# Structural Biochemistry/RNA World Hypothesis

The RNA World Hypothesis speculates that the origin of life began with ribonucleic acid (RNA) because of its ability to serve both as a storage for genetic information and enzymatic activity. It is proposed that RNA preceded the current genetic material, deoxyribonucleic acid (DNA), and led the evolution of the DNA → RNA → protein world.
There are two schools of thought that both support the RNA World hypothesis:
1. According to the Genetic Takeover Hypothesis, an earlier form of life on earth used RNA as its only genetic component. This proposes that there may be a pre-RNA molecule that used RNA or by change created RNA as a side product.
2. The first form of life on earth used RNA as its only genetic component. This theory requires that RNA came from inanimate matter.

## History

The book, The Genetic Code, written in 1967 by Carl Woese, was the first published material that supported RNA World Hypothesis. Francis Crick and Leslie Orgel proposed the idea that RNA once did the work of DNA and proteins in 1968. Their theories were not validated until the work of Nobel Prize laureate Thomas R. Cech. In the 1970s, Cech was studying the splicing of RNA in a single-celled organism, Tetrahymena thermophila, when he discovered that an unprocessed RNA molecule could splice itself. He announced his discovery in 1982 and became the first to show that RNA has catalytic functions. The phrase “RNA World Hypothesis” was then coined later in 1986 by Harvard molecular biologist and Nobel Prize laureate Walter Gilbert as he commented on the recent observations of the catalytic properties of RNA. Another major milestone occurred in 2000 when it was published in Science that "The Ribosome is a Ribozyme" and the proteins in the ribosomes exist primarily on the periphery.

## Theory

The primordial soup that made up Earth had compounds including nucleotides. These nucleotides sequenced spontaneously and randomly, eventually forming an RNA molecule (or a similar molecule) with catalytic characteristics. RNA has properties of autocatalytic self-replication and assembly, contributing to its exponential increase in number. Scientists today have assumed that replication was not perfect in the time of primitive life, and therefore variations of RNA developed. RNA's catalytic properties do not only apply to itself, but it also catalyzes transesterification—a process necessary for protein synthesis that allows specific peptide sequences and proteins to arise. Some of the peptides formed may have supported the self-replication of RNA and provided the possibility of undergoing modifications. Those modifications led to more efficient sequences of RNA molecules.
A possible model for forming purine and pyrimidine bases was proposed by Urey and Miller's experiment. This experiment brought about evidence that organic molecules originated from inorganic ingredients such as carbon dioxide, ammonia, and water etc. Such products were mixed under a reduced environment and subjected to electric shock (to simulate lightning), which led to the creation of more reactive molecules, such as hydrogen, cyanide, and aldehydes, as well as some amino acids and organic acids over a certain period of time. These amino acids contributed to the formation of peptide sequences.
http://www.smithlifescience.com/MillersExp.htm (see apparatus)
Further evidence that the RNA World Hypothesis has clout is found through the function of present day ribosomes. RNA is the ribosome's tool for synthesizing proteins and catalyzing the formation of peptide bonds. A form of RNA known as transfer RNA (tRNA) is responsible for delivering free amino acids to the ribosome and growing peptide chain. Therefore, this points to the fact that RNA is multifunctional, and can act as a synthesizer, transporter, messenger, and ribosome molecule.
One may ask, if RNA was the precursor of DNA and proteins, how did this evolution occur? DNA complements the RNA sequence and stores genomic information. Since DNA is a more stable molecule than RNA, it makes sense for DNA to adapt to the environment and take over this job of RNA. And how is DNA more stable than RNA? The difference between the general structure of DNA and RNA is found in the sugar. DNA has a deoxyribose sugar while RNA has a ribose sugar. The missing 2'-OH group on the deoxyribose sugar is what makes DNA more stable, since there is no hydroxyl group for other molecules to react with. Otherwise, RNA does not remain in a helical ring, as does DNA, since the chain of nucleotides would be easily broken apart. Another possibility scientists are exploring is the idea that reverse transcriptase (RT) played a role in the transformation from RNA to DNA. Reverse transcriptase catalyzes the formation of DNA from an RNA template, and RT is the defining feature of retroviruses like HIV. RT, along with RNA replicase, may be the enzyme that performed this transition. Furthermore, the combination of cyanoacetaldehyde and urea formed uracil (U) and cytosine (C)-- components of the primordial soup. This belief was supported by another of Miller's experiments. There is no evidence at this time that thymine (T), the nitrogenous base in DNA that takes the place of uracil (U) in RNA, was formed from this atmosphere. This infers that RNA was a predecessor of DNA. In addition, proteins that had formed from RNA were found to be versatile structures, allowing them to take over what was initially RNA's catalytic functioning.

