Nucleic Acids and Central Dogma

Nucleic Acids

• Macromolecules composed of monomers called nucleotides
• Carry genetic information and instructions for cell function
• Two main types: Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA)
• DNA constitutes the genetic material in most living organisms and viruses.
• RNA is the genetic material of certain viruses and is involved in protein synthesis.

Central Dogma

• The process of genetic information transfer from DNA to RNA to proteins.

Reverse Transcription

• Synthesis of DNA from an RNA template.
• Driven by reverse transcriptase.
• Occurs in retroviruses.

Nucleotides

• Organic molecules composed of a nitrogenous base, a pentose sugar, and a phosphate group.
• Combine to form nucleic acids (DNA or RNA).

Nitrogenous Bases

• Organic molecules containing nitrogen that act as bases in chemical reactions.
• Purines: Double-ring structure (Adenine (A) and Guanine (G)).
• Pyrimidines: Single-ring structure (Cytosine (C), Thymine (T), and Uracil (U)).

Phosphate Group

• Bridge between the 3’-hydroxyl group of the previous unit and the 5’-hydroxyl group of the following unit.
• Forms phosphodiester linkages, essential for stabilizing DNA and RNA structures.

Important Nucleotides

• Ribonucleotide: Monomeric building block of RNA (bases: A, U, C, G).
• Deoxyribonucleotide: Monomeric building block of DNA (bases: A, T, C, G).
• Adenosine Triphosphate (ATP): The primary energy source for most cellular processes.
• Cyclic Adenosine Monophosphate (cAMP): A second messenger involved in intracellular signal transduction.
• Nicotinamide Adenine Dinucleotide (NAD): A coenzyme central to metabolism.

Functions of Nucleotides

• Nucleic acid synthesis: Precursors for DNA and RNA.
• Energy transfer: ATP is the universal energy currency.
• Coenzymes: Adenine nucleotides are components of major coenzymes.
• Metabolism: Nucleotide derivatives act as activated intermediates in biosynthetic reactions and regulate metabolism.

Deoxyribonucleic Acids (DNA)

• Molecule carrying genetic information for organism development and functioning.
• Long, double-stranded, helical molecule composed of deoxyribonucleotides.

Properties of DNA

• Solubility: Polar in nature, soluble in water.
• Absorption: DNA bases absorb UV light at 260 nm.
• Denaturation: Melting of double-stranded DNA into single strands by breaking hydrogen bonds (caused by temperature, pH, or chemical agents).
• Renaturation: Formation of base pairs and complementary strands coming back together.

DNA Structure

• Two linked strands winding around each other, forming a double helix.
• Each strand has a backbone of deoxyribose sugar and phosphate groups.
• The two strands are connected by hydrogen bonds between paired bases (A-T, G-C).
• 5' carbon has a phosphate group, 3' carbon has a hydroxyl group.
• The strands are antiparallel, running in opposite directions (5' to 3' and 3' to 5').

Major Groove vs. Minor Groove

• The major groove is wider, allowing more space for protein binding and specific interactions.
• The minor groove is narrower, limiting access and interactions.

DNA Packaging in Eukaryotes

• Histones: Positively charged proteins that bind to negatively charged DNA, facilitating packaging into chromatin fibers.
• Nucleosome: Basic structural unit of DNA packaging, consisting of DNA wrapped around eight histone proteins.
• Chromatin: Coiled nucleosomes forming fibers, further packaged into chromosomes during cell division.

Genomic DNA Structures

• Linear: Found in eukaryotic organisms.
• Circular: Found in bacteria, mitochondria, and chloroplasts.

DNA Supercoiling

• Higher-order structure of DNA involving additional twisting.
• Reduces space for efficient packaging.
• Facilitates DNA replication and transcription.

Topoisomerases

• Enzymes modulating DNA supercoiling and topology.
• Type I: Cuts single strand of DNA to relieve tension.
• Type II: Cuts double strands of DNA to introduce or remove supercoils.

