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Puria Rafsanjani Mehrshad Nourani Ashkan Novin Nucleotides, Nucleic acids, and Heredity While these dogs might appear to be a normal mother and puppy, the latter is really the first cloned dog, Snuppy. The larger dog is a male Afghan whose DNA was used to create the clone. History of Genes discovery From about the end of the nineteenth century, biologists suspected that the transmission of hereditary information from one generation to another took place in the nucleus of the cell. More precisely, they believed that structures within the nucleus, called chromosomes, have something to do with heredity. Different species have different numbers of chromosomes in the nucleus. The information that determines external characteristics (red hair, blue eyes) and internal characteristics (blood group, hereditary diseases) was thought to reside in genes located inside the chromosomes. What does DNA do? The information that tells the cell which proteins to manufacture is carried in the molecules of DNA. We now know that not all genes lead to the production of protein, but all genes do lead to the production of another type of nucleic acid, called ribonucleic acid (RNA). What Are Nucleic Acids Made Of? Two kinds of nucleic acids are found in cells: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Each has its own role in the transmission of hereditary information. As we just saw, DNA is present in the chromosomes of the nuclei of eukaryotic cells. RNA is not found in the chromosomes, but rather is located elsewhere in the nucleus and even outside the nucleus, in the cytoplasm. Nucleic Acids => A. Bases The bases found in DNA and RNA are chiefly those shown in Figure. All of them are basic because they are heterocyclic aromatic amines. Purines Pyrimidines The five principal bases of DNA and RNA. The hydrogens shown in blue are lost when the bases bond to monosaccharides. What the hell are purines and Pyrimidines Pyrimidine is a heterocyclic aromatic organic compound similar to benzene and pyridine, containing two nitrogen atoms at positions 1 and 3 of the six-member ring. What the hell are purines and Pyrimidines A purine is a heterocyclic aromatic organic compound. It consists of a pyrimidine ring fused to an imidazole ring. Purines are the most widely occurring nitrogencontaining heterocycle in nature. Imidazole is an organic compound with the formula (CH)2N(NH)CH. Nucleic Acids => A. Bases adenine (A) and guanine (G)—are purines the other three—cytosine (C), thymine (T), and uracil (U)— are pyrimidines. Purines Pyrimidines The five principal bases of DNA and RNA. The hydrogens shown in blue are lost when the bases bond to monosaccharides. The two purines (A and G) and one of the pyrimidines (C) are found in both DNA and RNA, but uracil (U) is found only in RNA, and thymine (T) is found only in DNA. Note that thymine differs from uracil only in the methyl group in the 5 position. Nucleic Acids => B. Sugars The sugar component of RNA is D-ribose. In DNA, it is 2-deoxy-D-ribose (hence the name deoxyribonucleic acid). Nucleoside: The combination of sugar and base is known as a nucleoside. The purine bases are linked to C-1 of the monosaccharide through N-9 (the nitrogen at position 9 of the fivemembered ring) by a b-N-glycosidic bond: Nucleoside: The pyrimidine bases are linked to C-1 of the monosaccharide through their N-1 by a b-N-glycosidic bond. Nucleic Acids => C. Phosphate The third component of nucleic acids is phosphoric acid. When this group forms a phosphate ester bond with a nucleoside, the result is a compound known as a nucleotide. For example, adenosine combines with phosphate to form the nucleotide adenosine 5’ -monophosphate (AMP): The ‘ sign in adenosine 5’-monophosphate is used to distinguish which molecules the phosphate is bound to. Numbers without primes refer to positions on the purine or pyrimidine base. Numbers on the sugar are denoted with primes. 14 we will see how DNA and RNA are chains of nucleotides. In summary: A nucleoside : Base 1 Sugar A nucleotide : Base 1 Sugar 1 Phosphate A nucleic acid : A chain of nucleotides What Is the Structure of DNA and RNA? A. Primary Structure Nucleic acids are polymers of nucleotides. Their primary structure is the sequence of nucleotides. Note that it can be divided into two parts: (1) the backbone of the molecule and (2) the bases that are the side-chain groups. The backbone in DNA consists of alternating deoxyribose and phosphate groups. Each phosphate group is linked to the 3’ carbon of one deoxyribose unit and simultaneously to the 5’ carbon of the next deoxyribose unit. The primary structure of RNA is the same except that each sugar is ribose (so an -OH group appears in the 2’ position) rather than deoxyribose and U is present instead of T. What Is the Structure of DNA and RNA? B. Secondary Structure of DNA DNA is composed of two strands entwined around each other in a double helix. The sugar–phosphate backbone is on the