Biochem 8.2 Nucleic Acids PDF

# Nucleic Acids ## Introduction Just as amino acids polymerize to form peptides and monosaccharides polymerize to form polysaccharides, nucleotides polymerize to form nucleic acids. DNA and RNA are two distinct types of nucleic acids used by all known living organisms. They consist of deoxyribonucl...

# Nucleic Acids ## Introduction Just as amino acids polymerize to form peptides and monosaccharides polymerize to form polysaccharides, nucleotides polymerize to form nucleic acids. DNA and RNA are two distinct types of nucleic acids used by all known living organisms. They consist of deoxyribonucleotides and ribonucleotides, respectively. This lesson explains the process of nucleotide polymerization and describes interactions between and within nucleic acids. ## 8.2.01 Nucleic Acid Formation Concept 8.1.03 explains that the carbons in the sugar component of a nucleotide are numbered using the prime symbol ('). This numbering system is important in understanding the structure of nucleic acids. Just as peptides form when the carboxyl group of one amino acid reacts with the amino group of another, nucleic acids form when the hydroxyl group on the 3' carbon of one nucleotide reacts with a phosphate group on the 5' carbon of another nucleotide. The result is a phosphodiester bond linking the two nucleotides. When the 3' hydroxyl group of one nucleoside triphosphate (NTP or dNTP) attacks the a phosphate at the 5' end of the other NTP, the ẞ and y phosphates are released together as pyrophosphate (PP₁). Therefore, nucleic acid synthesis is a condensation reaction in which pyrophosphate is released instead of water. A condensation reaction between two nucleotides is shown in Figure 8.24. The resulting dinucleotide has one free 5' triphosphate and one free 3' hydroxyl. The 5' and 3' groups that were involved in the reaction, however, are no longer free (ie, they are unavailable to react with other nucleotides). The end of the nucleic acid corresponding to the free 5' phosphate is called the 5' end, and the end corresponding to the free 3' hydroxyl is called the 3' end. This is analogous to the N- and C-termini of a peptide or the nonreducing and reducing ends of a complex carbohydrate (see Figure 8.25). By convention, the sequence of a nucleic acid is always written from the 5' end to the 3' end, unless specified otherwise. The 3' end of a dinucleotide can react with the 5' end of another NTP to form a trinucleotide. This process can continue to form nucleic acids of any length. Nucleic acids containing a few nucleotides are often called oligonucleotides. However, the length of a nucleic acid can be extensive. For example, RNA molecules commonly contain hundreds or even thousands of individual nucleotide components. DNA is commonly even longer: each DNA strand of the human X chromosome consists of approximately 155 million individual nucleotides. These large molecules may be called polynucleotides but are often simply called nucleic acids. ## Concept Check 8.3 Describe the reaction that must occur for ATP and CTP to form a dinucleotide with the sequence CA. Then describe how a reaction between CA and UTP could form the trinucleotide CAU. - Solution: Each nucleic acid has a repeating backbone structure consisting of a sugar (eg, ribose, deoxyribose) linked to a phosphate, which is linked to another sugar, which is linked to another phosphate, and so on. This is known as a sugar-phosphate backbone. At physiological pH, each phosphate has a negative charge, and therefore the backbones of DNA and RNA are highly negatively charged. Eukaryotes use positively charged histone proteins to neutralize and store DNA (see Biology Lesson 1.4). Figure 8.26 shows the repeating sugar-phosphate structure and negative charges in a nucleic acid backbone. Nucleic acid synthesis is facilitated by the enzymes DNA polymerase (for DNA synthesis) and RNA polymerase (for RNA synthesis). Different types of RNA are synthesized by different RNA polymerases, as detailed in Biology Chapter 2. The nucleotide at the 5' end of a growing nucleic acid is the first to have been incorporated into the chain, and the nucleotide at the 3' end is incorporated last (Figure 8.27). As the nucleic acid grows, the 3' end of the growing strand attacks the 5' end of the next nucleotide to be added. Once a nucleic acid is synthesized, it may be modified. For example, DNA polymerase occasionally inserts the wrong nucleotide. Various mechanisms exist to detect such errors, and many DNA polymerase enzymes include 3'-5' exonuclease activity. Exonucleases are hydrolases that remove nucleotides from the ends of a nucleic acid strand (in this case the 3' end) by hydrolysis. Lesson 8.1 discusses how the nitrogenous bases in nucleotides may undergo chemical changes such as deamination. When this occurs, the affected base may be removed by a glycosylase enzyme, which hydrolyzes specific glycosidic bonds. This leaves a ribose or deoxyribose sugar with no nitrogenous base. The sugar can then be removed by an endonuclease, which hydrolyzes both phosphodiester bonds in the middle of a nucleic acid. DNA polymerase can then insert a new nucleotide into the DNA strand in place of the removed one. However, this process leaves a gap, called a nick, at the 3' end of the