotides could be modified in a number of ways in the ancestor of modern cells (Landweber, 1999).

  In collections of random RNA sequences (such as would have existed in the primordial soup), some RNA molecules can be isolated which perform certain functions (such as catalyzing reactions).  RNA enzymes (ribozymes) have been isolated from these random sequences that help to copy existing RNA molecules using the same reaction that proteins use in modern cells.  These ribozymes can also undergo a “natural selection” of sorts in which researchers favored a certain type of ribozyme—over time these random sequences produced better and better ribozymes until they could catalyze a reaction 7 million times faster than the rate observed without the ribozyme.   During the course of this “selection”, the catalytic efficiency of these random RNA sequences increases several hundred times (Bartel, 1993; Wright, 1997).

    Artificial ribozymes have been isolated which require as few as two specific nucleotides to catalyze a reaction (Landweber, 1999).  The ability of ribozymes to catalyze reactions is actually enhanced by higher temperatures and salt concentrations which might have existed in the early earth (Lathe, 2004). 

         RNA chains are not the only nucleic acids which can catalyze chemical reactions.  DNA sequences generated in the laboratory have even been shown to catalyse reactions in lab and can even perform better than RNA ribozymes (Landweber, 1999).  A deoxyribozymes are capable of catalyzing ligase reactions (Levy, 2002).  DNA deoxyribozymes are capable of catalyzing RNA molecules (Jaschke, 2001).  DNA molecules can function as enzymes which catalyze reactions such as those performed by ribonuclease, DNA ligase, and porphyrin metallation.  While most DNAzymes (and all natural ribozymes) require metal cofactors, some DNA enzymes can act independently of them (Sen, 1998).

 

 

c) Ribozymes in modern cells can catalyze a diversity of chemical reactions

     In the early earth, could RNA ribozymes have mediated the conversion of RNA to DNA, the conversion of RNA to protein, and the splicing of small coding units to form functional genetic messages?  This is not an unreasonable hypothesis, given that RNA molecules in modern cells perform these and other reactions.      RNA continues to perform diverse functions in living cells and is most active in the cellular activities that would have been the most ancient (such as the splicing of the genetic message and the synthesis of proteins). 

     The reactions that modern RNA molecules isolated from living cells perform include the cutting and splicing of RNA molecules (spliceosomes, self-editing introns; Sharp, 1985; Cech, 1987; Kruger, 1982), extending the ends of chromosomes (telomerase) (Poole, 1997), modification of the tRNAs used in protein construction (Rnase P), nucleotide insertion (Mueller, 1993), breaking triphosphate bonds for energy (srp RNA) (Jeffares, 1998) and the folding, cleavages, nucleotide modification, and assembly of ribosomal subunits (snoRNAs; Maxwell, 1995).  Proteins are synthesized at ribosomes which is actually a ribozyme (Steitz, 2003)

     The Varkud satellite RNA in Neurospora and the manganese-dependent ribozyme (the latter of which has only 7 nulceotides) can cleave RNA.  The ribozymes of group I and group II introns are known in both prokaryotic and eukaryotes (although group II introns in eukaryotes are limited to organelles which are thought to be derived from prokaryotes) (Landweber, 1999)..     Poorly understood RNA molecules called snoRNA and vault RNA also may be modern remnants of earlier RNA molecules which were essential to the first cells (Poole, 1997; Jeffares, 1998).

    Metals can stabilize RNA structure and help catalyze reactions, two characteristics that ribozymes share with modern protein enzymes. Most ribozymes are actually metallo-ribozymes similar to the protein enzymes which are more correctly called metalloenzymes (Landweber, 1999; Lilley, 2003). 

 

 

d) Ribozymes generated in the lab can catalyze a diversity of chemical reactions

     Can artificial ribozymes replicate themselves?  Yes and no.  Although ribozymes which perform the necessary reactions in self replication have been isolated, they do not function at the levels that a precursor to a living cell would require (Bartel, 2000).  Ribozymes can recognize a primer template and attach complimentary bases to the template.  The ability to join RNA nucleotides to a primer has been accomplished by different ribozymes with different structures and biochemical properties.  The accuracy of the addition of complemetary bases was 97% and could be increased to 99% by altering the nucleotide concentrations.  One ribozyme could extend the primer by 14 nucleotides (although the ribozyme itself was 189 nucleotides) (McGinness, 2003).  In one experiment, viral RNA was shown both to catalyze self-replication and serve as a template for it.  Selection experiments produced mutant RNAs which were 20x better at serving as a template (Doudna, 1991).    The following are images of ribozymes which possessed polymerase or ligase activity.

RNA

--after McGinness, 2003

      In experiments without cells, ribozymes can be selected for out of random RNA sequences that catalyze chemical reactions such as nucleotide synthesis, forming carbon-carbon bonds, forming bonds that modern ribosomes must form during protein synthesis, and forming the bonds that modern tRNA synthetases (which are proteins) perform in protein synthesis, cleave phosphodiester bonds,  act as RNA ligase, hydrolyze cyclic phosphates, phosphorylate RNA, transfer phosphate anhydride, perform acyl transfer, form amide bonds, form peptide bonds, form glycosidic bonds, and a number of other reactions.  (Bartel, 2000; Landweber, 1999, Ekland, 1996; Ekland, 1995. Green 1992, Illangasekare, 1995; Lohse, 1996;  Tarasow, 1997; Bartel, 1993;  Unrau, 1998). 

