The Chemical Nature of RNA
Ribonucleic acid (RNA) is a complex biological molecule that is classified along with DNA as a nucleic acid. Chemically, RNA is a polymer (long chain) consisting of subunits called ribonucleotides linked together by phosphodiester bonds. Each ribonucleotide consists of three parts: the sugar ribose (a five-carbon simple sugar), a negatively charged phosphate group, and a nitrogen-containing base. There are four types of ribonucleotides, and the differences among them lie solely in which of four possible bases each contains. The four bases are adenine (A), guanine (G), cytosine (C), and uracil (U).
The structures of DNA and RNA are very similar, with the following differences. The sugar found in the nucleotide subunits of DNA is deoxyribose, which differs slightly from the ribose found in the ribonucleotides of RNA. In addition, while DNA nucleotides also contain four possible bases, there is no uracil in DNA; instead, DNA nucleotides contain a different base called thymine (T). Finally, while DNA exists as a double-stranded helix in nature, RNA is almost always single-stranded. Like DNA, a single RNA strand has a 5′-to-3′ polarity. These numbers are based on which carbon atom is exposed at the end of the polymer, each of the carbon atoms being numbered around the sugar molecule.
The Folding of RNA Molecules
The function of an RNA molecule is determined by its nucleotide sequence, which represents information derived from DNA. This nucleotide sequence is called the primary structure of the molecule. Many RNAs also have an important secondary structure, a three-dimensional shape that is also important for the function of the molecule. The secondary structure is determined by hydrogen bonding between parts of the RNA molecule that are complementary. Complementary pairing is always between A and U ribonucleotides and C and G ribonucleotides. Hydrogen bonding results in double-stranded regions in the secondary structure.
Since RNA is single-stranded, it was recognized shortly after the discovery of some of its major roles that its capacity for folding is great and that this folding might play an important part in the functioning of the molecule. Base pairing often represents local interactions, and a common structural element is a “hairpin loop” or “stem loop.” A hairpin loop is formed when two complementary regions are separated by a short stretch of bases so that when they fold back and pair, some bases are left unpaired, forming the loop. The net sum of these local interactions is referred to as the RNA’s secondary structure and is usually important to an understanding of how the RNA works. All transfer RNAs (tRNAs), for example, are folded into a secondary structure that contains three stem loops and a fourth stem without a loop, a structure resembling a cloverleaf in two dimensions.
Finally, local structural elements may interact with other elements in long-range interactions, causing more complicated folding of the molecule. The full three-dimensional structure of a tRNA molecule from yeast was finally confirmed in 1978 by several groups independently, using X-ray diffraction. In this process, crystals of a molecule are bombarded with X-rays, which causes them to scatter; an expert can tell by the pattern of scattering how the different atoms in the molecule are oriented with respect to one another. The cloverleaf arrangement of a tRNA undergoes further folding so that the entire molecule takes on a roughly L-shaped appearance in three dimensions. An understanding of the three-dimensional shape of an RNA molecule is crucial to understanding its function. By the late 1990s, the three-dimensional structures of many tRNAs had been worked out, but it had proven difficult to do X-ray diffraction analyses on most other RNAs because of technical problems. More advanced computer programs and alternate structure-determining techniques like mass spectroscopy, nuclear magnetic resonance and cryo electron microscopy are enabling research in this field to proceed, with the RNA structures catalogued online in the Nucleic Acid Database and Protein Data Bank.
Synthesis and Stability of RNA
RNA molecules of all types are continually being synthesized and degraded in a cell; even the longest-lasting ones exist for only a day or two. Shortly after the structure of DNA was established, it became clear that RNA was synthesized using a DNA molecule as a template, and the mechanism was worked out shortly thereafter. The entire process by which an RNA molecule is constructed using the information in DNA is called transcription. An enzyme called RNA polymerase is responsible for assembling the ribonucleotides of a new RNA complementary to a specific DNA segment (gene). Only one strand of the DNA is used as a template (the sense strand), and the ribonucleotides are initially arranged according to the base-pairing rules. A DNA sequence called the “promoter” is a site RNA polymerase can bind initially and allows the process of RNA synthesis to begin. At the appropriate starting site, RNA polymerase begins to assemble and connect the nucleotides according to the complementary pairing rules, such that for every A nucleotide in the DNA, RNA polymerase incorporates a U ribonucleotide into the RNA being assembled. The remaining pairing rules stipulate that a T in DNA d an A in RNA and that a C in DNA represents a G in RNA (and vice versa). This process continues until another sequence, called a “terminator,” is reached. At this point, the RNA polymerase stops transcription, and a new RNA molecule is released.
