Nucleic Acids Learning Materials PDF

CHM 103 NUCLEIC ACIDS INTRODUCTION Nucleic acids and the central dogma of molecular biology are fundamental concepts that lie at the heart of life's genetic processes and inheritance. Nucleic acids, namely DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), serve as the carriers of genetic inf...

CHM 103 NUCLEIC ACIDS INTRODUCTION Nucleic acids and the central dogma of molecular biology are fundamental concepts that lie at the heart of life's genetic processes and inheritance. Nucleic acids, namely DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), serve as the carriers of genetic information in all living organisms. These macromolecules play a pivotal role in the transmission of hereditary traits from one generation to the next and govern the functioning of cells and organisms. Nucleic acids are composed of nucleotides, each consisting of a sugar molecule, a phosphate group, and a nitrogenous base. The unique sequence of these nitrogenous bases along the DNA strand encodes genetic instructions for the synthesis of proteins, which are essential for the structure, function, and regulation of all cellular activities. This coding capacity of DNA is the foundation of the central dogma of molecular biology, a principle that outlines the flow of genetic information in living systems. The central dogma of molecular biology describes the sequential process through which genetic information is transferred and expressed within cells. It begins with DNA replication, where the double- stranded DNA molecule unwinds and synthesizes two complementary strands, ensuring that each new cell retains an identical copy of the original DNA during cell division. The next step is transcription, in which a specific segment of DNA is transcribed into a single-stranded RNA molecule. This RNA, known as messenger RNA (mRNA), carries the genetic code from the DNA to the ribosomes, the cellular machinery responsible for protein synthesis. Finally, the process of translation occurs at the ribosomes, where mRNA is read, and amino acids are assembled into polypeptide chains, forming functional proteins. These proteins are essential for cellular structure, enzyme activity, and carrying out various biological functions. The central dogma thus represents the unidirectional flow of genetic information from DNA to RNA and ultimately to proteins, illustrating the molecular basis of life's complexity and diversity. Nucleic acids, such as DNA and RNA, hold the blueprints of life, encoding genetic information that shapes the characteristics and functions of all living organisms. The central dogma of molecular biology serves as the guiding principle in understanding the process by which this genetic information is transferred and expressed. This intricate relationship between nucleic acids and the central dogma provides a fundamental framework for biological research, medicine, and the pursuit of knowledge about the very essence of life itself. Nucleic acids are large biomolecules found in all living cells and are crucial for storing, transmitting, and expressing genetic information. There are two main types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA contains the genetic instructions for the development, functioning, growth, and reproduction of all living organisms, while RNA plays a central role in protein synthesis and various other cellular processes. In this lesson, we will explore the structure and function of nucleic acids in detail. Major purine and pyrimidine bases of nucleic acids. Some of the common names of these bases reflect the circumstances of their discovery. Guanine, for example, was first isolated from guano (bird manure), and thymine was first isolated from thymus tissue. Nucleotide and Nucleic Acid Nomenclature Base Nucleoside Nucleotide Nucleic acid Purines Adenine Adenosine Adenylate RNA Deoxyadenosine Deoxyadenylate DNA Guanine Guanosine Guanylate RNA Deoxyguanosine Deoxyguanylate DNA Pyrimidines Cytosine Cytidine Cytidylate RNA Deoxycytidine Deoxycytidylate DNA Thymine Thymidine Thymidylate RNA Deoxythymidine Deoxythymidylate DNA Uracil Uridine Uridylate RNA Structure of Nucleic Acids: 1. Nucleotides: Nucleic acids are made up of smaller building blocks called nucleotides. Each nucleotide consists of three components: - Sugar: In DNA, the sugar is deoxyribose, while in RNA, it is ribose. - Phosphate group: This provides a negative charge to the nucleotide. - Nitrogenous base: There are four types of nitrogenous bases in DNA: adenine (A), thymine (T), cytosine (C), and guanine (G). In RNA, thymine is replaced by uracil (U). These bases pair with each other to form the "rungs" of the DNA double helix. 