Lecture 3: Nucleic Acids PDF

Lecture 3 Nucleic acids Dr. Sophie SHI Ling [email protected] Department of Applied Science School of Science and Technology 1 Contents I. Nucleotides II. DNA III. RNA 2  What is nucleic acid? Macromolecules made up...

Lecture 3 Nucleic acids Dr. Sophie SHI Ling [email protected] Department of Applied Science School of Science and Technology 1 Contents I. Nucleotides II. DNA III. RNA 2  What is nucleic acid? Macromolecules made up of monomers called nucleotides Carry the genetic information of a cell and instructions for the functioning of the cell Information molecules that serve as blueprints for the proteins that are made by cells The hereditary material in cells, as reproducing cells pass the blueprints on to their offspring Two main types: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) DNA constitutes the genetic material in all free-living organisms and most viruses RNA is the genetic material of certain viruses, and involved in protein synthesis q Central dogma The process in which the genetic information flows from DNA to RNA, to make a functional product protein q Reverse transcription § Synthesis of DNA from an RNA template § Driven by reverse transcriptase § Occurs in retroviruses such as HIV and hepatitis B to replicate their genomes 3 Nucleotides q Definition organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate The nucleotides combine with each other to form a nucleic acid, DNA or RNA q Structure Ribonucleotide Ribose (deoxyribose Deoxyribonucleotide or ribose) H Deoxyribose 4 Nitrogenous bases q Definition Organic molecules that contain the element nitrogen and acts as a base in chemical reactions q Purines A double-ring structure and consist of adenine (A) and guanine (G) q Pyrimidines A single-ring structure and consist of cytosine (C), thymine (T), and uracil (U) 5 Phosphate group q Phosphate group bridge 3’-hydroxyl group of previous unit 5’-hydroxyl group of following unit q Phosphodiester linkage Ester bonds that form between sugar and phosphate to form the backbone of nucleic acids Occurs by the removal of a water molecule when 2 hydroxyl groups from 2 different sugars bond with a phosphate group Form sugar-phosphate backbone, crucial to stabilize the structure of DNA and RNA 6 Important nucleotides q Ribonucleotide Monomeric building block of RNA Bases: A,U,C,G Synthesized de novo from simple building blocks or they are obtained from the recycling of preformed bases q Deoxyribonucleotide Monomeric building block of DNA Bases: A,T,C,G Synthesized by the reduction of ribonucleotides by ribonucleotide reductase 7 Important nucleotides q Adenosine triphosphate (ATP) Consists of three main structures: the nitrogenous base, adenine; the sugar, ribose; and a chain of three phosphate groups (triphosphate) bound to ribose The main source of energy for most cellular processes, such as muscle contraction, nerve impulse propagation, and chemical synthesis The stored energy in ATP is primarily contained within the high-energy phosphate bonds that connect its three phosphate groups ATP is hydrolyzed into ADP in the following reaction: ATP+H2O→ADP+Pi+free energy ADP is combined with a phosphate to form ATP in the following reaction: ADP+Pi+free energy→ATP+H2O 8 Important nucleotides q Cyclic adenosine monophosphate (cAMP) Synthesized from ATP by adenylate cyclase A second messenger, used for intracellular signal transduction Involved in many biological processes, such as activation of protein kinases and regulation ion channel functions cAMP structure 9 Important nucleotides q Nicotinamide adenine dinucleotide (NAD) A coenzyme central to metabolism Consists of two nucleotides joined through their phosphate groups One nucleotide contains an adenine nucleobase and the other, nicotinamide Two forms: an oxidized form NAD+ and a reduced form NADH Involved in cellular energy metabolism and various metabolic pathways such as glycolysis, fatty acid oxidation, and the citric acid cycle Lactate Fatty acids 10  Functions of nucleotides Ø Nucleotides play key roles in various biochemical processes: ü Nucleic acid synthesis Nucleotides are the activated precursors of DNA and RNA ü Energy transfer ATP is used as a universal currency of energy in biological systems and GTP is utilized in many systems involved in movements of macromolecules ü Coenzymes Adenine nucleotides are components of some major coenzymes ü Metabolism Nucleotide derivatives are activated intermediates in many biosynthetic reactions, and they also serve as mediators to regulate metabolism 11 Contents I. Nucleotides II. DNA III. RNA 12 Deoxyribonucleic acids (DNA) Ø Definition The molecule that carries genetic information for the development and functioning of an organism A long, double-stranded, helical molecule composed of building blocks called deoxyribonucleotides Ø History 