Much attention is rightfully focused on transcription, since it controls the rate of synthesis of each RNA. It has become increasingly clear, however, that the amount of RNA in the cell at a given time is also strongly dependent on RNA stability (the rate at which it is degraded). Every cell contains several enzymes called ribonucleases (RNases) whose job it is to cut up RNA molecules into their ribonucleotides subunits. Some RNAs last only thirty seconds, while others may last up to a day or two. It is important to remember that both the rates of synthesis (transcription) and degradation ultimately determine the amount of functional RNA in a cell at any given time.
Three Classes of RNA
While all RNAs are produced by transcription, several classes of RNA are created, and each has a unique function. By the late 1960s, three major classes of RNAs had been identified, and their respective roles in the process of protein synthesis had been identified. In general, protein synthesis refers to the assembly of a protein using information encoded in DNA, with RNA acting as an intermediary to carry information and assist in protein building. In 1956, Francis Crick, one of the scientists who had discovered the double-helical structure of DNA, referred to this information flow as the “central dogma,” a term that continues to be used, although exceptions to it are now known.
A messenger RNA (mRNA) carries a complementary copy of the DNA instructions for building a particular protein. Making up about 5 percent of the three RNA classes, in eukaryotes mRNA typically represents the information from a single gene and carries the information to a ribosome, the site of protein synthesis. The information must be decoded to make a protein. Nucleotides are read in groups of three (called codons). In addition, mRNAs contain signals that tell a ribosome where to start and stop translating.
Ribosomal RNA (rRNA) is part of the structure of the ribosome and makes up about 80 percent of the total RNA in a cell. Four different rRNAs interact with many proteins to form functional ribosomes that direct the events of protein synthesis. One of the rRNAs interacts with mRNA to orient it properly so translation can begin at the correct location. Another rRNA acts to facilitate the transfer of the growing polypeptide from one tRNA to another (peptidyl transferase activity).
Transfer RNA (tRNA) accounts for 15 percent of the three RNA categories and serves the vital role of decoding the genetic information. There are at least twenty and usually more than forty different tRNAs in a cell. On one side, tRNAs contain an “anticodon” loop, which can base-pair with mRNA codons according to their sequence and the base-pairing rules. On the other side, each contains an amino acid binding site, with the appropriate amino acid for its anticodon. In this way, tRNAs recognize the codons and supply the appropriate amino acids. The process continues until an entire new polypeptide has been constructed.
The attachment of the correct amino acids is facilitated by a group of enzymes called tRNA amino acyl synthetases. Each type of tRNA has a corresponding synthetase that facilitates the attachment of the correct amino acid to the amino acid binding site. The integrity of this process is crucial to translation; if only one tRNA is attached to an incorrect amino acid, the resulting proteins will likely be nonfunctional.
Split Genes and mRNA Processing in Eukaryotes
In bacterial genes, there is colinearity between the segment of a DNA molecule that is transcribed and the resulting mRNA. In other words, the mRNA sequence is complementary to its template and is the same length, as would be expected. In the late 1970s, several groups of scientists made a seemingly bizarre discovery regarding mRNAs in eukaryotes (organisms whose cells contain a nucleus, including all living things that are not bacteria): the sequences of mRNAs isolated from eukaryotes were not collinear with the DNA from which they were transcribed. The coding regions of the corresponding DNA were interrupted by seemingly random sequences that served no apparent function. These “introns,” as they came to be known, were apparently transcribed along with the coding regions (exons) but were somehow removed before the mRNA was translated. This completely unexpected observation led to further investigations that revealed that mRNA is extensively processed, or modified, after its transcription in eukaryotes.
After a eukaryotic mRNA is transcribed, it contains several introns and is referred to as immature, or a “pre-mRNA.” Before it can become mature and functional, three major processing events must occur: splicing, the addition of a 5′ cap, and a “tail.” The process of splicing is complex and occurs in the nucleus with the aid of “spliceosomes,” large complexes of RNAs and proteins that identify intervening sequences and cut them out of the pre-mRNA. In addition, spliceosomes rejoin the exons to produce a complete, functional mRNA. Splicing must be extremely specific, since a mistake causing the removal of even one extra nucleotide could change the final protein, making it nonfunctional. During splicing, capping and the addition of a poly-A tail take place. A so-called cap, which consists of a
modified G nucleotide, is added to the beginning (5′ end) of the pre-mRNA by an unconventional linkage. The cap appears to function by interacting with the ribosome, helping to orient the mature mRNA so that translation begins at the proper end. A tail, which consists of many A nucleotides (often two hundred or more), is attached to the 3′ end of the pre-mRNA. This so-called poly-A tail, which virtually all eukaryotic mRNAs contain, seems to be one factor in determining the relative stability of an mRNA. These important steps must be performed after transcription in eukaryotes to produce a functional mRNA.
