BIOC201 - Nucleic Acid DNA Protein Synthesis - Part 1.pdf

University of KwaZulu-Natal Westville Campus Faculty of Science and Agriculture Discipline of Biochemistry BIOC201 Introduction to Biomo...

University of KwaZulu-Natal Westville Campus Faculty of Science and Agriculture Discipline of Biochemistry BIOC201 Introduction to Biomolecules DNA and Protein Synthesis Minor groove Minor groove Major groove Major groove B-DNA A-DNA Z-DNA 1. Introduction - The discovery of the substance that proved to be deoxyribonucleic acid (DNA) was made in 1869 by Friedrich Miescher. - Thereafter, Hoppe-Seyler isolated a similar substance from yeast cells; this substance is now known to be ribonucleic acid (RNA). Both DNA and RNA are polymers of nucleotides, or polynucleotides. - By the beginning of the twentieth century, scientists generally recognized that physical traits are inherited as discrete units (later called genes) and that chromosomes within the nucleus are the repositories of genetic information. - Over the next few years, the structures of nucleotides were determined, and in 1953, James D. Watson and Francis H. C. Crick proposed their model of the structure of double-stranded DNA. - We now know that a living organism contains a set of instructions for every step required by the organism to construct a replica of itself. The information resides in the genetic material, or genome, of the organism. The genomes of all cells are composed of DNA. Some viral genomes are composed of RNA. - In the past five decades, in one of the most fascinating and complex investigations of the twentieth century, molecular biologists formulated a general outline of biological inheritance and information transfer. This work revealed the following principles: i. The information encoded within DNA, which directs the functioning of living cells and is transmitted to offspring, consists of a specific sequence of nitrogenous bases. DNA synthesis involves the complementary pairing of nucleotide bases on two strands of DNA. The physiological and genetic function of DNA requires the synthesis of relatively error-free copies. ii. The mechanism by which genetic information is decoded and used to direct cellular processes begins with the synthesis of another type of nucleic acid, ribonucleic acid (RNA). RNA synthesis occurs by complementary pairing of ribonucleotide bases with the bases in a DNA molecule. iii. Several types of RNA are involved in the synthesis of the enzymes, structural proteins, and other polypeptides required for the synthesis of all other biomolecules involved in organismal function. 1 - The flow of biological information is summarized by the following sequence: DNA RNA Protein - This concept is referred to as the "central dogma of molecular biology" because it describes the flow of genetic information from DNA through RNA and eventually to proteins. Figure1: Biological Information Technological advances have made previously unimaginable access to the genomes of a growing number of organisms possible. The current challenge for biochemists and other life scientists is how to interpret not only the massive amounts of genetic information in living cells (the genome), but also how this information is expressed at the level of transcription (the transcriptome), protein synthesis (the proteome), and metabolism (the metabolome) so that specific biological and health problems can be solved. - In general, the information that specifies the primary structure of a protein is encoded in the sequence of nucleotides in DNA. This information is enzymatically copied during the synthesis of RNA, a process known as transcription. Some of the information contained in the transcribed RNA molecules is translated during the synthesis of polypeptide chains, which are then folded and assembled to form protein molecules. Thus, we can generalize that the biological information stored in a cell's DNA flows from DNA to RNA to protein. - In this subset of lectures, we will discuss the structure DNA and protein synthesis. 