2. DNA Structure: - Double Helix: DNA has a double-stranded structure, with two polynucleotide chains running in opposite directions and twisted around each other in a helical fashion. The nitrogenous bases form hydrogen bonds with each other, specifically adenine with thymine (or uracil in RNA) and cytosine with guanine, stabilizing the helical structure. 3. RNA Structure: - Single-Stranded: Unlike DNA, RNA is single-stranded, but it can fold upon itself to form intricate secondary structures. Structure of nucleotides. (a) General structure showing the numbering convention for the pentose ring. This is a ribonucleotide. In deoxyribonucleotides the —OH group on the carbon (in red) is replaced with —H. (b) The parent compounds of the pyrimidine and purine bases of nucleotides and nucleic acids, showing the numbering conventions. Phosphodiester linkages in the covalent backbone of DNA and RNA. The phosphodiester bonds (one of which is shaded in the DNA) link successive nucleotide units. The backbone of alternating pentose and phosphate groups in both types of nucleic acid is highly polar. The and ends of the macromolecule may be free or may have an attached phosphoryl group. Some simpler representations of this pentadeoxyribonucleotide are , pApCpGpTpA, and pACGTA. Hydrogen-bonding patterns in the base pairs defined by Watson and Crick. Here, as elsewhere, hydrogen bonds are represented by three blue lines. Function of Nucleic Acids: 1. DNA Function: - Genetic Information Storage: DNA carries the genetic code that instructs cells to synthesize specific proteins and determines an organism's traits and characteristics. These instructions are encoded in the sequence of nitrogenous bases along the DNA molecule. - Replication: DNA undergoes replication during cell division, ensuring that each new cell receives an identical copy of the genetic information. 2. RNA Function: - Messenger RNA (mRNA): Transcribes genetic information from DNA and carries it from the cell nucleus to the ribosomes in the cytoplasm. The ribosomes then use the mRNA sequence to synthesize proteins through translation.  - Transfer RNA (tRNA): Helps in the translation process by bringing the correct amino acids to the ribosome according to the mRNA sequence. - Ribosomal RNA (rRNA): Forms an essential part of the ribosomes, where protein synthesis occurs. RNA and DNA are two types of nucleic acids that play critical roles in living organisms. While they share some similarities, they also have several key differences in their structure and function. Let's discuss these differences in detail: 1. Structure: - DNA (Deoxyribonucleic Acid): - Sugar: DNA contains deoxyribose as its sugar component in the nucleotides. - Bases: DNA has four nitrogenous bases: adenine (A), thymine (T), cytosine (C), and guanine (G). - Double Helix: DNA exists as a double-stranded molecule, forming a stable double helix structure due to complementary base pairing. Adenine pairs with thymine (A-T) through two hydrogen bonds, while cytosine pairs with guanine (C-G) through three hydrogen bonds. - RNA (Ribonucleic Acid): - Sugar: RNA contains ribose as its sugar component in the nucleotides. - Bases: RNA also has four nitrogenous bases: adenine (A), uracil (U), cytosine (C), and guanine (G). Unlike DNA, RNA lacks thymine; instead, it contains uracil, which pairs with adenine (A-U) during transcription. 2. Strandedness: - DNA: DNA is double-stranded, with two polynucleotide chains running in opposite directions and forming a stable helix. - RNA: RNA is generally single-stranded, although it can fold upon itself to form secondary structures, such as hairpin loops and stem-loop structures. 3. Location and Function: - DNA: DNA is primarily found in the cell nucleus and serves as the repository of genetic information. It stores the instructions required for the development, functioning, growth, and reproduction of all living organisms. During cell division, DNA replicates to ensure that each new cell receives an identical copy of the genetic information. - RNA: RNA is found both in the cell nucleus and the cytoplasm. It plays a crucial role in translating the genetic instructions encoded in DNA into functional proteins. There are three main types of RNA: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA). mRNA carries the genetic code from DNA to the ribosomes for protein synthesis, tRNA helps in bringing the correct amino acids to the ribosome, and rRNA forms an essential part of the ribosomes, where protein synthesis occurs. 4. Stability: - DNA: DNA is generally more stable than RNA due to the presence of deoxyribose, which is less susceptible to degradation and chemical modifications. - RNA: RNA is more prone to degradation and has a shorter lifespan compared to DNA, making it a more dynamic molecule in terms of gene regulation and cellular processes. 