## Properties Supporting Hypothesis

Properties that support the RNA Hypothesis became more clear in the 1980s when it was discovered that RNA can activate and deactivate other molecules by binding with them while folding into specific structures. Before this discovery, researchers believed that RNA only had a few functions. Consideration of RNA as the pre-component of cellular life has since been studied extensively.
Major evidence that has the scientific community believing that RNA predates DNA and proteins are as follows:
- RNA has the ability to store genetic data, and pass down hereditary information.
- It is the main component linking up DNA and gene formation to amino acids and protein synthesis via transcription and translation.
- RNA's ability to duplicate itself as well as the genetic information it carries, very much like DNA.
- RNA's complexity is less than that of DNA, and involves fewer types of molecules in order to self-replicate.
- DNA requires an RNA primer in order to replicate while RNA does not need any such primer. This shows how DNA seems to depend more on RNA for its continued existence rather than the other way around.
- RNA is able to catalyze reactions as proteins do. The formation of a protein is also administered by RNA which points heavily to its preexistence over the proteins.
- RNA's ability to form double helices similar to DNA, and tertiary structures similar to those of catalytic proteins.
- The structure of RNA, with an hydroxyl group in the 2' position of the sugar molecule, makes it a less stable molecule which is capable of attacking a phosphodiester bond near it as long as the RNA molecule is in a flexible position and not constrained. This made it susceptible to breakdown and allowed an adaptation of different conformations which perhaps was beneficial to early life.
- RNA's different set of bases such as Uracil, which is “1 product of damage to cytosine” made RNA more prone to mutations thus making it more suitable to primitive life in early times.
An experiment supporting the RNA World Hypothesis was, the Miller-Urey Experiment. The Miller-Urey Experiment was an experiment tested by Stanley L. Miller and Harold C. Urey in 1953 to see which molecules were present in the origin of life. This experiment specifically tested the hypothesis of Alexander Oparin's and J.B.S. Haldane's hypothesis that stated the conditions of prebiotic Earth favored chemical reactions, synthesizing inorganic compounds into organic ones. Both experiments helped scientists around the world better understand the evolution of the Earth and how organic compounds formed. The gases used by Miller and Urey were Methane (CH$_4$), Ammonia (NH$_3$), Hydrogen (H$_2$) and water (H$_2$O). After putting these gases (which were presumed to be present in prebiotic Earth), Miller and Urey continuously ran an electric current throughout the closed vessel system to stimulate lightning, which was thought to be extremely common on early Earth. These compounds were put inside a sterile array of glass tubes and flasks connected in a loop, with one flask half-full of liquid water and another flask containing a pair of electrodes. The liquid water was heated to induce evaporation, sparks were fired between the electrodes to simulate lightning through the atmosphere and water vapor, and then the atmosphere was cooled again so that the water could condense and trickle back into the first flask in a continuous cycle. Within a day, the mixture had turned pink in color,[1] and at the end of one week of continuous operation, Miller and Urey observed that as much as 10–15% of the carbon within the system was now in the form of organic compounds. Two percent of the carbon had formed amino acids that are used to make proteins in living cells, with glycine as the most abundant. Sugars and liquids were also formed. Nucleic acids were not formed within the reaction. But the common 20 amino acids were formed, in various concentrations. This experiment thus showed that organic compounds that are vital to cellular function and life were easily made under the conditions of prebiotic Earth.
In an interview, Stanley Miller stated: "Just turning on the spark in a basic pre-biotic experiment will yield 11 out of 20 amino acids."[2] This further supported the RNA World Hypothesis.

