Nucleotides and Nucleic Acids PDF

Module 2: Cell Nucleotides and Nucleic Acids Albert Tiotuyco, MD | August 27, 2024 | Onsite A purine-derived nitrogenous b...

Module 2: Cell Nucleotides and Nucleic Acids Albert Tiotuyco, MD | August 27, 2024 | Onsite A purine-derived nitrogenous base with NH2 TABLE OF CONTENTS Guanine substituent at carbon 2 and a carbonyl (C=O) at Learning Objectives C. Types of RNA carbon 6 Summary of Terms IV.VIDEO LECTURE A pyrimidine-derived nitrogenous base with Cytosine I.COMPONENTS OF A. Nucleases NH2 at carbon 4 and a carbonyl at carbon 2 NUCLEIC ACIDS B. Genome In Health And A pyrimidine-derived nitrogenous base A. Nucleosides Disease Thymine carbonyl group at carbon 2 and carbon 4, and B. Nucleotides V. LIVE LECTURE NOTES CH3 at carbon 5 A ribonucleotide replacing thymine in an RNA C. Nitrogenous Bases A. Biomedical Importance Uracil sequence D. Nucleic Acids and DNA Of Nucleic Acids Contains instructions for biomolecule E. Nomenclature B. Central Dogma Of Gene production II. DNA Molecular Biology Chromosome DNA storage found in the nucleus A. Avery, Macleod, And C. Cyclic AMP McCarty Experiment VI.Summary & Keypoints I. COMPONENTS OF NUCLEIC ACIDS B. DNA Structure VII.Review Questions Nucleic acids are large biomolecules containing genetic C. Denaturation VIII.References information. D. Renaturation IX.Freedom Space The two types of nucleic acids are DNA and RNA. E. Forms of DNA Nucleotides are considered monomers of nucleic acids F. DNA Replication and (DNA and RNA), that carry the genetic information which Transcription are “read” in cells to do functions such as make proteins, III. RNA with which living things function. A. RNA Structure Nucleotides consist of three components: a nitrogenous B. RNA vs. DNA base, a sugar, and a phosphate group. LEARNING OBJECTIVES 1. Differentiate a nucleic acid from a nucleoside and a nucleotide 2. Describe the chemical monomeric and polymeric structure of DNA and RNA 3. Describe the convention for writing out nucleic acid sequences in terms of the directional character of nucleic acid structure 4. Explain why genomic eukaryotic DNA is double stranded and highly negatively charged 5. Describe the major types of RNA molecules and their general functions 6. Explain how the chemical nature of DNA facilitates faithful Figure 1. Nucleic Acid Structure duplication and faithful transcription into various types of Meanwhile, nucleosides only consist of a pentose sugar RNA and a nitrogenous base (see Figure 2). 7. Describe the organization of the genome into A. NUCLEOSIDES chromosomes SUMMARY OF TERMS Large biomolecules containing genetic Nucleic Acid information and contribute to metabolic functions. Biomolecule found within the nucleus of cells. It DNA is the genetic material and blueprint for life. A nucleic acid and is similar to DNA, but with RNA Figure 2. Cytidine (pyrimidine) nucleoside and guanosine (purine) nucleoside. only a single strand of nitrogenous bases. Monomeric units of nucleic acids composed of Nucleosides are molecules containing a pentose sugar Nucleotide a 5-carbon sugar, a nitrogenous base, and a covalently bonded to a nitrogenous base via a phosphate group. β-N-glycosidic bond. It is without a phosphate group The bonded product of the nitrogenous base attached. Nucleoside and five-carbon sugar (without a phosphate β-N-glycosidic bond group) →The bond between the sugar's carbon (C1’) and the Component of a nucleotide which contains Nitrogenous Base nitrogen in the base. nitrogen and basic chemical properties. →For purines, the bond is between C1’ and N9. A purine-derived nitrogenous base with NH2 Adenine →For pyrimidines, the bond is between C1’ and N1. substituent at carbon 6 CELL 02.10 TG 7 | Barnes, Calingasan, Cruz, Dy, Fermin, Jopson, Lim, Manzanero, Quizon, Ricaforte, Villasan 1 of 14 CG 16 | Alvez, Buensuceso, Chua, Faminialagao, Florendo, Jao, Martin, Sueno Jr., Sy, Villanueva, Yap, Zabella  Sugar molecules are typically either ribose or deoxyribose →Ribose: has a hydroxyl (OH) group attached to C2 →Deoxyribose: has a hydrogen (H) atom attached to C2 Depending on the type of sugar linked to the nitrogenous base, compounds follow different nomenclature. →For purines: Retain the name of the base, then drop the “–ine” for “-osine” →For pyrimidines: Retain the root word name of the base and add “-idine” Examples: Table 1. Examples of RNA and DNA Nucleosides →Answer: Deoxyribonucleoside Nucleosides Examples It is a nucleoside because there is no phosphate Adenosine group attached. Guanosine It is deoxy because it lacks an -OH in the C2’ of the RNA Nucleosides sugar Cytidine Uridine Recall: You can produce ribose and deoxyribose in the PPP. Deoxyadenosine Deoxyguanosine DNA Nucleosides Nucleotides serve a variety of physiological functions Deoxycytidine Deoxythymidine Table 2. Physiological Functions of Nucleotide Derivatives (T02.09, 2026) Derivative Function B. NUCLEOTIDES Principal transducer of free energy Take Note! ATP Most abundant free nucleotide in mammalian cells To differentiate between a nucleoside and a nucleotide: → Nucleotide vs Nucleotide Allosteric regulator and energy source for ✦Nucleotide contains: GTP protein synthesis − Phosphate group − Nitrogenous base Second messenger in response to nitric − Pentose sugar cGMP oxide (NO) during relaxation of smooth ✦Nucleoside contains: muscle − Nitrogenous base − Pentose sugar Sugar epimerizations → Nucleosides often end with “-osine” for purine Biosynthesis of glycogen, glucosyl UDP-sugar disaccharides derivatives or “-idine” for pyrimidine derivatives derivatives Oligosaccharides of glycoproteins and ✦Examples: guanosine, adenosine, cytidine, thymidine, proteoglycans uridine Nucleotides are phosphorylated nucleosides (became Forms urinary glucuronide conjugates of UDP-glucuronic acid bilirubin and of many drugs (including attached to one or more phosphate groups) aspirin) Participates in biosynthesis of CTP phosphoglycerides, sphingomyelin, and other substituted sphingosines C. NITROGENOUS BASES A nitrogenous base is an organic molecule containing nitrogen and possesses the chemical properties of that of a base. It is attached to a pentose sugar which is attached to a phosphate group. Figure 3. Nucleotide Structure Purines and pyrimidines are aromatic, nitrogen-containing A phosphoester bond is formed between the phosphate heterocycles. group and the 5’ carbon of sugar unit of the nucleoside. →Heterocycles: cyclic structures that contain other atoms This is usually the case in most biological nucleotides. aside from carbon. →The flat, two-dimensional (planar) form of these Lecture Questions nitrogenous bases facilitates “stacking”, thus enabling 1. What is the structure shown below? stabilization of the double-stranded DNA via close association of its constituents. →The oxo and amino groups of nitrogenous bases exhibit keto-enol and amine-imine tautomerism. Tautomers: molecules with the same molecular formula but different connectivity. CELL 02.10 Nucleotides and Nucleic Acids 2 of 14  Figure 4. Tautomerism of oxo and amino functional groups of purines and pyrimidines Structure-wise: Purines have two heterocyclic rings fused together while pyrimidines only have one heterocyclic ring. →Purines: Guanine, Adenine Have double rings; one 6-membered ring bonded to a 5-membered ring Guanine and Adenine can be found in either DNA or RNA →Pyrimidines: Cytosine, Thymine, Uracil Has one 6-membered ring Figure 6. Complementary Base Pairing of (a) Adenine and Thymine; and (b) Cytosine can be found in either DNA or RNA Guanine and Cytosine Thymine is only found in DNA while Uracil is only found in RNA D. NUCLEIC ACIDS AND DNA DNA and RNA are Polynucleotides Nucleic acids are polymers of nucleotides. Phosphodiester bonds connect nucleotides together. These are formed when the C3’ sugar of the first nucleotide bonds esterifies to the C5’ phosphate group of the second nucleotide. Dinucleotides are formed where the pentose moieties are Figure 5. Structure of Purines and Pyrimidines linked by a 3′,5′-phosphodiester bond to form the →The six-membered rings of purines and pyrimidines are “backbone” of RNA and DNA. numbered in opposite directions. → Formed through the elimination of water (dehydration) between two mononucleotides. Table 3. Purines and Pyrimidines Purines Pyrimidines → This representation is completely different from Has two rings; one the biological reality because the reverse reaction, 6-membered ring Has one hydrolysis of the phosphodiester bond, is Number of Carbon (pyrimidine ring) 6-membered thermodynamically favored. rings bonded to a pyrimidine →Despite being thermodynamically favored, the process 5-membered ring ring takes a long time thus, a catalyst (e.g. (imidazole ring) phosphodiesterases) is required Cytosine, Thymine, Nitrogenous Base Guanine, Adenine The long period allows DNA to persist and can be Uracil detected in fossils. Purine and pyrimidine components of nucleotides RNA is far less stable than DNA. participate in complementary base pairing via hydrogen →The 2’-hydroxyl group of RNA functions as a nucleophile bonding (see Figure 6). during hydrolysis of 3’,5’-phosphodiester bonds. →Guanine binds to cytosine Nice To Know! The C-G bonding are connected by three hydrogen bonds → Posttranslational modifications performed on Stronger than A-T bonds and thus, requires a higher polynucleotides can generate additional structures such as pseudouridine, which is a nucleoside with the melting point to degrade D-ribose linked to C-5 of uracil by a carbon-to-carbon →Adenine binds to Thymine bond rather than a β-N-glycosidic bond. The A-T are connected by two hydrogen bonds → Pseudouridylic acid (ψ) arises by rearrangement of a Adenine binds to Uracil in RNA instead of Thymine UMP of a performed tRNA. → Methylation by S-adenosylmethionine of a UMP of preformed tRNA forms TMP (thymidine monophosphate), which contains ribose rather than deoxyribose. E. NOMENCLATURE Nomenclature for Nucleotides and Nucleosides Format: [Nucleoside/Nucleotide component] [type of linkage]-[number of phosphate groups] CELL 02.10 Nucleotides and Nucleic Acids 3 of 14 Figure 7. Nucleic Acid Nomenclature → Begin with sugar-base (nucleoside) →Identify the type of linkage that exists between the sugar and phosphate groups. Figure 9. 