outside, exposed to the aqueous environment, and the bases point inward. The bases are hydrophobic, so they try to avoid contact with water. Through their hydrophobic interactions, they stabilize the double helix. The bases so paired form hydrogen bonds with each other, two for A-T and three for G-C, thereby stabilizing the double helix. A-T and G-C are complementary base pairs. The bases of DNA cannot stack properly in the double helix if a purine is opposite a purine or if a pyrimidine is opposite a pyrimidine. Only one hydrogen bond is possible for TG or CA. These combinations are not found in DNA. The form of the DNA double helix shown in Figure is called B-DNA. It is the most common and most stable form. Other forms become possible where the helix is wound more tightly or more loosely, or is wound in the opposite direction. With B-DNA, a distinguishing feature is the presence of a major groove and a minor groove, which arise because the two strands are not equally spaced around the helix. Interactions of proteins and drugs with the major and minor grooves of DNA serve as an active area of research. What Is the Structure of DNA and RNA? C. Higher-Order Structures of DNA If a human DNA molecule were fully stretched out, its length would be perhaps 1 m. However, the DNA molecules in the nuclei are not stretched out, but rather coiled around basic protein molecules called histones. The acidic DNA and the basic histones attract each other by electrostatic (ionic) forces, combining to form units called nucleosomes. In a nucleosome, eight histone molecules form a core, around which a 147-base-pair DNA double helix is wound. Nucleosomes are further condensed into chromatin when a 30-nm-wide fiber forms in which nucleosomes are wound in a solenoid fashion, with six nucleosomes forming a repeating unit. Chromatin fibers are organized still further into loops, and loops are arranged into bands to provide the superstructure of chromosomes. let us summarize the three differences in structure between DNA and RNA: 1. DNA has four bases: A, G, C, and T. RNA has three of these bases—A,G, and C—but its fourth base is U, not T. 2. In DNA, the sugar is 2-deoxy-D-ribose. In RNA, it is Dribose. 3. DNA is almost always double-stranded, with the helical structure shown. There are several kinds of RNA, None of them has a repetitive double-stranded structure like DNA, although base-pairing can occur within a chain. When it does, adenine pairs with uracil because thymine is not present. Other combinations of hydrogenbonded bases are also possible outside the confines of a double helix. 17.4 What Are the Different Classes of RNA? =>1. Messenger RNA (mRNA) mRNA molecules are produced in the process called transcription, and they carry the genetic information from the DNA in the nucleus directly to the cytoplasm, where most of the protein is synthesized. Messenger RNA consists of a chain of nucleotides whose sequence is exactly complementary to that of one of the strands of the DNA. This type of RNA is not long-lived, however. It is synthesized as needed and then degraded, so its concentration at any given time is rather low. The size of mRNA varies widely, with the average unit containing perhaps 750 nucleotides. central dogma of molecular biology: The fundamental process of information transfer in cells. (1) Information encoded in the nucleotide sequence of DNA is transcribed through synthesis of an RNA molecule whose sequence is dictated by the DNA sequence. (2) As the sequence of this RNA is read (as groups of three consecutive nucleotides) by the protein synthesis machinery, it is translated into the sequence of amino acids in a protein. 17.4 What Are the Different Classes of RNA? =>2. Transfer RNA (tRNA) Containing from 73 to 93 nucleotides per chain, tRNAs are relatively small molecules. There is at least one different tRNA molecule for each of the 20 amino acids from which the body makes its proteins. Transfer RNA molecules contain not only cytosine, guanine, adenine, and uracil, but also several other modified nucleotides, such as 1methylguanosine. 17.4 What Are the Different Classes of RNA? =>2. Transfer RNA (tRNA) 17.4 What Are the Different Classes of RNA? =>3.Ribosomal RNA (rRNA) Ribosomes, which are small spherical bodies located in the cells but outside the nuclei, contain rRNA. They consist of about 35% protein and 65% ribosomal RNA (rRNA). These large molecules have molecular weights up to 1 million. As you already know, protein synthesis takes place on the ribosomes. Ribosomes consist of two subunits, one larger than the other. In turn, the smaller subunit consists of one large RNA molecule and about 20 different proteins; the larger subunit consists of two RNA molecules in prokaryotes (three in eukaryotes) and about 35 different proteins in prokaryotes (about 50 in eukaryotes). 