DNA and RNA Synthesis

FIELDS OF STUDY: Biochemistry; Genetics; Molecular Biology

ABSTRACT

The basic process of DNA and RNA synthesis is described, and its importance in living biochemical systems is discussed. Also described are modern advances such as artificial methods for the synthesis of DNA and RNA and their applications.

Understanding DNA and RNA Synthesis

It is tempting to oversimplify the formation of deoxyribonucleic acid (DNA) by likening it to zipping up a zipper, but that is perhaps the easiest way to visualize the process. Indeed, DNA synthesis is diagrammed in virtually all biochemistry texts as such. The analogy is even further simplified by associating the "teeth" of the zipper with the purine and pyrimidine nucleotides from which the molecular structures of DNA and ribonucleic acid (RNA) are formed.

A nucleotide is formed when a purine or pyrimidine base and a phosphate group are chemically bonded to a sugar molecule. Only five different purine and pyrimidine bases are utilized in constructing DNA or RNA nucleotides. In DNA nucleotides, these are the bases adenine, cytosine, guanine, and thymine, while RNA uses the base uracil instead of thymine in its nucleotides. The different bases are indicated by the first letter of their names: A, C, T, G, and U. There are different mnemonic devices for recalling the complementarity of the different bases. One is that the curved letters C and G go together, as do the pointed letters A and T. Another is an easily remembered phrase such as "Cary Grant Ate Tacos." In fact, any number of such devices can be used to suit an individual’s personal preference.

The second major difference between DNA and RNA is the nature of the sugars with which the nucleotides are constructed. In RNA nucleotides, the sugar molecule is ribose, a five-carbon simple sugar related to fructose. Sugar molecules are carbohydrates, indicating that each carbon atom in the molecule is chemically bonded to both an H atom and an –OH group. These are the components of the water molecule, so the term indicates that each carbon (carbo-) atom in the molecule is hydrated (-hydrate). In DNA nucleotides, however, the sugar is deoxyribose. The name indicates that the molecule lacks an oxygen atom that is part of the ribose sugar molecular structure. This difference in the structure of the sugar portion of the nucleotide is subtle, but of essential importance because it alone determines whether the molecule, and its role in the biochemical process of life, is DNA or RNA. In both DNA and RNA, the sugar molecules are in the form of a five-member ring structure made up of one oxygen atom and four of the five carbon atoms.

The third component of DNA/RNA nucleotides is the phosphate group, PO43−, or Pi, generally referred to as "inorganic phosphate" when in that form and "phosphate" when bonded to another biomolecule, such as adenosine in adenosine triphosphate (ATP). The bonds between an oxygen atom in the phosphate group and other molecules are stable, even though they are deemed "high energy" bonds. In respiration and glycolysis (the decomposition of glucose), the bond between the third and second phosphate group in the triphosphate component is utilized both to store energy by its formation and to release energy when that bond is cleaved.

Both DNA and RNA are large molecules. Their respective molecular structures consist of a long, biopolymer chain of alternating sugar molecules and phosphate groups. In both the DNA and RNA structures, each sugar component has a purine or pyrimidine base molecule bonded to the carbon atom on one side of the ring oxygen atom, and the phosphate group bonded to a carbon atom on the other side of the ring oxygen atom. It is at this point that the difference between ribose and deoxyribose sugar becomes vitally important. The RNA molecule consists of a single strand made up of adenine, cytosine, guanine, and uracil nucleotides. The DNA molecule, however, consists of two complementary strands of adenine, cytosine, guanine, and thymine nucleotides. These match up with each other as the base portions of the nucleotides in one strand and connect to the corresponding nucleotides in the other strand. The end result is that a DNA molecule is considerably bigger than an RNA molecule, and it has the form of a double helix as the two component strands coil around each other. The RNA molecule consists of a single nucleotide strand that assumes different shapes according to its role in transcription and gene expression.

Discovery and Analysis of DNA and RNA Molecules

DNA was isolated from cell nuclei as early as 1869, but the fact that it bears genetic information was not known until 1943, when Oswald Avery, Colin MacLeod, and Maclyn McCarty demonstrated that introducing DNA from a virulent strain of the pneumococcus bacterium into a nonvirulent strain could produce the virulent strain. In 1953, James D. Watson and Francis Crick, with significant assistance from their laboratory manager Maurice Wilkins and using the analytical and theoretical work of Rosalind Franklin, first published the discovery that the DNA molecule has a double helix form, an image now so well recognized. Since then, methods and techniques for the manipulation and analysis of DNA have advanced, permitting biochemists and geneticists to obtain a better understanding of the role of genes and chromosomes in the DNA molecule. In 2001, the journal Nature published the first complete analysis of the human genome, which demonstrated, among other things, that all humans alive today are descended from a very few human populations that originated in Africa in the distant past. As the genome was deciphered over time, an understanding of the mechanisms by which DNA and RNA are synthesized was also obtained.