5’ and 3’ ends of Nucleic Acids Depends on what is attached to the carbon number of II. DNA the sugar moiety. Deoxyribonucleic acid (DNA) is a biomolecule found ○ Carbon atoms of the sugar moiety are numbered within the nucleus of cells. It is the genetic material and with prime (‘). blueprint for life (T02.11, 2028). →Identify the number of phosphate groups present. DNA contains around 3 billion nucleotides per cell. Single phosphate group = monophosphate Organized into genes, the fundamental unit of genetic Two = diphosphate information Three = triphosphate → Genes do not function autonomously. Example: Deoxyguanosine 3’- Monophosphate Their replication and function are controlled by various →Since the sugar is a deoxyribose combined with a gene products often in collaboration with components guanine (purine = -osine) nitrogenous base of various signal transduction pathways “deoxyguanosine” Their fate is mostly to be translated into amino acids that →There is only 1 linkage between the 3’ carbon will turn into proteins, which have important roles and “3’” functions in the body. →Only 1 phosphate group →However, DNA still needs to be read and turned into RNA “Monophosphate” before they turn into proteins. →Shorthand notation: 3’-dGMP d= deoxy A. AVERY, MACLEOD, AND MCCARTY EXPERIMENT G= Guanosine Their experiment showed that DNA contained genetic M= Mono information. P= Phosphate →Built upon Frederick Griffith’s experiment, where they Polynucleotides are Directional Macromolecules used a virulent S- strain bacteria of Streptococcus pneumoniae and the R- strain (non-virulent). Nucleic acids are polar and charged They treated the extracts of each strain with different →From attached phosphate groups enzymes to determine what agent caused pneumonia. Naming nucleotide sequence of DNA molecule: −2 − →Proteases: To destroy protein →5’ end is polar with a phosphate (P𝑂4 ) group with a 2 →RNase: To destroy RNA charge →DNase: To destroy DNA →3’ end is polar with a hydroxyl (-OH) group Result: The DNase was the only one that did not transform 5′-base is written at the left and the 3′-base (C) at the right the R strain, thus they concluded that it was DNA that was Represent the different nitrogenous bases with letters ﹣ responsible for transforming the R-strain (see Figure 10). 5’-N1 N2 N3-3’ DNA was eventually coined as the “Transforming Factor.” Figure 8. Short 5’ and 3’ Strand Example →Shortcut for naming: 5’- AGC-3’ Directional 3’ → 5’ phosphodiester bonds link monomers of polynucleotides. →Since all phosphodiester bonds are 3’ → 5’, the representation pGpGpApTpCpA indicates that the terminal 5’-hydroxyl is phosphorylated. →The representation GGATC, is conventionally written with the 5’-base (G) at the left and 3’-base (C) at the right. CELL 02.10 Nucleotides and Nucleic Acids 4 of 14  Nucleotides are arranged in specific linear sequences that contain the genetic information of the DNA, forming one strand out of the two. →Anti-parallel strands: The 5’ end of one strand is paired with the 3’ end of its complementary strand, and vice versa. 5’ end: contains the phosphate group. 3’ end: contains the sugar’s hydroxyl group. The four DNA nucleotide bases are flat and planar →This allows them to stack closely within the duplex DNA Hydrogen bonds link purine and pyrimidine nitrogenous bases. See broken lines in Figure 6. The 2 strands wind around a central axis in the form of a double helix Bases are also highly polarizable and contains partial charges → Allows bases to form van der Waals and electrostatic interactions. → These 2 forces collectively are called Base-stacking forces/Base-stacking interactions. Figure 10. Avery, Macleod,and McCarty Experiment Because there are 3 hydrogen bonds between C-G, and only 2 between A-T, the base-stacking interaction B. DNA STRUCTURE between C-G are stronger than A-T Linear Structure C-G rich DNA sequences are more resistant to The linear structure of DNA corresponds to one DNA denaturation compared to A-T rich DNA sequences. strand (see Figure 11). Watson and Crick Model DNA linear structure contains 4 distinct deoxynucleotides at random [Building blocks of DNA]. →deoxyadenylate →deoxyguanylate →deoxycytidylate →thymidylate Deoxynucleotide consists of: →Sugar: deoxyribose →Phosphate group →Nitrogenous base: A, C, G, T These monomeric units of DNA are held in polymeric form by 3’,5’-phosphodiester bonds constituting a single strand The phosphate group in the 5th carbon attaches to the 3rd carbon of another nucleotide’s sugar molecule. Figure 12. Watson and Crick B- form DNA model A diagrammatic representation of the double-helical structure of the B form of DNA Base pairing between complementary deoxynucleotides involves the formation of hydrogen bonds A-T and G-C base pairs are often referred to as Watson- Crick base pairs Figure 11. Linear structure of DNA Characteristics of a DNA Strand Double Stranded Helix Width: 20 A (Angstrom) DNA has a double-stranded helix, with the two strands connected by hydrogen bonds. 