## Other Experiments

The Miller-Urey and Oparin experiments helped launch other experiments to further show that organic compounds formed in early Earth and to also confirm the RNA World Hypothesis.
In 1961, Juan Oro conducted an experiment that concluded amino acids could be formed from HCN, hydrogen cyanide, and Ammonia (NH$_3$) in an aqueous solution. This experiment also produced Adenine, one of the nucleotide bases. This became a major breakthrough because adenine is one of the four bases in DNA and RNA, the genetic material of a cell. Adenine is also used during the process of ATP (adenosine triphosphate), which is an energy releasing molecule in cells.
This experiment led to more showing that the other three RNA and DNA bases could be formed through similar experiments of simulated chemical environments with reducing atmospheres.

## Properties Opposing Hypothesis

Most of the opponents of the RNA World concentrate on dispelling the idea that RNA was the first form of genetic material, although they do agree that there may have been some other pre-RNA form of genetic material. In summary:
1. Ribose is relatively unstable and difficult to form in a prebiotic mixture. Despite the favorable and controllable conditions that are available in laboratory settings, pre-cellular life has never been created from inanimate matter.
2. The origin of life began roughly 300 million years ago. Some believe that this is too short of a time period for the prebiotic soup to evolve in to a pre-RNA or RNA World.
3. Lack of evidence of large amounts of polyphosphates in primitive Earth makes it unlikely that it was the source of prebiotic energy or that it was involved in the first genetic material. .
Others believe that RNA is not a likely pre-DNA form of genetic material. Their arguments include:
1. The limited catalytic capabilities of RNA. Theorists say that RNA needed to have had a multitude of catalytic abilities to be able to survive the prebiotic world, but RNA has not shown this. Proteins, on the other hand, do have those catalytic abilities via their varying, enzymatic abilities.
2. The prebiotic simulation of the formation of the RNA molecule has shown some difficulty in that the bases and the sugar molecule do not readily react in water.
3. Opponents advocate proteins over RNA because they are easily formed.
4. The probability of the right components of pre-cellular life to exist at the same place and time, without contaminates, and with the correct catalytic reactions is next to improbable .
5. Recent research shows that non-coding RNA regions have well-adapted and very specialized roles in the cell. Examples include siRNA and miRNA—they work well in an environment where RNAi and mRNA already exist. Because of their usefulness that we are just beginning to understand, it makes it less likely that there are "relics" of the RNA World present in our DNA as Gilbert originally mentioned in 1986 .

## Alternative Theories

The difficulty of RNA formation has caused other propositions of alternative theories on precursor materials for cellular life:
- Peptide Nucleic Acid theory (PNA), a nucleic acid with a backbone of peptide bonds, made a likely theory because it overcame the problem in RNA theory regarding the difficulty of RNA to attach ribose and phosphate groups together.
- Threose nucleic acids are proposed as a more likely starting material than RNA.
- Glycold nucleic acids are proposed as precursors rather than RNA because they are easily formed.
- Double origin theory suggests that both RNA and proteins existed around the same time independently.

## Support

The RNA World hypothesis is supported by RNA's ability to store, transmit, and duplicate genetic information, as DNA does. RNA can also act as a ribozyme (an enzyme made of ribonucleic acid). Because it can reproduce on its own, performing the tasks of both DNA and proteins (enzymes), RNA is believed to have once been capable of independent life. Further, while nucleotides were not found in Miller-Urey's origins of life experiments, they were found by others' simulations, notably those of Joan Oro. Experiments with basic ribozymes, like the viral RNA Q-beta, have shown that simple self-replicating RNA structures can withstand even strong selective pressures (e.g., opposite-chirality chain terminators) (The Basics of Selection (London: Springer, 1997)).
Additionally, in the past a given RNA molecule might have survived longer than it can today. Ultraviolet light can cause RNA to polymerize while at the same time breaking down other types of organic molecules that could have the potential of catalyzing the break down of RNA (ribonucleases), suggesting that RNA may have been a relatively common substance on early Earth. This aspect of the theory is still untested and is based on a constant concentration of sugar-phosphate molecules.