5. Functionality: - DNA: DNA's primary function is to store and transmit genetic information from one generation to the next, providing the blueprint for the formation and functioning of all living organisms. - RNA: RNA functions as a key player in protein synthesis, facilitating the translation of genetic information into functional proteins, which are essential for cellular structure and function. Watson-Crick model for the structure of DNA. The original model proposed by Watson and Crick had 10 bp, or 34 Å (3.4 nm), per turn of the helix subsequent measurements revealed 10.5 bp, or 36 Å (3.6 nm),per turn. (a) Schematic representation, showing dimensions of the helix. (b) Stick representation showing the backbone and stacking of the bases. (c) Space-filling model. DNA Replication DNA replication occurs during the S-phase of the cell cycle, preceding cell division. It involves the unwinding of the DNA double helix and the synthesis of two complementary strands using each original strand as a template. This semi-conservative replication ensures that each new cell receives one old and one newly synthesized DNA strand. Key Enzymes in DNA Replication: a. Helicase: At the beginning of replication, helicase enzymes unwind the DNA double helix by breaking hydrogen bonds between base pairs. This creates a replication fork, a Y-shaped structure where the DNA strands separate. b. Single-Strand Binding Proteins (SSBs): As the DNA strands separate, SSBs bind to and stabilize the single-stranded DNA, preventing them from re-forming the double helix. c. Topoisomerases: During DNA replication, tension can build up ahead of the replication fork. Topoisomerases relieve this tension by introducing temporary breaks in the DNA, allowing it to relax and prevent supercoiling. d. Primase: Primase synthesizes short RNA primers that provide a starting point for DNA synthesis. These primers are essential because DNA polymerases can only add nucleotides to an existing 3'- OH group. e. DNA Polymerases: DNA polymerases are the main enzymes responsible for adding nucleotides to the growing DNA strands. In prokaryotes, DNA polymerase III is the primary replicative polymerase, while eukaryotes have several DNA polymerases, with DNA polymerase δ and ε playing major roles in replication. Leading and Lagging Strand Synthesis: DNA replication occurs bidirectionally from the replication fork. The two DNA strands are synthesized differently due to their antiparallel nature. a. Leading Strand: On the leading strand, DNA polymerase continuously adds nucleotides in the 5' to 3' direction, following the replication fork. b. Lagging Strand: On the lagging strand, DNA synthesis occurs in short, discontinuous segments called Okazaki fragments. Primase synthesizes RNA primers on the lagging strand, and DNA polymerase adds nucleotides in the 5' to 3' direction away from the replication fork. DNA ligase then joins the Okazaki fragments, forming a continuous strand. Defining DNA strands at the replication fork. A new DNA strand (light red) is always synthesized in the direction. The template is read in the opposite direction,. The leading strand is continuously synthesized in the direction taken by the replication fork. The other strand, the lagging strand, is synthesized discontinuously in short pieces (Okazaki fragments) in a direction opposite to that in which the replication fork moves. The Okazaki fragments are spliced together by DNA ligase. In bacteria, Okazaki fragments are ∼1,000 to 2,000 nucleotides long. In eukaryotic cells, they are 150 to 200 nucleotides long. Elongation of a DNA chain. (a) DNA polymerase I activity requires a single unpaired strand to act as template and a primer strand to provide the free hydroxyl group at the end to which the new nucleotide unit is added. Each incoming complementary nucleotide is bound selectively, in part by base-pairing to the appropriate nucleotide in the template strand. The reaction product has a new free hydroxyl, allowing the addition of another nucleotide. The newly formed base pair translocates to make the active site available to the next pair to be formed. (b) The core of most DNA polymerases is shaped like a human hand that wraps around the active site. The structure shown here is DNA polymerase I of Thermus aquaticus, bound to DNA. (c) A cartoon interpretation shows the insertion site, where the nucleotide addition occurs, and the postinsertion site, to which the newly formed base pair translocates. [(b) Data from PDB ID 4KTQ, Y. Li et al., EMBO J. 17 7514, 1998.] Termination of Replication: Replication is complete when the entire DNA molecule is duplicated. The replication forks meet at specific regions called replication termini, and the process is finalized. In prokaryotes, a protein complex called the Tus-Ter system helps regulate replication termination. Proofreading and Repair Mechanisms: DNA polymerases have proofreading abilities that help correct errors during replication. If an incorrect nucleotide is added, the 3' to 5' exonuclease activity