Formation of DNA and RNA in Living Cells: Four Basic Rules

Both DNA and RNA are produced by copying a preexisting DNA strand according to the base pairings of adenine to thymine and cytosine to guanine. In order to carry out this process, the DNA molecule must first "unzip," allowing nucleotide fragments to form a complementary RNA strand in which uracil nucleotides replace thymine nucleotides. From this complementary RNA strand, a duplicate of the original DNA strand is assembled from other fragments. This process can be thought of as making a mold from one half of the DNA molecule and then using it to cast a copy of the original.

Second, both RNA and DNA strands grow in one direction only. The phosphate group of each nucleotide is situated at the 5′ position of the sugar molecule, the number indicating a specific location in the molecular structure according to the conventions for naming organic molecules. At the 3′ position of each sugar molecule, there is a free hydroxyl (–OH) substituent that can form the phosphate ester bond with another nucleotide. Formation of a DNA or an RNA strand always proceeds from the 5′-position in one nucleotide to the 3′-position in the next nucleotide as nucleotides are added in sequence.

Third, both DNA and RNA are synthesized by very specific enzymes in polymerase chain reactions. New strands of RNA are produced only by RNA polymerases, and new DNA strands are produced only by DNA polymerases in DNA/RNA polymerase reactions. Through the process of transcription, a new RNA strand is produced as RNA polymerases transcribe the nucleotide pattern of the parent DNA strand. RNA polymerase enzymes are able to initiate the formation of a new strand by coordinating to an appropriate site on a duplex strand of DNA (the double helix form of the molecule), where they temporarily separate the two strands of the DNA molecule and begin the process of assembling a new RNA strand from the corresponding nucleotides. DNA polymerases are not able to initiate the formation of a new strand directly. Instead, the process requires the formation of a primer, a DNA or an RNA segment that is bound to the parent DNA strand and is acting as the template for the new DNA strand. Both RNA and DNA polymerases comprise several different proteins, each of which carries out a specific function or transformation.

Fourth, synthesis of a new duplex DNA strand proceeds only from a particular formation known as a "growing fork." Specific enzymes function to open the duplex strand, allowing other enzymes to assemble the matching complementary strands from the appropriate nucleotides. As the new strands are formed, still other enzymes function to rejoin the strands as the growing fork progresses along the length of the template duplex DNA molecule. An important aspect of duplex DNA replication is that, because the strands only grow in one direction, the directions of growth on the two branches of the growing fork are opposite to each other. The new strand on the "leading" branch of the fork grows continuously, nucleotide by nucleotide. The new strand on the "trailing" branch of the fork is assembled instead in bits and pieces from various nucleotide segments.

Amplifying DNA for Analysis

Among the many techniques and methods that have developed for the manipulation of DNA samples—and that are especially important for the science of DNA "fingerprinting"—probably the most important is DNA amplification. By this method, an extremely minute sample of DNA, as might be obtained from just a few hair follicles found at a crime scene, for example, undergoes repeated replications so that enough of the DNA is present to produce a clear fragmentation pattern that is the "fingerprint" of that particular DNA. This methodology has been used to convict criminals who might otherwise have gone free, as well as to free individuals from prison who had been wrongfully convicted of crimes they did not commit.

PRINCIPAL TERMS

• complementary strand: one of the two strands of nucleotides that make up a DNA molecule, with each nucleotide in one strand corresponding to the position of its complementary nucleotide (cytosine for guanine, adenine for thymine, and vice versa) in the other.
• deoxyribonucleic acid (DNA): a large molecule formed by two complementary strands of nucleotides that encodes the genetic information of all living organisms.
• gene expression: the process by which RNA copies genes, which are specific segments of the DNA molecule, and uses the information to synthesize either proteins or other types of RNA.
• nucleotide: the basic structural component of DNA and RNA, consisting of a ribose (in RNA) or deoxyribose (in DNA) sugar molecule bonded to a phosphate group and one of five nucleobases: cytosine, adenine, guanine, thymine (DNA only), or uracil (RNA only).
• polymerase chain reaction: a laboratory method in which a very small amount of DNA can be replicated thousands or even millions of times, using free nucleotides and an enzyme called DNA polymerase.
• ribonucleic acid (RNA): a category of large molecules, typically consisting of a single strand of nucleotides, that perform various functions in cells, including the transcription of DNA molecules and the transfer of specific genetic information for protein synthesis.

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