Ribozymes
The traditional roles of RNA in protein synthesis were originally considered its only roles. RNA in general, while considered an important molecule, was thought of as a “helper” in translation. This all began to change in 1982, when the molecular biologists Thomas Cech and Sidney Altman, working independently and with different systems, reported the existence of RNA molecules that had catalytic activity. This means that RNA molecules can function as enzymes; until this time, it was believed that all enzymes were protein molecules. The importance of these findings cannot be overstated, and Cech and Altman ultimately shared the 1989 Nobel Prize in Chemistry for the discovery of these RNA enzymes, or “ribozymes.” Both of these initial ribozymes catalyzed reactions that involved the cleavage of other RNA molecules, which is to say they acted as nucleases. Subsequently, many ribozymes have been found in various organisms, from bacteria to humans. Some of them are able to catalyze different types of reactions, and there are new ones reported every year. Thus ribozymes are not a mere curiosity but play an integral role in the molecular machinery of many organisms. Their discovery also gave rise to the idea that at one point in evolutionary history, molecular systems composed solely of RNA, performing many roles, existed in an “RNA world.”
RNAi Pathway and Small RNAs
At around the same time as these momentous discoveries, still other classes of RNAs were being discovered, each with its own specialized functions. In 1981, Jun-ichi Tomizawa discovered interference RNA whereby RNA has the ability to interfere with protein production. This hindering RNA is known as “antisense RNA” because it is the opposite complement of, and thus can bind to, the “sense” strand of protein-coding mRNA, resulting in the prevention or regulation of the mRNA’s translation.
In 1990, the power of antisense RNA showed itself in the form of an unexpected white flower. Researcher Richard Jorgensen was hoping to intensify the color of petunias with the introduction of a transgene to overexpress the pigmentation enzyme when instead he produced a white flower. This tipped off scientists to the existence of the post-transcriptional gene silencing (PTGS) cellular mechanism and the idea that adding genes to an organism can affect its phenotype.
In 1998, Craig Mello and Andrew Fire uncovered another significant use of interference RNA, for which they were awarded the 2006 Nobel Prize in Physiology or Medicine. The researchers noticed that the addition of mRNA and antisense RNA did not turn off their targeted gene in Caenorhabditis elegans roundworms as effectively as an injection of double-stranded RNA (dsRNA). Putting the current name to this interference phenomenon, RNAi, the duo had uncovered a crucial piece of a cell’s regulation capability and immune response known as the RNAi pathway. When dsRNA enters the cell, the Dicer enzyme chops it into small nucleotides of 20 to 25 bases. These small pieces, known as small interfering RNA (siRNA), are separated and fed into the
RNA-induced silencing complex (RISC) where they are bound to their complementary mRNA for the pairs’ dismantled demise.
Much excitement and attention toward these protein-regulating small RNAs has unveiled numerous different subspecies and the varying methods in which they are initiated within a cell. siRNA are generated in the cytoplasm from dsRNA and match almost completely to their doomed mRNA. Another small RNA, microRNA (miRNA), is also termed “noncoding RNA” because it initiates from the cell’s own gene, but is not created for the traditional purpose of protein-coding, but instead with the intent to restrain protein production. Once made, miRNA gets enzymatically processed before being sent into the cytoplasm where it is transported into the RISC mechanism. In humans, miRNA tend to only moderately complement their targeted mRNA, resulting in a partial restriction of the mRNA’s protein translation. This class of small RNA is practically absent in cancerous cells, and a recent study showed that its addition can profoundly reverse cancer cell growth.
This power to subdue out-of-control cells has revolutionized the search for therapeutic treatments of several human diseases and disorders. By delivering synthetic small RNA to trigger the RNAi pathway or fulfill other policing roles, researchers hope to silence genes that cause not only cancer but also neurodegenerative diseases, diabetes, asthma, and even infectious diseases such as hepatitis, influenza, and HIV. The act of delivering these nuggets of authority is easier said than done; nucleases and immune responses are always at the ready to pounce on these foreign nucleotides. Studies are being conducted to fuse them to delivery vehicles like lipids, antibodies, or polymers in order to transport small RNAs across the cellular membrane.