of the polymerase removes the mismatched base, allowing the correct nucleotide to be inserted. Additionally, cells possess various DNA repair mechanisms to fix replication errors and damage that may occur during the process. Transcription: DNA transcription is the process by which an RNA molecule is synthesized using one strand of the DNA double helix as a template. Transcription occurs in the nucleus of eukaryotic cells and in the cytoplasm of prokaryotic cells. It is carried out by a multi-subunit enzyme called RNA polymerase. Transcription initiation and elongation by E. coli RNA polymerase. Initiation of transcription requires several steps generally divided into two phases binding and initiation. In the binding phase, the initial interaction of the RNA polymerase with the promoter leads to formation of a closed complex, in which the promoter DNA is stably bound but not unwound. A 12 to 15 bp region of DNA — from within the region to position or — is then unwound to form an open complex. Additional intermediates (not shown) have been detected in the pathways leading to the closed and open complexes, along with several changes in protein conformation. The initiation phase encompasses promoter binding, transcription initiation, and promoter clearance (steps through here). Once elongation commences, the subunit is released and is replaced by the protein NusA. The polymerase leaves the promoter and becomes committed to elongation of the RNA (step ). When transcription is complete, the RNA is released, the NusA protein dissociates, and the RNA polymerase dissociates from the DNA (step ). Another subunit binds to the RNA polymerase and the process begins again. Key Steps of DNA Transcription: a. Initiation: Transcription initiation begins when RNA polymerase binds to a specific region on the DNA known as the promoter. Promoters are essential for determining which genes are transcribed and are recognized by specific transcription factors that help recruit RNA polymerase to the correct site. Once RNA polymerase is properly positioned, it unwinds the DNA double helix, exposing the template strand for transcription. b. Elongation: During the elongation phase, RNA polymerase adds ribonucleotides to the growing RNA strand, using the DNA template as a guide. The ribonucleotides are complementary to the DNA bases, with uracil (U) replacing thymine (T) in the RNA sequence. The RNA strand elongates in the 5' to 3' direction, while the DNA template is read in the 3' to 5' direction. c. Termination: Transcription termination occurs when RNA polymerase reaches a termination sequence on the DNA template. In prokaryotes, there are two types of termination: Rho-independent termination, where a hairpin structure in the RNA causes RNA polymerase to dissociate, and Rho-dependent termination, where the Rho protein helps detach the RNA polymerase from the DNA template. In eukaryotes, termination is more complex and involves specific sequences and proteins. Types of RNA Transcripts: Several types of RNA molecules can be transcribed from DNA: - Messenger RNA (mRNA): Carries the genetic information from the DNA to the ribosomes for protein synthesis. - Transfer RNA (tRNA): Transports amino acids to the ribosome during translation. - Ribosomal RNA (rRNA): Forms the structural and catalytic components of ribosomes. Post-Transcriptional Modifications: In eukaryotes, the initial RNA transcript, called pre-mRNA, undergoes post-transcriptional modifications before becoming a mature mRNA. These modifications include the addition of a 5' cap and a poly-A tail, as well as the removal of introns through a process called splicing. These modifications enhance the stability of the mRNA and facilitate its export to the cytoplasm for translation. Translation: Translation is the process by which the sequence of nucleotides in mRNA is decoded into a sequence of amino acids, forming a polypeptide chain, which will eventually fold into a functional protein. This process occurs in the ribosomes, which are complex molecular machines composed of ribosomal RNA (rRNA) and protein. Key Components of Translation: a. Ribosomes: Ribosomes consist of two subunits - a small subunit and a large subunit - that come together during translation. The small subunit binds to the mRNA, while the large subunit catalyzes the formation of peptide bonds between amino acids. b. Messenger RNA (mRNA): mRNA carries the genetic code from the DNA in the nucleus to the ribosomes in the cytoplasm. The sequence of codons in mRNA determines the order of amino acids in the polypeptide chain. c. Transfer RNA (tRNA): tRNA molecules act as adaptors, bringing the correct amino acids to the ribosome based on the codons in the mRNA. Each tRNA has an anticodon that pairs with the complementary codon on mRNA, ensuring the correct amino acid is added to the growing polypeptide