1869: DNA was first isolated by Swiss physician Friedrich Miescher 1929: Levene discovered deoxyribose sugar in DNA and proposed that it consists of four linked nucleotide units 1933: Jean Brachet suggested that DNA is found in the nucleus, while RNA is only in the cytoplasm, studying sea urchin eggs 1952: Rosalind Franklin used X-ray crystallography to capture images of DNA, building on ideas from Maurice Wilkins, which led to the discovery of the double helix structure 1953: Watson and Crick published their model of DNA's double helix, which is now widely accepted 1962: James Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize for discovering the molecular structure of DNA 13 Properties of DNA q Solubility DNA is polar in nature and thus soluble in water Negatively charged phosphate-sugar backbone gives its polarity q Absorption At 260 nm, DNA bases can absorb ultraviolet light (UV) q Denaturation DNA denaturation refers to the melting of double-stranded DNA to generate two single strands by breaking the hydrogen bonds Denaturation can occur when nucleic acids are subjected to: – Elevated temperature – Extremes of pH – Non-physiological concentrations of salt, organic solvents, urea, or other chemical agents q Renaturation The formation of base repairs and complementary strands of DNA come back together Occurs when the denatured DNAs are cooled in suitable conditions Depends on temperature, pH, length and constituents of the DNA structure 14 DNA denaturation and renaturation 15  DNA Structure DNA is made of two linked strands that wind around each other to resemble a twisted ladder — a shape known as a double helix Each strand has a backbone made of deoxyribose (sugar) and phosphate groups Attached to each sugar is one of four bases: adenine (A), cytosine (C), guanine (G) or thymine (T) The two strands are connected by hydrogen bonds between the paired bases Adenine (A) forms two hydrogen bonds with Thymine (T), and Guanine (G) forms three hydrogen bonds with Cytosine (C) The 5' carbon has a phosphate group attached to it and the 3' carbon a hydroxyl (-OH) group The two DNA strands are antiparallel to each other One strand runs in the 5' to 3' direction, the complementary strand runs in the 3' to 5' direction 16 https://biomodel.uah.es/en/model3/adn.htm  DNA Structure q Major groove vs. Minor groove DNA is structured as a double helix with two grooves: major and minor These grooves play crucial roles in DNA-protein interactions The major groove is 22 Angstroms wide, while the minor groove is 12 Angstroms wide Ø Major Groove Structure: Wider than the minor groove, allowing more space for protein binding Accessibility: Contains more hydrogen bond donors and acceptors exposed for specific interactions Function: ü Serves as a primary site for the binding of regulatory proteins (e.g., transcription factors) ü Facilitates recognition of specific DNA sequences, influencing gene expression Ø Minor Groove Structure: Narrower than the major groove, limiting access to the bases Accessibility: Fewer hydrogen bond interactions available due to its tighter structure Function: ü Some proteins can bind here, but interactions are generally less specific than in the major groove ü Important for certain DNA-binding proteins and small molecules 17 https://www.youtube.com/watch?v=o_-6JXLYS-k  DNA Packaging in Eukaryotes Ø Histone Proteins that facilitate the DNA packaging into chromatin fibers Positively charged and have many arginine and lysine amino acids that bind to the negatively charged DNA Core histones: H2A, H2B, H3 and H4 Two H3, H4 dimers and two H2A, H2B dimers form an octamer DNA Packaging Histones facilitate the coiling and folding of DNA, allowing it to fit within the nucleus Gene Regulation Histone modifications (e.g., acetylation, methylation) influence gene expression by altering chromatin structure Acetylation: Generally associated with gene activation Methylation: Can be associated with either activation or repression, depending on context 18 DNA Packaging in Eukaryotes Ø Nucleosome The basic structural unit of DNA packaging in eukaryotes Consists of a segment of DNA wound around eight histone proteins Ø Chromatin DNA can be further packaged by forming coils of nucleosomes, called chromatin fibers Chromatin fibers can unwind for DNA replication and transcription Ø Chromosome Chromatin fibers are condensed into chromosomes during mitosis, or the process of cell division 19 DNA Packaging in Eukaryotes https://www.youtube.com/watch?v=hYTVQqVLADU&t=41s 20 Genomic DNA may be linear or circular Most DNAs exist as double-helical complexes Depending on the source, the complexes can be linear or circular Circular DNA results from the formation of phosphodiester bonds between the 3 ‘ and 5’ termini of linear polynucleotides Formation of phosphodiester bonds between the 3' and 5' ends