## Difficulties

The base cytosine does not have a plausible prebiotic simulation method because it easily undergoes hydrolysis.
Prebiotic simulations making nucleotides have conditions incompatible with those for making sugars (lots of formaldehyde). So they must somehow be synthesized, then brought together. However, they do not react in water. Anhydrous reactions bind with purines, but only 8% of them bind with the correct carbon atom on the sugar bound to the correct nitrogen atom on the base. Pyrimidines, however, do not react with ribose, even anhydrously.
Then phosphate must be introduced, but in nature phosphate in solution is extremely rare because it is so readily precipitated. After being introduced, the phosphate must combine with the nucleoside and the correct hydroxyl must be phosphorylated, in order to create a nucleotide.
For the nucleotides to form RNA, they must be activated themselves (meaning that they must be combined with two more phosphate groups, as in adenosine triphosphate). Activated purine nucleotides form small chains on a pre-existing template of all-pyrimidine RNA. However, this does not happen in reverse because the pyrimidine nucleotides do not stack well.
Additionally, the ribose must all be the same enantiomer, because any nucleotides of the wrong chirality act as chain terminators.
A.G. Cairns-Smith in 1982 criticized writers for exaggerating the implications of the Miller-Urey experiment. He argued that the experiment showed, not the possibility that nucleic acids preceded life, but its implausibility. He claimed that the process of constructing nucleic acids would require 18 distinct conditions and events that would have to occur continually over millions of years in order to build up the required quantities.

## Alternative Hypothesis

As mentioned above, a different version of the same hypothesis is "Pre-RNA world", where a different nucleic acid is proposed to pre-date RNA. A candidate nucleic acid is peptide nucleic acid (PNA), which uses simple peptide bonds to link nucleobases.53 PNA is more stable than RNA, but its ability to be generated under prebiological conditions has yet to be demonstrated experimentally.
Threose nucleic acid (GNA), and like PNA, also lack experimental evidence for their respective abiogenesis.
An alternative—or complementary— theory of RNA origin is proposed in the PAH world hypothesis, whereby 57
The iron-sulfur world theory proposes that simple metabolic processes developed before genetic materials did, and these energy-producing cycles catalyzed the production of genes.
Yet another alternative theory to the RNA world hypothesis is the panspermia hypothesis. It discusses the possibility that the earliest life on this planet was carried here from somewhere else in the galaxy, possibly on meteorites similar to the Murchison meteorite.58 This does not invalidate the concept of an RNA world, but posits that this world was not Earth but rather another, probably older, planet.

## Implications of the RNA World

The RNA world hypothesis, if true, has important implications for the very definition of life. For the majority of the time following the elucidation of the structure of DNA by Watson and Crick, life was considered as being largely defined in terms of DNA and proteins: DNA and proteins seemed to be the dominant macromolecules in the living cell, with RNA serving only to aid in creating proteins from the DNA blueprint.
The RNA world hypothesis places RNA at center-stage when life originated. This has been accompanied by many studies in the last ten years demonstrating important aspects of RNA function that were not previously known, and support the idea of a critical role for RNA in the functionality of life. In 2001, the RNA world hypothesis was given a major boost with the deciphering of the 3-dimensional structure of the ribosome, which revealed the key catalytic sites of ribosomes to be composed of RNA and for the proteins to hold no major structural role, and be of peripheral functional importance. Specifically, the formation of the peptide bond, the reaction that binds amino acids together into proteins, is now known to be catalyzed by an adenine residue in the rRNA: the ribosome is a ribozyme. This finding suggests that RNA molecules were most likely capable of generating the first proteins. Other interesting discoveries demonstrating a role for RNA beyond a simple message or transfer molecule include the importance of small nuclear ribonucleoproteins (SnRNPs) in the processing of pre-mRNA and RNA editing and reverse transcription from RNA in Eucaryotes in the maintenance of telomeres in the telomerase reaction.

## References

* ^ Gesteland, R.F., Cech, T.R., Atkins, J.F., 2006, The RNA World: the nature of modern RNA suggests a prebiotic RNA, Cold Spring Harbor Laboratory Press, United States of America, 768 p.
* ^ Gilbert, W., "The RNA World". Nature 618.
* ^ Altman, S. The RNA World
* ^ Lazcano, A, Miller, S.L., "The Origin and Early Evolution of Life: Prebiotic Chemistry, the Pre-RNA World, and Time" Cell, Vol. 85, 7930798, June 14, 1996.
* ^ RNA World Hypothesis at NationMaster.com
* ^ RNA World Hypothesis at ExperienceFestival
* ^ Eddy, S.R., "Non-Coding RNA Genes and the Modern RNA World" Nature Reviews: Genetics Vol. 2, Dec. 2001.
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