2 2. SINGLE STANDED (s/s) DNA STRUCTURE - Nucleotides the building blocks of DNA - Each nucleotide monomer in DNA is composed of a nitrogenous base [either a purine (adenine and guanine) or a pyrimidine (thymine and cytosine)], a deoxyribose sugar, and phosphate (figure 2). Figure 2: Nucleotide building blocks of DNA - The mononucleotides are linked to each other by 3' to 5’ phosphodiester bonds. These bonds join the 5'-hydroxyl group of the deoxyribose of one nucleotide to the 3'-hydroxyl group of the sugar unit of another nucleotide through a phosphate group (Figure 3). - The primary structure of a nucleic acid is the sequence of residues that are connected by 3', 5’-phosphodiester bonds linkages. 3 Figure 3: DNA strand structure In a DNA strand the deoxyribonucleotide residues are connected to each other by 3'-5’-phosphodiester linkages. The sequence of the illustrated strand is 5’-pATGC-3’. 4 - Polynucleotide chains have directionality i.e., one end of a linear polynucleotide chain is said to be 5' (because no residue is attached to its 5'-carbon) and the other is said to be 3' (because no residue is attached to its 3'-carbon). - By convention, the forward direction is 5' Æ 3'. Therefore, structural abbreviations are assumed to read 5' Å 3' when not otherwise specified. Because phosphates can be abbreviated as p, the tetra-nucleotide in Figure 4 can be referred to as 5' pApGpTpC 3', or even AGTC. - Each phosphate group that participates in a phosphodiester linkage has a pKa of about 2 and bears a negative charge at neutral pH. Consequently, nucleic acids are polyanions under physiological conditions. 3. DOUBLE STRANDED (d/s) DNA STRUCTURE - Erwin Chargaff deduced certain regularities in the nucleotide compositions of DNA samples obtained from a wide variety of prokaryotes and eukaryotes. - Chargaff observed that in the DNA of a given cell, A and T are present in equimolar amounts, as are G and C (Table.1). - The percentage of purine bases always equals the percentage of pyrimidine bases in DNA that is the ratio of purines to pyrimidines in DNA is always 1:1. - Note that total mole percent of (G + C) may differ considerably from that of (A + T). - The DNA of some organisms, such as yeast, is relatively deficient in (G + C), whereas the DNA of other organisms, such as the bacterium Mycobacterium tuberculosis, is rich in (G + C). - In general, the DNAs of closely related species, such as cows, pigs, and humans, have similar base compositions. Table 1: Base composition of DNA (mole %) and ratios of bases *Purine/ Source A G C T A/T* G/C* G+C Pyrimidine Escherichia coli 26.0 24.9 25.2 23.9 1.09 0.99 50.1 1.04 Mycobacterium tuberculosis 15.1 34.9 35.4 14.6 1.03 0.99 70.3 1.00 Yeast 31.7 18.3 17.4 32.6 0.97 1.05 35.7 1.00 Cow 29.0 21.2 21.2 28.7 1.01 1.00 42.4 1.01 Pig 29.8 20.7 20.7 29.1 1.02 1.00 41.4 1.01 Human 30.4 19.9 19.9 30.1 1.01 1.00 39.8 1.01 *Deviations from a 1:1 ratio are due to experimental variations. 1 - The Watson-Crick model accounted for the equal amounts of purines and pyrimidines by suggesting that DNA was double-stranded and that bases on one strand paired specifically with bases on the other strand, A with T and G with C. Watson and Crick's proposed structure is now referred to as the B conformation of DNA, or simply B-DNA. - Essentially DNA consists of two polynucleotide strands wound around each other to form a right-handed double helix (Figure 4). A B 2.4 nm Figure 4: A) DNA double helix as a spiral ladder. B) Space filing model of the helix. - The antiparallel orientation of the two polynucleotide strands allows hydrogen bonds to form between the nitrogenous bases that are oriented toward the helix interior (Figure 5). - There are two types of base pairs (bp) in DNA: i. adenine (a purine) pairs with thymine (a pyrimidine) ii. gaunine (a purine) pairs with cytosine (a pyrimidine) - Because each base pair is oriented at an angle to the long axis of the helix, the overall structure of DNA resembles a twisted staircase. 2 - The dimensions of crystalline DNA have been precisely measured. a) One turn of the double helix spans 3.4 nm and consists of approximately 10.4 base pairs. (Changes in pH and salt concentrations affect these values slightly). b) The diameter of the double helix is 2.4 nm. The interior space of the double helix is only suitable for base pairing a purine and a pyrimidine. Pairing two pyrimidines would create a gap, & pairing purines would destabilize the helix. c) The distance between adjacent base pairs is 0.34 nm. Figure 5: Structure of double-stranded DNA and hydrogen bonding interactions. The two strands run in opposite directions. Due to base pairing the order of bases in