1 Angstrom (Å)= 0.1 nm It has base pairs attached to a ribose backbone with a Length of a single turn: 34 A phosphate group in between. Equivalent to 10 base pairs →The phosphate group provides the negative charge. Distance between base pairs: 3.4 Å DNA’s polarity is dictated by each end of the DNA strand CELL 02.10 Nucleotides and Nucleic Acids 5 of 14  →5’ phosphate terminus: referring to the phosphate group ○ Joined by three hydrogen bonds. attached to the 5th carbon of the sugar (the “start” of the ○ Higher melting point. strand) The more H-bonds, the higher the melting point and →3’ hydroxyl terminus: Hydroxyl group attached to the 3rd the boiling point. carbon of the sugar ○ G-C pairs are harder to split apart due to the Anti-Parallel: number of hydrogen bonds. →One strand runs in the 5’-3’ direction, while the other Lecture Questions runs in the 3’-5’ direction Two Strands 1. While studying the structure of a small gene that was sequenced during the Human Genome Project, an → Template strand (non-coding strand): The strand investigator notices that one strand of the DNA that contains the genetic information molecule contains 25 A, 40 G, 30 C, and 20 T. How Strand that is copied or transcribed during RNA many of each base is found in the complete synthesis double-stranded molecule? → The opposite strand (coding strand): Called →Answer: total number for each base is as follows: coding strand because it matches the sequence of the Total of A = 25 + 20 = 45 RNA transcript (but contains Uracil instead of Thymine) Total of G = 40 + 30 = 70 Total of C = 40 + 30 = 70 Total of T = 25 + 20 = 45 →Rationale: There is base-pairing between guanine and cytosine (3 H-bonds), and adenine and thymine (2 H-bonds). DNA spirals like a twisted ladder (see Figure 22). Figure 13. DNA Template and RNA Transcript Strands. If you know the ratio of adenine, you will have an equal number of residues of thymine on the opposite Chargaff’s Rule side of the strand. →The amount of purines should be equal to the amount of ○ If adenine has 25, then thymine will have 25 on the pyrimidines! other side. The same is true for thymine, guanine, Adenine Bases = Thymine Bases and cytosine. Guanine Bases = Cytosine Bases ○ Add the bases to know how many of each base is →In RNA, Uracil replaces Thymine found in the complete double-strand molecule. Grooves ○ Major and minor grooves wind along the molecule, parallel to the phosphodiester backbone ○ In these grooves, proteins often interact specifically with exposed atoms of the nucleotides Grooves allow regulatory proteins to recognize and bind to specific sequences in the DNA Base-Pairing in DNA Figure 15. Helical Model of DNA C. DENATURATION Denaturation happens when hydrogen bonds and van der Waals interactions within the double helix are weakened, causing it to lose its structure and, eventually, the strands to separate. DNA strands can be denatured by: →Increasing temperature →Decreasing salt concentrations of the solution Disrupts the electrostatic interactions that aid in stabilizing the DNA →Adding chaotropic agents Figure 14. Base-Pairing in the DNA Structure Forms competing H-bonds with individual Adenine (A) and Thymine (T) deoxynucleotide bases ○ Joined by two hydrogen bonds. Hyperchromicity ○ Lower melting point. Guanine (G) and Cytosine (C ) CELL 02.10 Nucleotides and Nucleic Acids 6 of 14  →When absorbance of the purine and pyrimidine bases in →Hybridization is paired with gel electrophoresis to the ultraviolet spectrum (260 nm) increases after separate nucleic acids by size. denaturation →Radioactive or fluorescent complementary probes are →The bases are more exposed because it is unwound, used for detection. which leads to more absorbance Analytical Techniques Melting Temperature Hybridization with sophisticated techniques enables: It is also important to consider the primer melting → Specific detection and identification of nucleic acids. temperature (Tm) of DNA. This is the midpoint → Determination of sequences from very small amounts of temperature where bonds begin to separate. nucleic acids. Factors Influencing DNA Melting Temperature] E. FORMS OF DNA 1. Base Composition of DNA Closed circular DNA →More G-C Pairs (3 H-bonds): Higher Tₘ →DNA ends join to form a closed circle, retaining polarity. →More A-T Pairs (2 H-bonds): Lower Tₘ →3’ and 5’ hydroxyl and phosphoryl groups are removed. 2. Salt Concentration →Exists in relaxed or supercoiled forms (see Figure 14). →Higher Salt (e.g., NaCl): Increases Tₘ Example: NaCl increase from 0.01 M to 0.1 M → Tₘ Relaxed and Supercoiled Forms rises by ~16.6°C 1. Relaxed 3. Chaotropic Agents (e.g., Urea, Formamide) → DNA is not twisted beyond its normal helical turns. →Effect: Decrease Tₘ 2. Supercoiled →Mechanism: Disrupt hydrogen bonding between bases →DNA twists around its own axis, creating supercoils that →Benefits of Lower Tₘ: induce stress. Easier strand separation at lower temps →Preferred form in biological systems, requiring energy. Prevents phosphodiester bond breakage and chemical →Negative Supercoils damage at high temperatures. DNA is "underwound," twisted in the opposite direction of the right-handed double helix (B-form). D. RENATURATION →Topoisomerase Renaturation or reannealing is when separated strands of Enzymes that catalyze changes in DNA's topological DNA can come back together. state. Conditions of Reassociation Can relax or insert supercoils using ATP. 1. Temperature and Salt Conditions →DNA strands renature under specific temperature and salt conditions. →Optimal conditions allow strands to find and bind to their complementary pairs. 