DNA Replication

• Step 1: Initiation: Unwinding of DNA helix at specific origins of replication.
• Step 2: Priming: Synthesis of RNA primers by Primase, providing starting points for DNA synthesis.
• Step 3: Elongation: DNA Polymerase adds nucleotides to the 3' end of the growing strand, synthesizing continuously (leading strand) or discontinuously (lagging strand) with Okazaki fragments.
• Step 4: Termination: Completion of synthesis, removal of RNA primers, and ligation of fragments.

DNA Proofreading and Repair

• Proofreading: DNA Polymerases remove incorrectly paired nucleotides during synthesis.
• Post-Replication Repair: Mechanisms that correct errors after replication, including mismatch repair, base excision repair, nucleotide excision repair, and double-strand break repair.

Functions of DNA

• Storage of genetic information in base sequences.
• Transmission of genetic material through replication.
• Instruction of protein synthesis through transcription and translation.

RNA Definition

• RNA is a single-stranded nucleic acid composed of ribonucleotides.

History of RNA

• Friedrich Miescher discovered nucleic acids in 1868.
• Severo Ochoa won the Nobel Prize in Medicine in 1959 for discovering an enzyme that can make RNA in the lab.
• Alex Rich and David Davies created the first RNA crystal in 1956, allowing its structure to be studied using X-ray crystallography.
• Robert W. Holley determined the sequence of 77 nucleotides in yeast tRNA in 1965, earning the Nobel Prize in Medicine in 1968.
• The discovery of introns and RNA splicing in viruses and genes led to the Nobel Prize for Philip Sharp and Richard Roberts in 1993.
• Katalin Karikó and Drew Weissman received the Nobel Prize in Physiology or Medicine in 2023 for their work on mRNA vaccines, which made effective COVID vaccines possible.

Types of RNA

• mRNA (messenger RNA)
• rRNA (ribosomal RNA)
• tRNA (transfer RNA)

Properties of RNA

• RNA is more reactive than DNA due to the presence of a ribose sugar.
• RNA is not stable in alkaline conditions.
• RNA strands are constantly made, broken down, and reused within cells.
• RNA has a relatively higher mutation rate compared to DNA.
• RNA is more versatile than DNA and performs numerous diverse tasks in organisms.

RNA Structure

• RNA is typically a single-stranded helix.
• It has a 5' end with a phosphate group and a 3' end with a hydroxyl group.
• RNA is composed of ribonucleotides linked by phosphodiester bonds.
• The nitrogenous bases in ribonucleotides are adenine (A), cytosine (C), guanine (G), and uracil (U).
• Adenine pairs with Uracil (A-U), and Cytosine pairs with Guanine (C-G).

Structural Differences between RNA and DNA

• Thymine (T) in DNA is replaced by uracil (U) in RNA.
• The sugar in RNA is ribose, while DNA contains deoxyribose.
• RNA is usually single-stranded, whereas DNA is a double-stranded helix.

RNA Secondary Structure

• Most RNA molecules are single-stranded, but some regions can form complementary base pairing, leading to double-stranded regions.
• Ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs) exhibit significant secondary structure, as do some messenger RNAs (mRNAs).

Messenger RNA (mRNA)

• mRNA is a single-stranded RNA involved in protein synthesis.
• It is made from a DNA template during transcription.
• mRNA carries protein information from the DNA in the cell's nucleus to the cytoplasm.
• The protein-making machinery reads the mRNA sequence, translating each three-base codon into its corresponding amino acid in a growing protein chain.

Transfer RNA (tRNA)

• tRNA is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides long.
• It acts as a physical link between mRNA and the amino acid sequence of proteins.
• tRNA transports amino acids to the ribosome, which is responsible for protein synthesis.
• tRNA's anticodon, a three-nucleotide sequence, complements a three-nucleotide codon in mRNA, resulting in protein synthesis based on the mRNA code.
• tRNAs are essential for translation, the process of synthesizing new proteins according to the genetic code.

Ribosomal RNA (rRNA)

• rRNA is a non-coding RNA that is the primary component of ribosomes, essential for all cells.
• It is transcribed from ribosomal DNA (rDNA) and binds to ribosomal proteins to form small and large ribosome subunits.
• rRNA provides structural support for ribosomal proteins, forming the ribosome's architecture.
• It also catalyzes peptide bond formation between amino acids.
• rRNA is crucial for protein synthesis, playing a key role in translating mRNA into proteins.
• It is the most abundant form of RNA in most cells, making up about 80% of cellular RNA despite not being translated into proteins itself.