     Ribozymes can act as a polynucleotide kinase, form cabon-sulfur bonds, cause alkylation, insert copper into porphyrin, aminoacyl transfer to form ester bonds, peptide bonds (Cui, 2004).  Ribozymes have been generated which can create 30 different kinds of dipeptides.  The creation of uncoded dipepetides could thus have preceded the synthesis of coded peptides (Sun, 2002).  An in-vitro selected ribozyme not only formed peptide bonds in a way similar to that performed by the rRNA of ribosomes, it also structurally resembled the rRNA of a ribosome (Zhang, 1998). Although in modern cells, the enzymes which attach amino acids to tRNAs are proteins, ribozymes have been isolated in vitro which can perform this aminoacylation.  Thus this important step for protein synthesis could have occurred without protein (Martinez Gimenez, 2002).  Artificial RNAs can polymerize nucleotides and catalyze some of the steps of nucleotide synthesis (Joyce, 2002).  Genetic recombination can occur between RNA strands mediated by a ribozyme (Riley, 2003).

    DNA molecules can also catalyze chemical reactions; in vitro selection produced a DNA enzyme which could cleave RNA at a very high catalytic efficiency (Santoro, 1997).

HOW MIGHT LIFE HAVE EVOLVED?  The following is a possible pathway:

RNA

A RNA WHICH CATALYZES THE SYNTHESIS OF RNA MOLECULES USING EXISTING TEMPLATES DEVELOPS AND IS INCORPORATED INTO A LIPID MEMBRANE

 

All of the basic biomolecules found in living things have been synthesized in the absence of life; in a world without microbes and with very little atmospheric oxygen, these molecules would have accumulated in the oceans over time.

   Spherical membrane balls do form spontaneously from synthetic organic molecules, even those of extraterrestrial origin.  Other organic molecules, such as RNA, do collect inside these spheres.

   Random RNA (and protein) sequences often include some molecules with catalytic properties, even self-replicating RNA and peptide sequences have been generated.

PRECELL PRECELL PRECELL
THE RNA WORLD: PRE-CELLS ACQUIRE A DIVERSE ARRAY OF RNA MOLECULES  

--RNA could have served as both the genetic code and the biomolecule which performed vital functions.  Modified RNA bases could have provided a diversity of functional groups comparable to that found in amino acids.

 

 

LATERAL TRANSFER

PRECELL

: A PRECELL ABSORBING RNA FROM ITS ENVIRONMENT

PRECELL

    How could the first protocells acquire additional catalytic RNA molecules?  Mutations occur today in all organisms and certainly could have added to the repertoire of these enzymes.  In modern organisms, lateral transfer can also occur in which genes can move from one species into another.  For example, both human and bacterial cells can take up DNA from the environment and incorporate it into their chromosome.  It is possible that in human intestines, fragments of chromosomes are interchanged between human and bacterial cells.  Gene clusters of tightly linked photosynthesis genes (such as those in purple photosynthetic organisms) may have radiated among bacterial groups through lateral transfer (Xiong, 2000).  Viruses occasionally introduce DNA fragments from their previous host into their next host. 

     Early genetic systems would have experienced a very high mutation rate and a very high lateral transfer rate.  As a result, “organisms” as the term is currently understood did not exist since genes could be exchanged regardless of lineage.  It has been proposed that the last universal common ancestor of modern organisms was more of a community of cells rather than a single cell. Lateral gene transfer was the primary mechanism of evolution in these earliest cells, not descent with modification (Woese, 1998).

The magnitude of lateral gene transfer among microbes may be such that it is impossible to approximate the gene content of the earliest cells by studying the distribution of genes in modern organisms (Nesbo, 2001).

 

 

     THE TRANSITION TO PROTEINS

      The pre-cells which began to form chains of amino acids would obviously not have been able to create a specific sequence and therefore could not have required the proteins for any vital functions. Then what use could proteins possibly serve?  In modern cells, there are a number of examples known in which RNA molecules interact with proteins to form a complex (such as the ribosome and spliceosome).  The proteins function in stabilizing the three-dimensional shape of the RNA, which actually performs the vital function.  It seems that the original function of proteins in ancestral cells was to stabilize RNA.  As time went on, mechanisms were developed to control the amino acid sequence of proteins.  Proteins have the advantage of being able to form more stable 3 dimensional shapes than RNA molecules can.  Over time, proteins replaced most of the functions performed by ancestral RNA molecules and became the primary determinants of the phenotype of cells.  However, proteins did not completely replace RNA.  RNA molecules continue to perform functions in living cells such as the synthesis of proteins and the splicing of genetic messages.  Although cells conceivably use only proteins for these tasks, catalytic RNAs display what may be a conserved function from the RNA world.

     Some RNA molecules form complexes with multiple proteins (ribosome subunits, U2B”-U2A-U2 hairpin, S15, S6, S18-rRNA site), single proteins (tRNA synthetase-tRNA complexes, EF-Tu-tRNA, Met transformylase-fMET tRNA, L11-23S rRNA site, L25-5S rRNA site, S15 rRNA site, L30-autoregulatory RNA, MS2 coat protein-operator, MS2 coat protein-aptamer, TRAP-ssRNA site, Rho-polyC, Ffh-SRP RNA), single domains (RNP, Nova KH-gly receptor pre mRNA, ds RBD-ds RNA), peptides (N-box, and Tat-TAR), and amino acids (arginine aptamer) (Frankel, 2000).  The Sm/Lsm proteins form ribonucleoprotein complexes which function in RNA splicing, mRNA degradation, and the maintanence of telomeres.   They are known in eukaryotes and archaea (Collins, 2001).