17.4 What Are the Different Classes of RNA? =>4. Small Nuclear RNA (snRNA) A recently discovered RNA molecule is sn- RNA, which is found, as the name implies, in the nucleus of eukaryotic cells. This type of RNA is small, about 100 to 200 nucleotides long, but it is neither a tRNA molecule nor a small subunit of rRNA. In the cell, it is complexed with proteins to form small nuclear ribonucleoprotein particles, sn- RNPs, pronounced “snurps.” Their function is to help with the processing of the initial mRNA transcribed from DNA into a mature form that is ready for export out of the nucleus. This process is often referred to as splicing, and it is an active area of research. 17.4 What Are the Different Classes of RNA? =>5. Micro RNA (miRNA) A very recent discovery is another type of small RNA, miRNA. These RNAs are only 20–22 nucleotides long but are important in the timing of an organism’s development. They inhibit translation of mRNA into protein and promote the degradation of mRNA. It was recently discovered, however, that these versatile RNAs can also stimulate protein production in cells when the cell cycle has been arrested. They play important roles in cancer, stress respsonses, and viral infections. 17.4 What Are the Different Classes of RNA? =>6. Small Interfering RNA (siRNA) Short stretches of RNA (20–30 nucleotides long), called small interfering RNA, have been found to have an enormous control over gene expression. This process serves as a protective mechanism in many species, with the siRNAs being used to eliminate expression of an undesirable gene. siRNAs lead to the degradation of specific mRNA molecules. In what has become an explosion of new biotechnology, many companies have been created to produce and market designer siRNAs to knock out hundreds of known genes. This technology also has medical applications, as siRNA has been used to protect mouse liver from hepatitis and to help clear infected liver cells of the disease. 17.5 What Are Genes? A gene is a stretch of DNA, containing a few hundred nucleotides, that carries one particular message—for example, “make a globin molecule” or “make a tRNA molecule.” One DNA molecule may have between 1 million and 100 million bases. Therefore, many genes are present in one DNA molecule. In bacteria, this message is continuous; in higher organisms, it is not. That is, stretches of DNA that spell out (encode) the amino acid sequence to be assembled are interrupted by long stretches that seemingly do not code for anything. The coding sequences are called exons, short for “expressed sequences,” and the noncoding sequences are called introns, short for “intervening sequences.” In other words, the introns function as spacers and, in rare instances, as enzymes, catalyzing the splicing of exons into mature mRNA. Figure shows the difference between prokaryotic and eukaryotic production of proteins. In humans, only 3% of the DNA codes for proteins or RNA with clear functions. Introns are not the only noncoding DNA sequences, however. Satellites are DNA molecules in which short nucleotide sequences are repeated hundreds or thousands of times. Large satellite stretches appear at the ends and centers of chromosomes and provide stability for the chromosomes. Smaller repetitive sequences, called mini-satellites or microsatellites, are associated with cancer when they mutate. 17.6 How Is DNA Replicated? Introduction In a human cell, some 3 billion base pairs must be duplicated at each cell cycle, and a fully grown human being may contain more than 1 trillion cells. Each cell contains the same amount of DNA as the original single cell. 17.6 How Is DNA Replicated? Introduction Replication begins at a point in the DNA called an origin of replication. In human cells, the average chromosome has several hundred origins of replication where the copying occurs simultaneously. The DNA double helix has two strands running in opposite directions. The point on the DNA where replication is proceeding is called the replication fork. 17.6 How Is DNA Replicated? Replication is bidirectional and takes place at the same speed in both directions. An interesting detail of DNA replication is that the two daughter strands are synthesized in different ways. One of the syntheses is continuous along the 3’→5’ strand. It is called the leading strand. Along the other strand that runs in the 5’→3’ direction, the synthesis is discontinuous. It is called the lagging strand. Replication always proceeds from the 5’ to the 3’ direction from the perspective of the chain that is being synthesized. General features of the replication of DNA. The two strands of the DNA double helix are shown separating at the replication fork. The actual reaction occurring is a nucleophilic attack by the 3’ hydroxyl of the deoxyribose of one nucleotide against the first phosphate on the 5’ carbon of the incoming nucleoside triphosphate. One of the more interesting aspects of DNA replication is that the basic reaction of synthesis always requires an existing chain with a nucleotide that has a free 3’-hydroxyl to do the nucleophilic attack. DNA replication cannot begin without this preexisting chain to latch