one strand determines the sequence of bases of the other strand 3 - As befits its role in living processes, DNA is a relatively chemically inert and several types of non-covalent bonding interactions contribute to the stability of its structure namely; i. Hydrophobic interactions. The base ring S cloud of electrons between stacked purine and pyrimidine bases is relatively nonpolar. The clustering of the base components of nucleotides within the double helix is a stabilizing factor in the three-dimensional macromolecule because it minimizes their interactions with water. ii. Hydrogen bonds. The base pairs, on close approach, form a preferred set of hydrogen bonds, three between GC pairs and two between AT pairs. The cumulative "zippering" effect of these hydrogen bonds keeps the strands in correct complementary orientation. iii. Base stacking. The stacked base pairs form weak van der Waals contacts. Although the forces between individual stacked base pairs are weak they are cumulative, so in large DNA molecules, van der Waals contacts are a significant source of stability. iv. Electrostatic interactions. DNA's external surface, referred to as the sugar phosphate backbone, possesses negatively charged phosphate groups. Repulsion between nearby phosphate groups, a potentially destabilizing force, is minimized by the shielding effects of divalent cations such as Mg2+ and polycationic molecules such as the polyamines and histones. - In B-DNA, the base pairs are stacked one above the other and are nearly perpendicular to the long axis of the molecule. The cooperative, non-covalent interactions between the upper and lower surfaces of each base pair bring the pairs closer together and create a hydrophobic interior that causes the sugar- phosphate backbone to twist. It is these stacking interactions that create the familiar helix (Figure 4). Because the two hydrophilic sugar-phosphate backbones wind around the outside of the helix, they are exposed to the aqueous environment. In contrast, the stacked, relatively hydrophobic bases are located in the interior of the helix, where they are largely shielded from water. - The double helix has two grooves of unequal width because of the way the base pairs stack and the sugar-phosphate backbones twist. These grooves are called the major groove and the minor groove (Figure 4). Within each groove, functional groups on the edges of the base pairs are exposed to water and are chemically distinguishable. Because the base pairs are accessible in the grooves, molecules that interact with particular base pairs can identify them without disrupting the 4  helix. This is particularly important for proteins that must bind to double-stranded DNA and "read" a specific sequence. - The size of a DNA molecule can be expressed in term of its molecular weight. However, due to rapid buildup of the molecular weights, it is convenient to express the size of DNA as the number of nucleotide base (nt) or base pairs (bp) per molecule. The genome of E. coli is 4.6 megabase (million) pairs. 4. Conformations of Double stranded DNA - X-ray crystallographic studies of various synthetic oligo-deoxyribonucleotides of known sequence have suggested that DNA molecules inside the cell do not exist in a "pure" B conformation. Instead, DNA is a dynamic molecule whose exact conformation changes as it bends in solution or binds to protein. - Two important alternative conformations are A-DNA, which forms when DNA is dehydrated, and Z-DNA, which can form when certain sequences are present (Figure 6). - A-DNA is more tightly wound than B-DNA, and the major and minor grooves of A- DNA are similar in width. Z-DNA differs even more from B-DNA. There are no grooves in Z-DNA, and the helix is left-handed, not right-handed. Both alternative conformations exist in vivo, but they are confined to short regions of DNA. Table 2: Comparison of some A-, B- and Z-DNA structural properties Helix type Parameter A-DNA B-DNA Z-DNA Shape Broadest Intermediate Narrowest Helix diameter 2.6 nm 2.4nm 1.8nm Base pairs per turn of helix 11 10.4 12 Rise per base pair 2.3 Å 3.4 Å 3.8 Å Glycosidic bond anti anti Alternating anti and syn Pitch per turn of helix 25.3 Å 35.4 Å 45.6 Å Helix rotation Right-handed Right-handed Left-handed Minor groove Minor groove Major groove Major groove B-DNA A-DNA Z-DNA Figure 6: A-DNA, B-DNA and Z-DNA 5