Transcription

• Transcription is the process of copying information from DNA sequences into RNA sequences.
• It is performed by enzymes called RNA polymerases.
• RNA polymerase uses only one strand of DNA, called the template strand, of a gene to catalyze the synthesis of a complementary, antiparallel RNA strand.
• RNA is synthesized in the 5' to 3' direction.
• RNA polymerases begin transcription at DNA sequences called promoters and end at sequences called terminators.

Stages of Transcription

• Initiation: RNA polymerase binds to the DNA of the gene at the promoter region.
• Elongation: As RNA polymerase moves along the DNA, an RNA chain complementary to the template strand of DNA is synthesized.
• Termination: Transcription ends when the RNA polymerase releases the DNA template and the newly synthesized RNA.

RNA Splicing

• RNA splicing removes introns from precursor mRNA (pre-mRNA) and joins exons together.
• This process mainly occurs in eukaryotic cells and is absent in prokaryotic cells.
• Exon: A section of a gene that contains protein-coding information.
• Intron: A non-coding section of a gene.

RNA Splicing Process

• Pre-mRNA is synthesized from DNA during transcription.
• Introns are removed, and exons are joined together to form mature mRNA.

Spliceosome

• The splicing process is carried out by a complex called the spliceosome, which consists of proteins and small nuclear RNAs.

Significance of RNA Splicing

• Splicing allows for the generation of multiple protein isoforms from a single gene through alternative splicing, increasing protein diversity and functional complexity.

Alternative Splicing

• Alternative splicing allows for multiple different proteins to be produced from a single gene.
• This is done by combining different combinations of exons in the mature mRNA.
• Alternative splicing increases the diversity of proteins that can be produced from a given genome.

Translation

• Translation is the process of using information in mRNAs to direct the synthesis of proteins.

Initiation of Translation

• 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.
• The large ribosomal subunit then binds to the initiator tRNA, forming a complete ribosomal complex at the start codon.

Elongation & Translocation in Translation

• The large ribosomal subunit has three tRNA binding sites:
  • The initiator tRNA binds to the central P site.
  • 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 becomes deacylated (no amino acid), while the tRNA in the A site carries the peptide chain.
  • The ribosome moves along the mRNA strand, continuing the cycle of elongation and translocation.

Termination of Translation

• Elongation and translocation continue until the ribosome reaches a stop codon.
• Stop codons recruit a release factor (protein) that signals the end of translation.
• The polypeptide is released, and the ribosome disassembles back into its two independent subunits.

Amino Acid Codons

• 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 acts as both the codon for methionine and the start codon for translation.
• The amino acid codon is universal, meaning the same codons specify the same amino acids across almost all organisms.

Other RNA

• MicroRNA (miRNA)

  • Small, single-stranded, non-coding RNA molecules containing 21 to 25 nucleotides.
  • They regulate gene expression by binding to complementary sequences in target mRNA.
  • This binding can lead to mRNA degradation or translational repression.
  • They are produced endogenously (naturally) from longer primary transcripts.

• Small interfering RNA (siRNA)

  • Double-stranded RNA molecules (20–25 bp length) that mediate RNA interference (RNAi).
  • They perfectly pair with target mRNA, leading to mRNA degradation and silencing of gene expression.
  • They are often derived from exogenous sources (e.g., viral RNA) or synthesized artificially.

• Short hairpin RNA (shRNA)

  • Typically 20-30 base pairs in a hairpin structure.
  • They function similarly to siRNA.
  • They are processed by the protein Dicer into siRNA and used in RNA interference to silence genes.
  • shRNA is often artificially synthesized.

Function of RNA

• mRNA: Carries information specifying amino acid sequences of proteins from DNA to ribosomes. It codes for proteins.
• tRNA: Transports amino acids to the site of protein synthesis, serving as adaptors between mRNA and amino acids during protein synthesis.
• rRNA: Forms the core of the ribosome's structure and catalyzes protein synthesis.
• miRNA, siRNA & shRNA: Regulate gene expression.