chain. d. Amino Acids: The building blocks of proteins, amino acids are linked together to form the polypeptide chain during translation. An overview of the five stages of protein synthesis. Steps of Translation: a. Initiation: Translation initiation begins when the small ribosomal subunit binds to the 5' end of the mRNA, at a specific sequence called the Shine-Dalgarno sequence in prokaryotes or the 5' cap in eukaryotes. The small subunit then scans along the mRNA until it reaches the start codon, AUG (encoding methionine), where translation begins. An initiator tRNA carrying methionine binds to the start codon. b. Elongation: During elongation, the ribosome moves along the mRNA in a 5' to 3' direction, reading each codon and bringing in the corresponding tRNA with the appropriate amino acid. The ribosome catalyzes the formation of a peptide bond between the amino acids, transferring the polypeptide chain from the tRNA in the P site to the tRNA in the A site. The uncharged tRNA in the P site is then moved to the E site and exits the ribosome, while the tRNA in the A site carrying the growing polypeptide chain moves to the P site. c. Termination: Translation termination occurs when the ribosome reaches a stop codon (UAA, UAG, or UGA) on the mRNA. At the stop codon, release factors bind to the ribosome, causing it to release the newly synthesized polypeptide chain. The ribosomal subunits dissociate, and the completed protein is released into the cytoplasm. Post-Translational Modifications: After translation, the newly synthesized polypeptide chain may undergo various post-translational modifications, such as folding into its three-dimensional shape, cleavage of signal sequences, addition of chemical groups (e.g., phosphorylation, glycosylation), and assembly into multimeric protein complexes. Genetic Mutations: Genetic mutations are alterations in the DNA sequence that can occur spontaneously or be induced by various factors. They are the driving force of genetic diversity and evolution and can have significant implications for health and disease. In this lesson, we will explore the types of genetic mutations, their causes, consequences, and the role they play in biology. Types of Genetic Mutations: a. Point Mutations: Point mutations involve changes in a single nucleotide base pair. There are three main types of point mutations: - Substitution: One base is replaced with another, which can result in synonymous (silent), missense, or nonsense mutations, depending on whether the amino acid encoded is the same, different, or a stop codon. - Insertion: One or more bases are inserted into the DNA sequence, shifting the reading frame and often leading to frameshift mutations. - Deletion: One or more bases are deleted from the DNA sequence, also causing a frameshift mutation. b. Chromosomal Mutations: Chromosomal mutations involve changes in the structure or number of chromosomes. These mutations include: - Deletion: A segment of a chromosome is lost. - Duplication: A segment of a chromosome is duplicated. - Inversion: A segment of a chromosome is reversed in orientation. - Translocation: A segment of one chromosome is transferred to another non-homologous chromosome. Causes of Genetic Mutations: a. Spontaneous Mutations: Spontaneous mutations occur naturally during DNA replication, recombination, or repair due to errors in cellular processes. b. Induced Mutations: Induced mutations are caused by external factors, such as radiation, chemicals (mutagens), and certain viruses. Consequences of Genetic Mutations: a. Silent Mutations: Silent mutations do not result in any change in the amino acid sequence of a protein due to the redundancy of the genetic code. b. Missense Mutations: Missense mutations lead to the substitution of one amino acid with another, potentially altering the protein's structure and function. c. Nonsense Mutations: Nonsense mutations create premature stop codons, resulting in the production of truncated and often non-functional proteins. d. Frameshift Mutations: Frameshift mutations alter the reading frame, leading to a completely different protein sequence downstream of the mutation. e. Chromosomal Mutations: Chromosomal mutations can disrupt the function of genes or lead to gene dosage imbalances, which may cause developmental disorders or cancer. Importance of Genetic Mutations: a. Genetic Diversity: Mutations are essential for generating genetic diversity within populations, enabling organisms to adapt to changing environments and contributing to evolution. b. Disease and Health: Some mutations can cause genetic disorders or increase the risk of diseases such as cancer. Conversely, certain mutations can confer resistance to diseases or beneficial traits. c. Evolutionary Innovation: Mutations play a key role in driving evolutionary innovations and the emergence of new traits in populations.