2. Specificity →Nucleic acid strands associate specifically with their exact complementary sequences. →The process ensures precise recognition between complementary sequences. Figure 16. Forms of DNA →Renatured DNA hybrids can be detected and quantified, F. DNA REPLICATION AND TRANSCRIPTION even with a single base pair mismatch. DNA as a template Factors Influencing Reassociation Rate →Functions: Concentration of complementary strands Serves as a source of genetic information for protein →Higher concentration of complementary strands synthesis. increases the reassociation rate. Provides inherited information to daughter cells or →Increased concentration boosts the likelihood of strands offspring. encountering and binding with their complementary DNA Template roles pairs. →Transcription Formation of Hybrid Molecules DNA is transcribed into RNA. → Replication Different types of hybrid molecules can form under DNA replicates to form daughter DNA molecules. suitable conditions, particularly when hybridization is combined with gel electrophoresis and probe labeling Semiconservative Replication techniques (T02.10, 2027). 1. Strand Separation Applications: →Parental DNA strands separate, each serving as an →DNA-DNA Hybrids: Southern Blotting independent template. →DNA-RNA Hybrids: Northern Blotting 2. New Strand Synthesis →RNA-RNA Hybrids: RNA analysis techniques →A complementary strand is synthesized on each Technique: template strand. 3. Formation of Daughter DNA Molecules CELL 02.10 Nucleotides and Nucleic Acids 7 of 14  →Two double-stranded daughter DNA molecules are formed, each containing one parental and one newly synthesized strand. Mitosis and DNA Distribution Sorting of DNA →During mitosis, the daughter DNA molecules are distributed to the daughter cells. Results in daughter cells →Each daughter cell inherits DNA molecules with information identical to the parent cell's. Figure 18. Segment of RNA with purine and pyrimidine bases held by phosphodiester bonds. →The DNA from the parent cell is semi-conserved in each daughter cell. B. RNA vs. DNA Structural Difference of RNA from DNA: →RNA is often single-stranded, with its sequence representing the primary structure. →The RNA sequence is complementary to the template strand of the gene it was transcribed from. →RNA binds specifically to its template DNA via base-pairing rules. →RNA’s sequence matches the gene's coding strand (except U replaces T, see Figure 13). Specific Differences of RNA from DNA: →Sugar Moiety RNA contains ribose, while DNA contains Figure 17. DNA Synthesis maintains the sequence and structure of the original 2’-deoxyribose. template DNA →Base Composition In Figure 15 above, the double-stranded structure of DNA RNA has uracil (U) instead of thymine (T). and the template function of each old parental strand →Strand Configuration (orange) on which a new complementary daughter strand RNA typically exists as a single strand but can fold (blue) is synthesized. back on itself in a hairpin loop if complementary base sequences with opposite polarity are present (refer to Figure 18). →Base Pair Content RNA’s guanine (G) content doesn't necessarily equal its cytosine (C) content, and adenine (A) doesn't equal uracil (U). →Alkali Hydrolysis RNA can be hydrolyzed by alkali to form 2′, 3′ cyclic diesters of mononucleotides due to the presence of a 2′-hydroxyl group, a process not possible in DNA. Figure 18. DNA replication is semiconservative In the replication process, each strand of DNA serves as a template for a new complementary strand, highlighting the semiconservative nature of DNA replication, which impacts gene expression control. III. RNA Ribonucleic acid (RNA) is also a nucleic acid and is similar Figure 19. Hairpin-like folding of RNA to DNA, but with only a single strand of nucleotides. C. TYPES OF RNA Among its numerous functions, it is vital in making proteins based off of DNA's genetic information. A. RNA STRUCTURE Chemical Nature of RNA →A polymer of purine and pyrimidine ribonucleotides linked together by 3′,5′-phosphodiester bonds. Figure 20. Three Main Types of RNA CELL 02.10 Nucleotides and Nucleic Acids 8 of 14 1. Messenger RNA (mRNA) →Characteristics: →Function: Carries genetic information from DNA to the ~70% of total cellular RNA. protein-synthesizing machinery. Packaged in the nucleolus with specific ribosomal →Template: Guides the sequence of amino acids in protein proteins. synthesis. →Ribosome Structure: →Characteristics: Mammalian Ribosomes: Composed of two Most heterogeneous in abundance, size, and stability. subunits—60S (with 5S, 5.8S, and 28S rRNA) and 40S 2-5% of total eukaryotic RNA. (with 18S rRNA). 5' Cap: 7-methylguanosine triphosphate cap at the 5’ All rRNAs except 5S rRNA (independently transcribed) terminus for mRNA recognition and stabilization are processed from a 45S precursor RNA in the against exoribonucleases (see Figure 20). nucleolus. 