repaired nucleotide. DNA ligase uses ATP to provide the energy needed to repair the nick. Figure 8.28 shows the mechanism of nucleotide removal and substitution within a nucleic acid. ## 8.2.02 Base Pairing and Secondary Structure in Nucleic Acids The sequence of nucleotides in a nucleic acid constitutes the primary structure of that nucleic acid. Just as peptides can form secondary structures, so can nucleic acids. This concept outlines the secondary structures commonly found in both DNA and RNA and the forces that hold them together. Perhaps the best-known secondary structure in nucleic acids is the double helix. In living cells, DNA exists as a double-stranded molecule (ie, two distinct DNA strands held together noncovalently). These two strands twist around each other to form the helical structure. The process of double-strand formation is called annealing. The annealed strands align in an antiparallel orientation (see Figure 8.29), meaning that the 3' end of one strand aligns with the 5' end of the other. Several forces help hold the two strands together. Lesson 8.1 introduces the concept of hydrogen bonding between particular nitrogenous bases to form base pairs. When aligned properly, guanine pairs with cytosine and adenine pairs with thymine in DNA. Note that each base pair consists of one purine and one pyrimidine. A purine and a pyrimidine that pair with each other in this way are said to be complementary. Combined with the antiparallel structure of a double helix, the two strands of a double-stranded DNA molecule are said to be the reverse complements of each other. The hydrogen bonds between complementary bases on opposite DNA strands provide a significant portion of the force that drives double helix formation. DNA strands that do not have complementary bases do not anneal, and even a single mismatch (ie, alignment of noncomplementary bases) can significantly reduce the favorability of annealing. In addition to hydrogen bonding between bases within a double helix, the structure is also stabilized by a hydrophobic effect similar to that seen in protein folding. Compared to the negatively charged backbone, the bases in DNA are relatively hydrophobic. Therefore, when these bases are exposed to an aqueous environment, water must form a more organized solvation layer around them than around the more hydrophilic backbone groups. Double helix formation allows the nitrogenous bases to "hide" from the surrounding water, while the water can interact favorably with the exposed sugar and phosphate groups. Several interactions that stabilize double helices are shown in Figure 8.30. At the same time, within a double helix, the base pairs of each strand are stacked almost on top of each other. These bases are aromatic and have pi orbitals above and below their planes. In this arrangement, each base can therefore participate in a noncovalent interaction called pi stacking with the bases that are adjacent to it. Pi stacking helps stabilize the specific DNA conformation. RNA can also form secondary structures. During transcription, for example, the RNA molecule being synthesized temporarily pairs with the DNA strand that serves as the template and may briefly form a DNA-RNA double helix. More importantly, many RNA molecules perform biological functions that require specific structures (eg, tRNA, ribosomal RNA). These molecules tend to be single-stranded but nevertheless form secondary and tertiary structures. Commonly, the bases in one portion of a functional RNA strand are complementary to another portion of the same strand. In such a case, the strand may fold on itself and allow the complementary bases to pair with each other. This pairing gives rise to a helical structure within a single RNA strand. Like proteins, RNA may further fold into complex three-dimensional shapes that correspond to their functions. RNA structure is often depicted in two-dimensional form, with phosphodiester bonds between adjacent nucleotides shown as solid lines and hydrogen bonds between complementary bases indicated by dashed lines. These depictions often result in structures that resemble hairpins and are consequently named hairpin structures. Figure 8.31 shows both two-dimensional and three-dimensional depictions of tRNA. Note that the 2D depiction clearly shows complementary base pairing in a three-leafed clover shape; however, the true 3D structure of tRNA is more L-shaped. ## 8.2.03 Chargaff's Rules Base pairing in nucleic acids gives rise to a set of rules known as Chargaff's rules, which dictate certain aspects of the composition of paired nucleic acid strands. Chargaff's rules state the following: - The number of adenine bases in double-stranded DNA equals the number of thymine bases, and the number of guanine bases equals the number of cytosine bases. This is because every adenine base in one strand is paired with a thymine base in the complementary strand, and every guanine base is paired with a cytosine base. - The sum of purines equals the sum of pyrimidines because every purine is paired with a pyrimidine. Note that Chargaff's rules apply only to double-stranded nucleic acids, specifically DNA. Each individual strand may have any number of adenine, guanine, cytosine, and thymine bases. Only when a single strand is paired with its complementary