Summary

• Nucleotides are the basic building blocks of nucleic acids, which include DNA and RNA.
• DNA is a double-stranded helix, while RNA is typically single-stranded.
• DNA supercoiling and packaging are important processes in DNA structure and function.
• ATP, cAMP, and NAD are important nucleotides involved in cellular energy and signaling pathways.
• DNA replication is the process of making a copy of DNA, while transcription is the process of copying information from DNA into RNA.
• Translation is the process of using information in mRNA to direct the synthesis of proteins.

Nucleic Acids

• Nucleic acids are macromolecules made up of monomers called nucleotides.
• They carry genetic information and instructions for cellular functions.
• They serve as blueprints for protein synthesis.
• They are the hereditary material in cells, passed on to offspring during reproduction.

Central Dogma

• Describes the flow of genetic information: DNA to RNA to protein.

Reverse Transcription

• Synthesis of DNA from an RNA template.
• Catalyzed by reverse transcriptase.
• Occurs in retroviruses such as HIV and hepatitis B for genome replication.

Nucleotides

• Composed of a nitrogenous base, a pentose sugar, and a phosphate group.
• Combine to form DNA or RNA.

Nitrogenous Bases

• Organic molecules containing nitrogen, acting as bases.
• Purines: Adenine (A) and Guanine (G), with a double-ring structure.
• Pyrimidines: Cytosine (C), Thymine (T), and Uracil (U), with a single-ring structure.

Phosphate Group

• Bridges nucleotides through 3’-hydroxyl and 5’-hydroxyl groups.
• Forms phosphodiester linkages, which stabilize the backbone of nucleic acids.

Important Nucleotides

• Ribonucleotides: Building blocks of RNA, containing A, U, C, G bases.
• Deoxyribonucleotides: Building blocks of DNA, containing A, T, C, G bases.
• Adenosine Triphosphate (ATP): Major energy source for cellular processes; consists of adenine, ribose, and three phosphate groups.
• Cyclic Adenosine Monophosphate (cAMP): Second messenger in intracellular signal transduction, synthesized from ATP.
• Nicotinamide Adenine Dinucleotide (NAD): Coenzyme in metabolism, with oxidized (NAD+) and reduced (NADH) forms.

Functions of Nucleotides

• Nucleic acid synthesis: Precursors for DNA and RNA.
• Energy transfer: ATP is the universal energy currency; GTP is used in macromolecule movement.
• Coenzymes: Adenine nucleotides are components of coenzymes.
• Metabolism: Nucleotide derivatives are activated intermediates and regulators in metabolic reactions.

Deoxyribonucleic Acid (DNA)

• The molecule carrying genetic information for development and function.
• Long, double-stranded, helical molecule composed of deoxyribonucleotides.
• First isolated in 1869 by Friedrich Miescher.

Properties of DNA

• Solubility: Polar, soluble in water due to the negatively charged backbone.
• Absorption: Bases absorb ultraviolet (UV) light at 260nm.
• Denaturation: Separation of double-stranded DNA into single strands by breaking hydrogen bonds.
• Renaturation: Re-association of complementary strands upon cooling.

DNA Structure

• Two strands wound around each other, forming a double helix resembling a twisted ladder.
• Backbone made of deoxyribose (sugar) and phosphate groups.
• Bases: Adenine (A), Cytosine (C), Guanine (G), and Thymine (T).
• A pairs with T via two hydrogen bonds; G pairs with C via three hydrogen bonds.
• Strands run antiparallel (5' to 3' and 3' to 5').

Major Groove vs. Minor Groove

• Major Groove: Wider, allows for protein binding.
• Minor Groove: Narrower, limits access to bases.

DNA Packaging in Eukaryotes

• Histones: Positively charged proteins that bind to DNA to facilitate packaging.
• Nucleosome: Basic structural unit of DNA packaging, consisting of DNA wrapped around eight histone proteins.
• Chromatin: Coils of nucleosomes.
• Chromosome: Condensed chromatin fibers during cell division.