3' Poly(A) Tail: 20-250 adenylate residues added 4. Small RNA (sRNA) post-transcriptionally to maintain stability against Involved in rRNA and mRNA processing and gene exoribonucleases as well. regulation. In mammalian cells, pre-mRNA is processed into →E.g., U1, U2, U4, U5, U6 snRNAs in mRNA splicing; U7 mature mRNA before entering the cytoplasm. snRNA in histone mRNA processing. → Heterogenous nuclear RNA (hnRNA) Non-Coding Regulatory RNAs (NcRNAs): unprocessed mRNA (pre-mRNA) →Small NcRNAs: miRNAs: ~500-1000 nucleotides, involved in gene silencing. siRNAs: Derived from large dsRNAs, also involved in gene silencing. →Large NcRNAs: LncRNAs: Long noncoding RNAs, 300 to thousands of nucleotides, involved in gene regulation. circRNAs: Circular RNAs, detected in eukaryotes and metazoans, involved in RNA splicing. 5. Bacterial sRNA →Range from 50 to 500 nucleotides. →Regulate gene activity by binding to mRNA, often repressing or activating protein synthesis. 6. RNA in Animal and Plant Viruses Figure 21. Cap structure of 5’ Prime End for Eukaryotic mRNA →RNA serves as the genetic material. 2. Transfer RNA (tRNA) →Retroviruses: E.g., HIV, use reverse transcriptase to →Function: Serves as an adapter for translating mRNA into produce DNA from RNA, which integrates into the host specific amino acids. genome. →Characteristics: →Viral mRNAs are transcribed and translated by the host 74-95 nucleotides long. into viral proteins. ~20% of cellular RNA. 7. Non-coding RNA At least 20 tRNA molecules per cell, each →RNA’s that are not important in synthesis of a protein in corresponding to an amino acid. the translation process. Structure: Cloverleaf-like secondary structure with four →Includes snRNA, miRNA, etc. main double-stranded arms with single stranded loops. Table 4. The Types of RNA and its Roles Amino Acid Attachment: Amino acids are "charged" The Roles of Different Kinds of RNA onto the 3’-OH group of the acceptor arm by RNA Type Size Function aminoacyl tRNA synthetases. Transports amino Transfer RNA Small acids to site of protein synthesis Combines with Several Kinds– proteins to form Ribosomal RNA variable in size ribosomes, the site of protein synthesis Directs amino acid Messenger RNA Variable sequence of proteins Processes initial Small nuclear RNA Small mRNA to its mature form in eukaryotes Affects gene expression; used by Small interfering RNA Small Figure 22. Structure of a mature functional tRNA. scientists to knock out a gene being studied 3. Ribosomal RNA (rRNA) Affect gene expression; important →Function: Essential for ribosome assembly, binding and Micro RNA Small in growth and translation of mRNA into proteins. development CELL 02.10 Nucleotides and Nucleic Acids 9 of 14  Ribozymes → CRISPR-Cas utilizes a short “guide sequence” to target RNA molecules with intrinsic catalytic activity. specific parts of DNA and cuts the targeted location Involved in RNA splicing and peptide bond formation using the Cas9 enzyme. ✦After the DNA is cleaved, scientists leverage the cell's during translation. DNA repair mechanisms to insert or remove genetic ACTIVE RECALL BOX fragments, or to modify the DNA by substituting a 1. Type of RNA responsible for assembly of ribosomes and specific segment with a tailored DNA sequence. binding of mRNA to ribosomes and mRNA translation? 2. T/F: Bacterial sRNA range in size from 5 to 50 B. GENOME IN HEALTH AND DISEASE nucleotides. 1. T/F: The poly (A) tail at the 3’ end maintains intracellular Genes stability of the mRNA by preventing nucleolytic attack by →Segment of DNA that contains instructions for the 5′-exoribonucleases. production of biological molecules, usually proteins. Answers: 1 rRNA, 2F, 3F →Humans have 20,000 genes, all containing the information needed to build one or more proteins. IV. VIDEO LECTURE Some genes determine physical characteristics. ○ Example: eye color A. NUCLEASES Some genes influence our risk of developing certain Enzymes that degrade nucleic acids. conditions. Vital in nucleic acid metabolism. ○ Example: diabetes mellitus Essential tool in modern molecular genetics and molecular →Genes only account for 2% of all our genetic information. medicine. Which is made up of more than 3 BILLION letters Specific Nucleases (bases). Deoxyribonucleases: specifically hydrolyze DNA We do not yet understand the exact function of all the Ribonucleases: specifically hydrolyze RNA other 98%, we do know changes to this DNA can also Endonucleases impact our health. →Degrade both DNA and RNA. Where is all this DNA stored? →Cleaving internal phosphodiester bonds to produce →Almost all of our DNA is arranged into tightly coiled either 3′-hydroxyl and 5′-phosphoryl terminals or 5′- structures called chromosomes. hydroxyl and 3′-phosphoryl terminals. →Humans have 23 pairs of chromosomes, with one of Varying capabilities each pair inherited from our father and the other from →Some are capable of hydrolyzing both strands of our mother. double-stranded molecules or only cleave single →Chromosomes are located in the nucleus of a cell. strands of nucleic acids. A very small amount of DNA is found on the →Some can hydrolyze unpaired single strands or single mitochondria strands participating in the formation of a ○ Structure which provides energy for the cell double-stranded molecule. Genome Classes of endonucleases that recognize specific →All the DNA contained in one cell. sequences in DNA: →We have one copy of our genome in nearly every cell in →Restriction Endonucleases our body. a.k.a. restriction enzyme. →Our genomes are approximately 99.8% identical to that Directly binding specific (usually) contiguous DNA of every other human being. base pairs (typically 4, 5, 6, or 8 bp). The 0.2% variation in our genome that interests us in Cleave both strands of DNA (usually within the our healthcare. binding/recognition sequence element). Understanding it can help in the prediction, prevention, →CRISPR-Cas diagnosis, and treatment of disease. Family of enzymes - Ribonucleoprotein complexes. V. LIVE LECTURE NOTES Utilizes a “guide RNA” of specific nucleotide sequence. that targets a nuclease to cleave distant A. BIOMEDICAL IMPORTANCE OF NUCLEIC ACIDS DNA or RNA sequences. Nucleic Acids play roles in metabolic functions (protein Adopted by researchers from bacteria’s immune synthesis, energy production, enzyme activity regulation, defense system for genome editing technology. and signal transduction). Exonucleases →ATP is used in energy transductions that accompany →Hydrolyzing a nucleotide only when it is present at a metabolic interconversions. terminal of a molecule. →Act in one direction (3’ → 5’ or 5’ → 3’) only. Nice! → In bacteria, 3’ → 5’ exonucleases are integral parts of the DNA replication machinery. ✦Serves to edit or proofread the most recently added deoxynucleotide for base-pairing errors. CELL 02.10 Nucleotides and Nucleic Acids 10 of 14  3.Which RNA can contain thymine? →Answer: none of them 4.Which RNA can contain methionine? →Answer: tRNA 5.Which RNA will contain the codon? →Answer: mRNA Figure 23. ATP Structure 6.Which RNA will contain the anticodon? →cAMP and cGMP are secondary messengers in →Answer: tRNA hormonally regulated events of the body, aiding in C. CYCLIC AMP pathways of signal transduction. Nucleotides may be linked to other biomolecules to perform unique functions: →When nucleotides and vitamins (or vitamin derivatives) can join to form portions of coenzymes used to ease reaction processes. →When linked to carbohydrates or lipids, nucleotides constitute biosynthetic intermediates of metabolic pathways. Figure 25. Cyclic Adenosine Monophosphate (cAMP) Synthetically produced purine and pyrimidine have medical applications: →Synthetic base analogs are used in the chemotherapy of Cyclic Adenosine Monophosphate (cAMP) cancer and AIDS →nucleotide consisting of Adenosine, a ribose sugar →Purine and pyrimidine analogs are used as group, and a single phosphate group. immunosuppressants in organ transplants. →Contains a cyclical structure between the ribose and phosphate group. B. CENTRAL DOGMA OF MOLECULAR BIOLOGY →Acts as a secondary messenger, activating multiple cellular processes involved in: Cell signaling Cell division Gene expression Metabolism (e.g., Glycogenolysis) Synthesis and Degradation of cAMP Figue 24. The Central Dogma of Molecular Biology Flow of genetic information from DNA to RNA to protein. Replication: duplicating DNA Transcription: copying DNA into RNA Reverse Transcription: making RNA into DNA →Example: Some viruses can create DNA from RNA via reverse transcriptase enzyme. Certain viruses are able to create a DNA strand from an mRNA template. Translation: Transferring the transcription into another “language” or form Lecture Questions 1. Is there such a thing as Reverse translation? But if it became possible, how would it affect the way we can manipulate our genes? →Answer: No. If it were possible, it could have technological benefits. Let’s say you have a protein like insulin molecule, then you can create an RNA from the insulin. Then you can put the DNA into a patient who is Figure 26. Adenylyl Cyclase and cAMP Phosphodiesterase not producing enough insulin. Caveat: If it were a poison or toxin (cholera toxin), Adenylyl Cyclase and you reverse translate it into a gene. Then, your →Synthesizes cAMP from ATP and releases cell will die after incorporating it into its genome pyrophosphate (PPi). →Activated by primary messengers such as hormones 2.Which RNA can contain uracil? (e.g., epinephrine, glucagon). →Answer: mRNA, tRNA, and rRNA (all of them) cAMP Phosphodiesterase CELL 02.10 Nucleotides and Nucleic Acids 11 of 14  →An enzyme that converts cAMP to AMP. #1: Which of the following statements is TRUE? I. DNA and RNA are polynucleosides. Lecture Questions II. Anticodons can be seen in mRNA. 1. What happens if adenylyl cyclase is inhibited? III. What differentiates nucleosides and nucleotides apart →Answer: ↓cAMP, ↑ATP is the presence of a phosphate group. Decrease in glycogenolysis and decrease in IV. Phosphodiester bonds can be seen between effectiveness of glucagon in low-blood glucose state. nitrogenous bases in DNA. 2.What happens if phosphodiesterase is inhibited? A. I, II, II, and IV are true →Answer: ↑cAMP, ↓AMP B. I, III, and IV are true This can exacerbate the secondary messenger roles C. III is true of cAMP, resulting in physiological effects such as D. IV is true