of each strand by DNA ligase produces a covalently closed circle Most DNA in bacteria exists solely as closed circles Mitochondria and chloroplasts in higher eukaryotic cells contain circular DNAs which encode unique proteins used by the organelles DNA ligase 21  DNA supercoiling DNA supercoiling describes a higher-order DNA structure A double helix DNA that has undergone additional twisting in the same direction as or in the opposite direction from the turns in the original helix Supercoiling functions to reduce the space required for DNA packaging, allowing for more efficient storage of DNA In eukaryotic cells, nucleosomes help to supercoil DNA q Importance of Supercoiling DNA Compaction ü Facilitates the packing of long DNA molecules into the nucleus Accessibility for Processes ü Replication: Supercoiling helps with the unwinding of DNA strands for replication ü Transcription: Enhances accessibility for RNA polymerase during gene expression https://www.youtube.com/watch?v=DXznYpXZu3M 22  DNA supercoiling Ø Topoisomerase——Enzyme in DNA supercoiling Topoisomerases are enzymes that modulate the supercoiling and topology of DNA, allowing it to maintain its structural integrity during cellular processes q Type I Topoisomerases Cuts single strand of DNA Relieves tension by allowing DNA to twist and unwind q Type II Topoisomerases Cuts double strands of DNA Introduces or removes supercoils by allowing one DNA segment to pass through another q Importance of Topoisomerase in Cellular Processes DNA Replication ü Prevents tangling and supercoiling during DNA unwinding Transcription ü Facilitates access to DNA for RNA polymerase by managing supercoiling DNA Repair ü Helps maintain genomic stability by resolving topological issues 23  DNA supercoiling Ø Topoisomerase——Enzyme in DNA supercoiling 24  DNA supercoiling Ø Topoisomerase——Enzyme in DNA supercoiling 25 DNA Replication Ø Step 1: Initiation Replication begins at specific locations on the DNA molecule called origins of replication DNA helicase unwinds the double DNA helix so that they can be used as a template for replication After DNA helix unwound, Y-shaped structures are formed, known as replication fork where the DNA replication will take place Ø Step 2: Priming The enzyme Primase synthesizes short RNA primers (about 10-15 nucleotides long) complementary to the DNA template These primers provide a free 3' hydroxyl (OH) group for the addition of DNA nucleotides 26 DNA Replication Ø Step 3: Elongation q Binding of DNA Polymerase DNA Polymerase attach to the template strands of DNA, then start to synthesize new strands of DNA to match the templates in 5’ to 3’ direction This means DNA polymerase add nucleotides to the 3' end of the growing DNA strand q Leading Strand Synthesis Leading strand: The continuously synthesized strand Single Primer: Initiates synthesis at the origin of replication q Lagging Strand Synthesis Lagging strand: The strand of DNA that is synthesized discontinuously Synthesized in short segments known as Okazaki fragments because it runs in the opposite direction to the replication fork Multiple RNA Primers: Each Okazaki fragment requires its own primer Fragment Synthesis: DNA polymerase synthesizes each Okazaki fragment in the 5' to 3' direction, leading to multiple fragments being created as replication progresses 27 DNA Replication Ø Step 3: Elongation 28 DNA Replication Ø Step 4: Termination The process of expanding the new DNA strands continues until there is either no more DNA template strand left to replicate q Completion of Synthesis Meeting of Replication Forks: Replication proceeds until replication forks from adjacent origins meet q Removal of RNA Primers Ribonuclease H (RNase H): Removes RNA primers from the lagging strand DNA Polymerase: Fills in gaps left by removed primers with DNA nucleotides q Ligation of Fragments DNA Ligase: Joins Okazaki fragments on the lagging strand, creating a continuous DNA strand 29 DNA Replication https://www.youtube.com/watch?v=TNKWgcFPHqw 30 DNA Proofreading and Repair 1. Proofreading During DNA Replication q DNA Polymerases with Exonuclease Activity Remove incorrectly paired nucleotides during synthesis Mechanism Detects mismatches Excises the incorrect base and resumes synthesis 2. Post-Replication Repair Mechanisms q Mismatch Repair (MMR) Corrects base pairing errors that escape proofreading q Base Excision Repair (BER) Repairs small base lesions (e.g., deaminated bases) q Nucleotide Excision Repair (NER) Repairs bulky DNA adducts (e.g., UV damage) Removes a segment of DNA around the damage and fills it in q Double-Strand Break Repair Homologous Recombination (HR): Uses a homologous template for accurate repair Non-Homologous End Joining (NHEJ): Directly joins broken ends, often resulting in small deletions 31 DNA Proofreading and Repair 32  Functions of DNA Ø Storage