Genomic DNA

• Exists as linear or circular double helices.
• Circular DNA: Found in bacteria, mitochondria, and chloroplasts.
• Supercoiling: Additional twisting of DNA to reduce space and facilitate packaging.

Topoisomerases

• Enzymes that regulate DNA supercoiling and topology.
• Type I: Cuts single strand of DNA, relieving tension.
• Type II: Cuts double strand of DNA, introducing or removing supercoils.

DNA Replication

• Step 1: Initiation: Unwinding of DNA at origins of replication by helicase.
• Step 2: Priming: Synthesis of RNA primers by primase.
• Step 3: Elongation: Synthesis of new DNA strands by DNA polymerase in a 5' to 3' direction.
  • Leading Strand: Continuous synthesis.
  • Lagging Strand: Discontinuous synthesis in Okazaki fragments.
• Step 4: Termination: Completion of synthesis, removal of RNA primers, and ligation of fragments.

DNA Proofreading and Repair

• Proofreading: DNA polymerase removes mismatched nucleotides during replication.
• Post-Replication Repair: Mechanisms for correcting errors that escape proofreading.
  • Mismatch Repair (MMR): Corrects base pairing errors.
  • Base Excision Repair (BER): Repairs small base lesions.
  • Nucleotide Excision Repair (NER): Repairs bulky DNA adducts.
  • Double-Strand Break Repair: Uses either homologous recombination (HR) or non-homologous end joining (NHEJ).

Functions of DNA

• Storage of genetic information: In the sequence of bases organized into genes.
• Transmission of genetic materials: Replication ensures accurate transfer of genetic information during cell division.
• Instruction of protein synthesis: Information is transferred to mRNA via transcription, and then to protein by translation.

Ribonucleic Acid (RNA)

• RNA is a single-stranded molecule made up of ribonucleotides.
• RNA is more reactive than DNA and less stable in alkaline conditions.
• RNA strands are constantly being made, broken down, and reused, making them more versatile than DNA.
• RNA has a higher mutation rate compared to DNA.

History of RNA

• Friedrich Miescher discovered nucleic acids in 1868, naming them "nuclein."
• Severo Ochoa won the Nobel Prize in Medicine in 1959 for discovering an enzyme that synthesizes RNA.
• Alex Rich and David Davies created the first RNA crystal in 1956, enabling structural analysis using X-ray crystallography.
• Robert W. Holley determined the sequence of 77 nucleotides in yeast tRNA in 1965, earning the Nobel Prize in Medicine in 1968 for his discovery.
• Philip Sharp and Richard Roberts won the Nobel Prize in 1993 for their discovery of introns and RNA splicing in viruses and genes.
• Katalin Karikó and Drew Weissman received the Nobel Prize in Physiology or Medicine in 2023 for their work on mRNA vaccines, which made effective COVID vaccines possible.

Major Types of RNA

• mRNA (Messenger RNA): Carries genetic information from DNA to ribosomes for protein synthesis.
• rRNA (Ribosomal RNA): Forms the structural core of ribosomes, essential for protein synthesis.
• tRNA (Transfer RNA): Acts as an adapter molecule, bringing amino acids to the ribosomes during protein synthesis.

RNA Structure

• RNA is typically a single-stranded helix with a 5' end (phosphate group) and a 3' end (hydroxyl group).
• Ribonucleotides are linked by phosphodiester bonds.
• Nitrogenous bases in ribonucleotides are adenine (A), cytosine (C), guanine (G), and uracil (U).
• Base pairing in RNA: A pairs with U, and C pairs with G.

Differences Between RNA and DNA

• Uracil (U) replaces thymine (T) in RNA.
• The sugar in RNA is ribose, while DNA contains deoxyribose.
• RNA is usually single-stranded, while DNA is a double-stranded helix.

RNA Secondary Structure

• Some RNA molecules can form complementary base pairing within their own strand, creating loops and double-stranded regions.
• rRNA and tRNA exhibit substantial secondary structure, as do some mRNA molecules.

Messenger RNA (mRNA)

• mRNA carries the protein information from the DNA in the nucleus to the cytoplasm.
• During protein synthesis, the ribosome translates the mRNA sequence, reading three-base codons to determine the corresponding amino acid sequence in a growing protein chain.