smooth muscle relaxation and decreased inflammatory responses. #2: T/F: The nucleoside Adenosine Triphosphate is the principle transducer of energy. Phosphodiesterase Inhibitors as Drugs True or False? Inhibitors of phosphodiesterase can be used #3 Why are the major and minor grooves seen in DNA pharmacologically, treating ailments caused by low levels important? of cAMP such as: A. It provides the 'kink' for DNA to form its double helix →Chronic Obstructive Pulmonary Disorder structure. →Erectile Dysfunction (e.g. Viagra) B. It allows regulatory proteins to access specific DNA →Pulmonary Arterial Hypertension sequences. →Benign Prostatic Hyperplasia C. Both statements are true. →Psoriasis D. Both statements are false. →Decompensation Heart Failure →Many other diseases #4: What interaction seen in DNA would need the highest These drugs aim to alleviate the diseases caused by low energy to separate? cAMP levels by inhibiting phosphodiesterase, increasing A. Hydrogen bond between Adenine and Cytosine cAMP levels. B. Hydrogen bond between Cytosine and Guanine C. Hydrogen bond between Thymine and Adenine VI. SUMMARY AND KEY POINTS D. Hydrogen bond between ribose and phosphate Nucleotides consist of three components: a nitrogenous base, a sugar, and a phosphate group #5: Which of the following molecules is a non-coding RNA? Nitrogenous bases in nucleotides may be purines or A. miRNA pyrimidines, which participate in complementary base B. mRNA pairing with each other. C. Both A and B are non-coding RNA In nucleoside formation, a nitrogenous base is attached to D. Neither A nor B are non-coding RNA a sugar molecule via a β-N-glycosidic bond, while ester #6: T/F: Guanine is a nucleoside. bonds bind phosphate groups to the sugar molecule to form nucleotides. True or False? Nucleic acids contain genetic information and have two #7: T/F: Viagra is an example of an adenylyl cyclase types: DNA and RNA. inhibitor. The nucleotide cyclic AMP (cAMP) is an important secondary messenger involved in the regulation of multiple True or False? cellular and metabolic processes, and the inhibition of Answers: 1C, 2F, 3B, 4B, 5A, 6F, 7F enzymes related to its synthesis and degradation has biochemical and pharmacological implications. A. RATIONALE TO ANSWERS OF REVIEW QUESTIONS There are different types of RNAs serving specific [C] - Nucleosides only contain a nitrogenous base and a functions in protein synthesis; mRNAs as messengers of sugar group. A is incorrect because DNA and RNA are not genetic information, tRNAs as adapters for translation, polynucleosides as they contain a phosphate group, rRNAs binders of mRNAs to ribosomes, and sRNAs for making them polynucleotides. B is incorrect because gene regulation and structural regulation of chromatins. anticodons are seen in tRNA, not mRNA. Lastly, D is Nucleases are enzymes vital in nucleic acid metabolism. incorrect because the bonds between the nitrogenous Deoxyribonucleases and ribonucleases hydrolyze DNA and bases in DNA are H-bonds. RNA respectively, while endonucleases degrade both and [F] - Adenosine triphosphate (ATP) is a nucleotide, for it exonucleases degrade nucleotides present at the terminal. contains a nitrogenous base, ribose sugar, and phosphate Genes account for 2% of humans’ genetic information and groups. contain instructions for production of biomolecules, [B] - The major and minor grooves arise due to the double specifically amino acids. helix structure of DNA, and it allows regulatory proteins to DNA is stored in chromosomes located, primarily, in the easily bind to specific sequences of DNA. nucleus. [B] - 3 hydrogen bonds are seen in the interaction between VII. REVIEW QUESTIONS cytosine and guanine bases in DNA. Compared to the 2 CELL 02.10 Nucleotides and Nucleic Acids 12 of 14  hydrogen bonds between adenine and thymine, a higher temperature is needed to separate the hydrogen bonds. [A] - microRNA are small, highly conserved non-coding RNA involved in gene expression regulation (Tiotuyco, 2023) [F] - Guanine is a purine nitrogenous base. A nucleoside should consist of a nitrogenous base and a pentose sugar. [F] - Viagra is a phosphodiesterase inhibitor, which is used to increase cAMP levels, alleviating erectile dysfunction. IV. REFERENCES A. REQUIRED RESOURCES ASMPH Batch 2026. [02.09] Nucleotides and Nucleic Acids. ASMPH Batch 2027. [02.10] Nucleotides and Nucleic Acids. ASMPH Batch 2028. [02.11] Nucleotides and Nucleic Acids. Rodwell, V.W. (2018). Nucleotides. Rodwell V.W., & Bender D.A., & Botham K.M., & Kennelly P.J., & Weil P(Eds.), Harper's Illustrated Biochemistry, 32. McGraw-Hill Education. Weil, P.A. (2018). Nucleic Acid Structure and Function. Rodwell V.W., & Bender D.A., & Botham K.M., & Kennelly P.J., & Weil P(Eds.), Harper's Illustrated Biochemistry, 34. McGraw-Hill Education. B. SUPPLEMENTARY RESOURCES Explain the transformation experiment of Avery, MacLeod and MC Carty. - steps | CK-12 foundation. (n.d.). https://www.ck12.org/flexi/life-science/dna/explain-t he-transformation-experiment-of-avery-mac-leod-a nd-mc-carty/ CELL 02.10 Nucleotides and Nucleic Acids 13 of 14  FREEDOM SPACE CELL 02.10 Nucleotides and Nucleic Acids 14 of 14