of genetic information DNA stores information in the sequence of its bases The information stored in the order of bases is organized into genes: each gene contains information for making a functional product In eukaryotes, DNA stores genetic information mainly in the nucleus Ø Transmit genetic materials through replication DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule Essential for cell division during growth and repair of damaged tissues Ensures that the genetic information of an organism is accurately passed on to its offspring during cell division Ø Instruction of protein synthesis In the first step, the information in DNA is transferred to a messenger RNA (mRNA) molecule by way of a process called transcription During the second step called translation, mRNA is "read" according to the genetic code, which relates the DNA sequence to the amino acid sequence in proteins The mRNA sequence is thus used as a template to assemble—in order—the chain of amino acids that form a protein 33 Contents I. Nucleotides II. DNA III. RNA 34  Ribonucleic acid (RNA) Ø Definition A single-stranded molecule composed of building blocks called ribonucleotides usually a single-stranded molecule Ø History 1868: Friedrich Miescher discovered nucleic acids and named them "nuclein" because they were found in the nucleus. 1959: Severo Ochoa won the Nobel Prize in Medicine for discovering an enzyme that can make RNA in the lab. 1956: Alex Rich and David Davies created the first RNA crystal, allowing its structure to be studied with X-ray crystallography. 1965: Robert W. Holley determined the sequence of 77 nucleotides in yeast tRNA, earning the Nobel Prize in Medicine in 1968 1977: The discovery of introns and RNA splicing in viruses and genes led to the Nobel Prize for Philip Sharp and Richard Roberts in 1993 2023: Katalin Karikó and Drew Weissman received the Nobel Prize in Physiology or Medicine for their work on mRNA vaccines, which made effective COVID vaccines possible 35 Ribonucleic acid (RNA) Ø Major types of RNA mRNA (messenger RNA) rRNA (ribosomal RNA) tRNA (transfer RNA) Ø Properties of RNA RNA, containing a ribose sugar, is more reactive than DNA and is not stable in alkaline conditions RNA strands are continually made, broken down and reused RNA’s mutation rate is relatively higher RNA is more versatile than DNA, capable of performing numerous, diverse tasks in an organism 36 RNA structure Ø Characteristics RNA is usually a single-stranded helix The strand has a 5′ end (with a phosphate group) and a 3′ end (with a hydroxyl group) Made of ribonucleotides that are linked by phosphodiester bonds The nitrogenous bases that compose the ribonucleotides include adenine (A), cytosine (C), guanine (G), and uracil (U) A pair with U, C pair with G Ø Structural difference between RNA & DNA Thymine (T) in DNA is replaced by uracil (U) in RNA The sugar in RNA is ribose rather than deoxyribose as in DNA RNA is usually single-stranded, while DNA is double-stranded helix 37 RNA secondary structure Ø Characteristics Most RNA molecules are single-stranded but an RNA molecule may contain regions which can form complementary base pairing where the RNA strand loops back on itself If so, the RNA will have some double-stranded regions Ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) exhibit substantial secondary structure, as do some messenger RNAs (mRNAs) 38 Messenger RNA (mRNA) A type of single-stranded RNA involved in protein synthesis Made from a DNA template during the process of transcription carry protein information from the DNA in a cell’s nucleus to the cell’s cytoplasm The protein-making machinery reads the mRNA sequence and translates each three-base codon into its corresponding amino acid in a growing protein chain https://www.youtube.com/watch?v=_Zyb8bpGMR0 39  Transfer RNA (tRNA) An adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length Serves as the physical link between the mRNA and the amino acid sequence of proteins tRNA functions by transporting an amino acid to the ribosome, which is the cellular machinery responsible for protein synthesis Complementation of a 3-nucleotide codon in a messenger RNA (mRNA) by a 3-nucleotide anticodon of the tRNA results in protein synthesis based on the mRNA code tRNAs are a necessary component of translation, the biological synthesis of new proteins in accordance with the genetic code 40 Ribosomal RNA (rRNA) A type of non-coding RNA which is the primary component of ribosomes, essential to all cells Transcribed from ribosomal DNA (rDNA) and then bound to ribosomal proteins to form small and large ribosome subunits Structural Role: Provides the framework for ribosomal proteins, forming ribosome architecture Catalytic Activity: Catalyzes peptide bond formation between amino acids Essential for protein synthesis, plays an important role in