strand do Chargaff's rules apply. Figure 8.32 shows an example of Chargaff's rules applied to a small, double-stranded DNA molecule. A double-stranded DNA molecule contains 200 base pairs (ie, 400 bases total-200 in each strand). If both strands combined contain 97 guanine bases, how many of the 400 total bases are thymine? - Solution: Base pairing within an RNA strand follows a variant of Chargaff's rules, in which the number of adenine bases equals the number of uracil bases instead of thymine. However, functional RNA molecules commonly exhibit some unusual base pairing called "wobble pairing" (eg, G paired with U), so Chargaff's rules do not universally apply to RNA. A tRNA molecule with a wobble pair and sections that obey Chargaff's rules is shown in Figure 8.33. As mentioned in Concept 8.2.02, the bases in a DNA strand can pair with the bases in an RNA strand. This occurs, for example, when RNA polymerase uses DNA as a template to synthesize RNA or when reverse transcriptase uses RNA as a template to synthesize DNA. When a DNA strand is paired with an RNA strand, yet another variation of Chargaff's rules applies. In this case, the RNA strand contains uracil, which pairs with adenine bases in the DNA strand. In contrast, the DNA strand contains thymine, which pairs with adenine in the RNA strand. Therefore, the total number of adenine bases in the complex is equal to the sum of the thymine and uracil bases in the complex. Figure 8.34 shows Chargaff's rules applied to a DNA-RNA complex. ## 8.2.04 Double Helix Stability As with higher levels of protein structure, nucleic acid secondary structure may be disrupted by environmental conditions. These conditions include pH, salt concentrations, and temperature. The optimal pH range for DNA annealing is near 7.4, because at this pH the hydrogen bond participants in each base are neutral and can form the expected hydrogen bonds with their complementary bases. If the pH is brought too far below or above this level, functional groups in bases can become protonated or deprotonated, making them positively or negatively charged, and acid- or base-catalyzed tautomerism of the bases (Concept 8.1.02) may also occur. This alters interactions between bases (see Figure 8.35). The sugar-phosphate backbone is negatively charged, so the phosphate groups repel each other, which can destabilize helix formation. Eukaryotes largely resolve this by providing DNA with positively charged histones. The phosphate groups may also be stabilized by the presence of salts because the cations from the salt (eg, Mg2+) interact well with the phosphate groups. The interaction between a cation and a phosphate group tends to be stronger than the interaction between water and a phosphate group. Consequently, higher salt concentrations tend to result in greater double helix stability (see Figure 8.36). Higher temperatures cause the bonds within a double helix to move more rapidly and allow the interactions holding strands together to break. This causes the two strands in a double helix to denature (ie, to come apart), as shown in Figure 8.37. The temperature that causes 50% of double helices in solution to denature is called the melting temperature, Tm, for that double helix. The composition of a double helix can significantly impact its Tm. A longer helix contains more hydrogen bonds, each of which must break for denaturation to occur. Consequently, a higher temperature is required to melt a long strand than to melt a short strand of similar composition. Guanine and cytosine pair through three hydrogen bonds, whereas adenine and thymine (or adenine and uracil) pair through only two hydrogen bonds. Correspondingly, a helix with a high percentage of guanine and cytosine (ie, high GC content) has a higher Tm than a helix of similar length with lower GC content. The effects of length and GC content on melting temperature are shown in Figure 8.38. The melting temperature of a double helix is particularly important in biotechnology contexts such as the polymerase chain reaction (PCR) (see Biology Lesson 4.1). In PCR, it is important to ensure that primers anneal at certain temperatures and denature at others. Consequently, PCR primers are typically designed to optimize Tm by manipulating GC content, and salt and pH conditions are carefully controlled. The melting temperature of a given double helix can be determined experimentally by generating a melting curve. A sample of DNA or RNA with at least some double helical portions is gradually heated and monitored for denaturation. Often this is done by including a fluorescent dye that binds to double-stranded DNA (or annealed RNA) but not to single-stranded nucleic acids. Upon binding, fluorescence intensity increases. As the sample is heated, the dye unbinds from the denatured nucleic acid and fluorescence decreases. An example of the resulting melting curve is shown in Figure 8.39. Note the sigmoidal shape of the curve. This shape indicates that nucleic acid denaturation is positively cooperative: as portions of the double helix separate, it becomes easier to separate additional portions. Similarly, as temperature decreases, complementary strands reanneal in a cooperative manner: as interactions between strands form, it becomes easier to form additional interactions.