Other Important Classes of RNA and Specialized Functions
Another major class of RNAs, the small nuclear RNAs (snRNAs), was also discovered in the early 1980’s. Molecular biologist Joan Steitz was working on the autoimmune disease systemic lupus when she began to characterize the snRNAs. There are six different snRNAs, now called U1-U6 RNAs. These RNAs exist in the nucleus of eukaryotic cells and play a vital role in mRNA splicing. They associate with proteins in the spliceosome, forming so-called ribonucleoprotein complexes (snRNPs, pronounced “snurps”), and play a prominent role in detecting proper splice sites and directing the protein enzymes to cut and paste at the proper locations.
It has been known since the late 1950’s that many viruses contain RNA, and not DNA, as their genetic material. This is another fascinating role for RNA. The viruses that cause influenza, polio, and a host of other diseases are RNA viruses. Of particular note are a class of RNA viruses known as retroviruses. Retroviruses, which include human immunodeficiency virus (HIV), the virus that causes acquired immunodeficiency syndrome (AIDS) in humans, use a special enzyme called reverse transcriptase to make a DNA copy of their RNA when they enter a cell. The DNA copy is inserted into the DNA of the host cell, where it is referred to as a “provirus,” and never leaves. This discovery represents one of
the exceptions to the central dogma. In the central dogma, RNA is always made from DNA, and retroviruses have reversed this flow of information. Clearly, understanding the structures and functions of the RNAs associated with these viruses will be important in attempting to create effective treatments for the diseases associated with them.
An additional role of RNA was noted during the elucidation of the mechanism of DNA replication. It was found that a small piece of RNA, called a “primer,” must be laid down by the enzyme primase, an RNA polymerase, before DNA polymerase can begin. RNA primers are later removed and replaced with DNA. Also, it is worth mentioning that the universal energy-storing molecule of all cells, adenosine triphosphate (ATP), is in fact a version of the RNA nucleotide containing adenine (A).
Impact and Applications
The discovery of the many functions of RNA, especially its catalytic ability, has radically changed the understanding of the functioning of genetic and biological systems and has revolutionized the views of the scientific community regarding the origin of life. The key to understanding how RNA can perform all of its diverse functions lies in elucidating its many structures, since structure and function are inseparable. Much progress has been made in establishing the three-dimensional structure of hundreds of RNA molecules.
Extensive research has been conducted on RNA folding, degradation, and regulation, as it has become clear that it plays a vital role in genetic disease, cancer, and retroviral infections. The revelation of the RNAi pathway has ignited hope for treatments of ailments ranging from AIDS to Parkinson disease. One of the first RNAi remedies, Macugen, to pass human trials involved the eye disease wet macular degeneration. This occurs when too many blood vessels are grown due to an overactive gene, and the vessels’ leakiness damages vision. The eye has a low population of nucleases, so when naked siRNAs are directly injected into the vitreous cavity, they successfully stunt the overactive gene, and vision is restored.
Additionally, plants, bacteria, and animals have been genetically engineered to alter the expression of some of their genes, in many cases making use of the new RNA technology. An example is a genetically engineered tomato that does not ripen until it is treated at the point of sale. This tomato was created by inserting an antisense RNA gene; when it is expressed, it inactivates the mRNA that codes for the enzyme involved in production of the ripening hormone.
One thing is clear: RNA is one of the most structurally interesting and functionally diverse of all the biological molecules.
Key terms
messenger RNA (mRNA)
:
a type of RNA that carries genetic instructions, copied from genes in DNA, to the ribosome to be decoded during translation
retrovirus
:
a special type of virus that carries its genetic information as RNA and converts it into DNA that integrates into the cells of the virus’s host organism
ribosomal RNA (rRNA)
:
a type of RNA that forms a major part of the structure of the ribosome
ribosomes
:
organelles that function in protein synthesis and are made up of a large and a small subunit composed of proteins and ribosomal RNA (rRNA) molecules
ribozyme
:
an RNA molecule that can function catalytically as an enzyme
RNAi
:
abbreviation for “interference RNA,” which hampers mRNA translation
small RNA
:
a class of RNA in which several subspecies are only twenty to twenty-five base pairs long and involved in the degradation or regulation of mRNA
transcription
:
the synthesis of an RNA molecule directed by RNA polymerase using a DNA template
transfer RNA (tRNA)
:
a form of RNA that acts to decode genetic information present in mRNA, carries a particular amino acid, and is vital to translation
translation
:
the synthesis of a protein molecule directed by the ribosome using information provided by an mRNA
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