translating mRNA into proteins The predominant form of RNA found in most cells; it makes up about 80% of cellular RNA despite never being translated into proteins itself 41 Transcription The process of copying information from DNA sequences into RNA sequences Performed by enzymes called RNA polymerases RNA polymerase uses only one strand of DNA, called the template strand, of a gene to catalyze synthesis of a complementary, antiparallel RNA strand RNA is synthesized from 5' to 3’ direction RNA polymerases begin transcription at DNA sequences called promoters, and end at sequences called terminators q Stages of transcription Initiation: RNA polymerase binds to the DNA of the gene at a region called the promoter Elongation: As RNA polymerase moves along DNA, an RNA chain complementary to the template strand of DNA is synthesized Termination: Occurs when a transcribing RNA polymerase releases the DNA template and the newly synthesized RNA 42  RNA Splicing RNA splicing is the process of removing introns from precursor mRNA (pre-mRNA) and joining exons together RNA splicing primarily occurs in eukaryotic cells, whereas it is absent in prokaryotic cells Exon: An exon is a part of a gene that contains the information needed to make a protein Intron: An intron is a section of a gene that does not code for a protein q Process Pre-mRNA is synthesized from DNA during transcription Introns are removed, and exons are joined together to form mature mRNA q Spliceosome The splicing process is carried out by a complex called the spliceosome, which consists of proteins and small nuclear RNAs q Significance Splicing allows for the generation of multiple protein isoforms from a single gene through alternative splicing Increasing protein diversity and functional complexity 43 RNA Splicing 44 Alternative Splicing 45 Translation The process by which information in mRNAs is used to direct the synthesis of proteins q Initiation The small ribosomal subunit binds to an initiator tRNA that recognizes the start codon (AUG) This complex attaches to the mRNA and moves to the start codon At the start codon, the large ribosomal subunit binds to the initiator tRNA, forming a complete ribosomal complex 46 Translation q Elongation & Translocation The large ribosomal subunit has three tRNA binding sites The initiator tRNA is bound to the central P site and a second tRNA molecule pairs with the next codon in the A site The amino acid in the P site is covalently attached via a peptide bond to the amino acid in the A site The tRNA in the P site is now deacylated (no amino acid), while the tRNA in the A site carries the peptide chain The ribosome moves along the mRNA strand 47 Translation q Termination Elongation and translocation continue in a repeating cycle until the ribosome reaches a stop codon Stop codon recruit a release factor (protein) that signals for translation to stop The polypeptide is released, and the ribosome disassembles back into its two independent subunits 48 Amino acid codon There are 64 different codons in the genetic code Three sequences, UAG, UGA, and UAA, known as stop codons, do not code for an amino acid The sequence AUG—read as methionine—can serve as a start codon to initiate translation The amino acid codon is universal, meaning that the same codons specify the same amino acids across almost all organisms https://www.youtube.com/watch?v=gG7uCskUOrA 49 Other RNA q MicroRNA (miRNA) Small, single-stranded, non-coding RNA molecules containing 21 to 25 nucleotides Regulates gene expression by binding to complementary sequences in target mRNA Leading to mRNA degradation or translational repression Endogenously (naturally) produced from longer primary transcripts q Small interfering RNA (siRNA) A class of double-stranded RNA molecules (20–25 bp length) Mediates RNA interference (RNAi) by perfectly pairing with target mRNA Leading to mRNA degradation and silencing of gene expression Often derived from exogenous sources (e.g., viral RNA) or synthesized artificially q Short hairpin RNA (shRNA) Typically 20-30 base pairs in a hairpin structure Functions similarly to siRNA It is processed by the protein Dicer into siRNA and used in RNA interference to silence genes shRNA is often artificially synthesized 50 Function of RNA q mRNA Carries information specifying amino acid sequences of proteins from DNA to ribosomes Code for proteins q tRNA Transports amino acids to site of protein synthesis Serve as adaptors between mRNA and amino acids during protein synthesis q rRNA Form the core of the ribosome’s structure & catalyze protein synthesis q miRNA, siRNA & shRNA Regulate gene expression 51 Summary Nucleotides Nucleic acids Basic structure DNA RNA Ribonucleotide Single strand Double helix mRNA Deoxyribonucleotide DNA supercoiling tRNA ATP DNA packaging rRNA cAMP DNA replication Transcription NAD Translation 52