onto. We call this chain a primer. In all known forms of replication, the primer is made out of RNA, not DNA. Replication is a very complex process involving a number of enzymes and binding proteins. A growing body of evidence indicates that these enzymes assemble their products in “factories” through which the DNA moves. Such factories may be bound to membranes in bacteria. In higher organisms, the replication factories are not permanent structures. Instead, they may be disassembled and their parts reassembled in ever-larger factories. These assemblies of enzyme “factories” go by the name of replisomes, and they contain key enzymes such as polymerases, helicases, and primases. Steps of DNA Replication : 1. Opening up the superstructure During replication, the very condensed superstructure of chromosomes must be opened so that it becomes accessible to enzymes and other proteins. A complicated signal transduction mechanism accomplishes this feat. One notable step of the signal transduction is the acetylation and deacetylation of key lysine residues of histones. When histone acetylase, an enzyme, puts acetyl groups on key lysine residues, some positive charges are eliminated and the strength of the DNA–histone interaction is weakened Steps of DNA Replication : 1. Opening up the superstructure Histone This Acetylation : process allows the opening up of key regions on the DNA molecule. When another enzyme, histone deacetylase, removes these acetyl groups, the positive charges are reestablished. That, in turn, facilitates regaining the highly condensed structure of chromatin. Steps of DNA Replication : 2. Relaxation of Higher-Order Structures of DNA Topoisomerases (also called gyrases) are enzymes that facilitate the relaxation of supercoiling in DNA. They do so during replication by temporarily introducing either singleor double-strand breaks in DNA. The transient break forms a phosphodiester linkage between a tyrosyl residue of the enzyme and either the 5’ or 3’ end of a phosphate on the DNA. Once the supercoiling is relaxed, the broken strands are joined together, and the topoisomerase diffuses from the location of the replicating fork. Topoisomerases are also involved in the untangling of the replicated chromosomes, before cell division can occur. Steps of DNA Replication : 3. Unwinding the DNA Double Helix The replication of DNA molecules starts with the unwinding of the double helix, which can occur at either end or in the middle. Special unwinding protein molecules, called helicases, attach themselves to one DNA strand and cause the separation of the double helix. Helicases of eukaryotes are made of six different protein subunits. The subunits form a ring with a hollow core, where the single-stranded DNA sits. The helicases hydrolyze ATP as the DNA strand moves through. The energy of the hydrolysis promotes this movement. Steps of DNA Replication : 4. Primers/Primases Primers are short—4 to 15 nucleotides long—RNA oligonucleotides synthesized from ribonucleoside triphosphates. They are needed to initiate the synthesis of both daughter strands. The enzyme catalyzing this synthesis is called primase. Primases form complexes with DNA polymerase in eukaryotes. Primers are placed about every 50 nucleotides in the lagging-strand synthesis. Steps of DNA Replication : 5. DNA Polymerase Once the two strands are separated at the replication fork, the DNA nucleotides must be lined up. All four kinds of free DNA nucleotide molecules are present in the vicinity of the replication fork. These nucleotides constantly move into the area and try to fit themselves into new chains. Wherever a cytosine, for example, is present on one of the strands of an unwound portion of the helix, all four nucleotides may approach, but three of them will be turned away because they do not fit. Only the nucleotide of guanine fits. Steps of DNA Replication : 5. DNA Polymerase In the absence of an enzyme, this alignment is extremely slow. The speed and specificity are provided by DNA polymerase. It surrounds the end of the DNA template– primer complex, creating a specifically shaped pocket for the incoming nucleotide. With such a close contact, the activation energy is lowered and the polymerase enables complementary base pairing with high specificity at a rate of 100 times per second. While the bases of the newly arrived nucleotides are being hydrogen-bonded to their partners, polymerases join the nucleotide backbones. Along the lagging strand 3’ to 5’, the enzymes can synthesize only short fragments because the only way they can work is from 5’ to 3’. These short fragments consist of about 200 nucleotides each, named Okazaki fragments after their discoverer. And Again : Steps of DNA Replication : 6. Ligation The Okazaki fragments and any nicks remaining are eventually joined together by another enzyme, DNA ligase. At the end of the process, there are two double-stranded DNA molecules, each exactly the same as the original molecule. Thank You forYour Attention!!!