Transfer RNA (tRNA)

• tRNA is a short RNA molecule, usually 76 to 90 nucleotides long, acting as an adapter between mRNA and amino acids.
• tRNA binds to a specific amino acid and brings it to the ribosome.
• tRNA recognizes complementary codons in mRNA, ensuring the correct amino acid sequence is incorporated into the growing polypeptide chain.

Ribosomal RNA (rRNA)

• rRNA is a major component of ribosomes, the protein synthesis machinery.
• rRNA is transcribed from rDNA and combines with ribosomal proteins to form small and large ribosome subunits.
• rRNA provides the structural framework for ribosome architecture and catalyzes peptide bond formation between amino acids.

Transcription

• Transcription is the process of copying genetic information from DNA into RNA.
• RNA polymerases are enzymes that catalyze transcription.
• RNA polymerase uses the template strand of DNA to synthesize a complementary RNA strand in the 5' to 3' direction.
• Transcription starts at promoters and ends at terminators.
• Stages of transcription:
  • Initiation: RNA polymerase binds to the promoter region of DNA.
  • Elongation: RNA polymerase moves along the DNA, synthesizing an RNA molecule complementary to the template strand.
  • Termination: Transcription stops at the terminator sequence, and the newly synthesized RNA is released from the DNA template.

RNA Splicing

• Splicing removes non-coding introns from pre-mRNA and joins the coding exons together, producing mature mRNA.
• Splicing primarily occurs in eukaryotic cells, but is absent in prokaryotes.
• Exons: Coding regions of a gene that contain protein information.
• Introns: Non-coding regions of a gene that are removed during splicing.
• The spliceosome, a complex of proteins and small nuclear RNAs, carries out the splicing process.
• Alternative splicing allows for the production of multiple protein isoforms from a single gene, increasing protein diversity and complexity.

Translation

• Translation is the process of using the genetic information in mRNA to synthesize proteins.
• Stages of translation:
  • Initiation: The small ribosomal subunit binds to the initiator tRNA, which recognizes the start codon (AUG). This complex binds to mRNA and moves to the start codon. The large ribosomal subunit then joins them, forming a complete ribosome.
  • Elongation & Translocation: The ribosome has three tRNA binding sites: the P site, the A site, and the E site. The initiator tRNA binds to the P site, and a second tRNA containing the next amino acid binds to the A site. Peptide bond formation links the amino acid in the P site to the amino acid in the A site. The ribosome then moves along the mRNA, shifting the tRNAs.
  • Termination: The ribosome encounters a stop codon. A release factor protein binds to the stop codon, causing the polypeptide chain to detach from the tRNA and the ribosome to disassemble.

Amino Acid Codon

• The genetic code consists of 64 codons, each containing three nucleotides.
• Three codons (UAG, UGA, and UAA) are stop codons, signaling the end of translation.
• The codon AUG codes for methionine and also serves as the start codon for translation.
• The genetic code is universal, meaning the same codons specify the same amino acids in virtually all organisms.

Other RNA Molecules

• MicroRNA (miRNA): Short, single-stranded non-coding RNA molecules (21-25 nucleotides) involved in gene regulation by binding to complementary sequences in target mRNA, either degrading or inhibiting translation of the mRNA.
• Small interfering RNA (siRNA): Double-stranded RNA molecules (20-25 nucleotides) that induce RNA interference (RNAi) by perfectly pairing with target mRNAs, triggering their degradation and silencing gene expression.
• Short hairpin RNA (shRNA): Hairpin-shaped RNA molecules (20-30 base pairs) that function similarly to siRNA. They are processed by Dicer into siRNA for RNA interference.

Function of RNA

• mRNA: Carries genetic information from DNA to ribosomes, serving as the template for protein synthesis.
• tRNA: Transports amino acids to the ribosomes during protein synthesis.
• rRNA: Forms the structural and catalytic core of ribosomes, essential for protein synthesis.
• miRNA, siRNA, and shRNA: Regulate gene expression by modulating mRNA stability and translation.