Nucleic Acids: Structure and Function

• The notes cover the structural and packaging aspects of nucleic acids, key topics in molecular genetics and genomics.
• The content is relevant to the BIOL310 course schedule for Spring 2025.

Introduction

• Different phenotypes arise from variations in genetic material.
• Nucleic acids include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), which affects genetic traits.
• Nucleic acids are larger units of the cell.
• Nucleic acids contain nucleotides.
• It transmits phenotypic characteristics from one generation to another and contains information about an organism's structure and function.
• It replicates accurately to ensure parent and daughter cells share genetic information and can change.
• Miescher discovered nucleic acids initially in white blood cells from pus in bandages in 1869.
• The substance was named nuclein and contains C, H, O, N and P atoms.
• Nucleic acids are not proteins.

Experiments on the Genetic Material

• Three main experiments confirmed that a cell's genetic material is DNA.
• The genetic material is DNA
• 1. Griffith's transformation experiment showed the transforming principle.
• 2. Avery's transformation experiment identified Griffith's transforming principle as DNA.
• 3. Hershey and Chase's bacteriophage experiment directly confirmed that DNA is the genetic material.

Griffith's Experiment

• S strain bacteria have a polysaccharide coat (capsule) and are virulent.
• R strain are generally identical to S but lack the capsule due to a mutation and are non-virulent.
• Griffith's conclusion showed live non-virulent bacteria acquired the ability to form capsules from dead virulent bacteria.
• Transformation is the acquisition of exogenous DNA.
• Transforming principle means an unknown agent responsible for the change in genotype via transformation in bacteria, which is now known to be DNA.

Avery's Experiment

• S strain cells are lysed using a detergent to extract cellular components.
• Lysate contains proteins, lipids, carbohydrates, nucleic acids, and cellular debris.
• Cell-free extract is fractionated into classes of purified molecules.
• The molecule carrying the heritable information is DNA.

Hershey-Chase Experiment

• Bacteriophages undergo a lytic life cycle to infect cells.
• Bacteriophages are made of proteins and DNA.
• To prove that DNA is the genetic material, Phosphorus and sulfur were used.
• Sulfur is found in protein but not in DNA, and phosphorus is in DNA but not protein.
• Only DNA is passed from parent to offspring in phage reproduction so DNA is the genetic material.

Genetic Material

• Genetic material in viruses = DNA or RNA.
• Genetic material of prokaryotes = DNA.
• Genetic material of eukaryotes = DNA.

Composition of Nucleic Acids

• Nucleotides are the building blocks of nucleic acids and consist of pentose sugar + nitrogenous base + phosphate group.
• Nucleoside = pentose sugar + nitrogenous base.
• DNA contains deoxyribose sugar and nitrogenous bases A, G, C, T, and a phosphate group.
• RNA contains ribose sugar and nitrogenous bases A, G, C, U and a phosphate group.
• Nitrogenous bases bind to C1 of pentose sugar via N9 of purines and N1 of pyrimidines.
• Phosphodiester bonds link nucleotides from 5' to 3'.
• Phosphate groups give DNA a negative charge.
• Nitrogenous bases bind to C1 of pentose sugar.
• Pentose sugars + Phosphate groups makeup the DNA backbone, which is stable and negatively charged.
• Polynucleotide chains have polarity with a 5' end which = C5 of deoxyribose attached to a phosphate group, and a 3' end: C3 of deoxyribose attached to a hydroxyl group.

DNA Structure

• Three studies proved that DNA is an antiparallel double helix.
• 1. Chargaff's study to understand DNA.
• 2. Franklin and Wilkins used X-ray.
• 3. Watson and Crick's is a continuation of study of Franklin and Wilkins.
• Chargaff's study showed the number of purines equals the number of pyrimidines and that A=T and G=C.
• Franklin and Wilkins relied on X-ray diffraction to show DNA is a double helix.
• Watson and Crick study demonstrated 1) DNA is a right-handed double helix, with one complete turn every 10 bp, 2) the two polynucleotide chains are antiparallel, 3) sugar-phosphate backbone is on the outside, and nitrogenous bases are located internally, 4) nitrogenous bases are linked by complementary base pairing via hydrogen bonds, and 5) complementary base paring results in an equal diameter helix.
• DNA can come in 3 forms: A-DNA, B-DNA, Z-DNA, according to x-ray diffraction.
• B-DNA is closest to the DNA in our cells, which are in solution, unlike X-ray diffraction.

Genes

• A gene = segment of DNA containing instructions to make a particular protein.
• Elements of a gene are Regulatory sequences (controlling gene expression), Exons (coding regions), and Introns (non-coding regions).
• Promoter = sequence of DNA at which RNA polymerase binds and where transcription starts.
• Terminator = sequence of DNA where transcription ends.

Structure of RNA

• RNAs are single-stranded molecules that fold into secondary structures.
• mRNA is a messenger RNA that codes for proteins.
• rRNA is a Ribosomal RNA that forms the basic structure of the ribosome and catalyzes protein synthesis.
• tRNA is a transfer RNA that transfers RNA central to protein synthesis as adaptors between mRNA and amino acids.
• snRNAs are small nuclear RNAs that function in various nuclear processes.
• snoRNAs are small nucleolar RNAs that help process and chemically modify rRNAs.
• miRNAs help regulate gene expression.
• siRNAs have gene expression directed by the degradation of selective mRNAs
• piRNAs bind to piwi proteins and protect the germline from transposable elements
• lncRNAs are long nonconding RNAs, many of which serve as scaffolds; they regulate diverse cell processes, like X-chromosome inactivation

Packaging of Genetic Material

• In viruses, DNA or RNA can be either circular or linear within one chromosome.
• Genomes may be segmented into several nucleic acid fragments.
• Prokaryotes have genetic material in one double-stranded circular DNA molecule known as the nucleoid and have plasmids.
• Plasmids = extra genetic material that help keep prokaryotes alive.
• Plasmids can be linear or circular in shape and are small in size.
• Extranuclear DNA is double-stranded circular DNA found in mitochondria and chloroplasts.

Eukaryotic Packaging of Genetic Material

• In eukaryotic cells, DNA needs to fit in the nucleus.
• Chromosomes are packaged differently during interphase vs. mitosis.
• High compaction of mitotic chromosomes aids sister chromatid separation and protect DNA.
• Beads on a string form are made of chromosomes and are made of chromatin.
• Chromatin = DNA + proteins.
• Nucleosome = Bead + Linker DNA.
• Histone octamer = 2 of each histones, that are H2A, H2B, H3 and H4.
• Duing mitosis, chromosomes are more condensed.
• Chromatin fiber folded into loops are loops of folded chromatin.

DNA Packaging Differences and Repetitive DNA

• There are differences in DNA and are based on phases of the cell cycle.
• Mitosis and Meiosis have two phases, which are S phase and Chromosomes.
• S phase = DNA is dispersed.
• Mitosis and Meiosis = DNA is condensed.
• There are different types of chromatin.
• Euchromatin = the changes can be seen in staining intensity because DNA undergoes transcription and has no repetitive sequences.
• Constitutive and facultative heterochromatin have condensed states during every phase.
• Constitutive heterochromatin = repetitive DNA sequences are present at the same position in homologous chromosomes like centromeres and telomeres.
• Centromeres = DNA sequences to which spindle fibers attach during mitosis or meiosis.
• Telomere = repetitive DNA sequences at chromosome extremities.
• Repetitive DNA sequences are interspersed repeated DNA which are called LINES and SINES.
• Tandemly repeated DNA are such as telomeric and centromeric DNA.
• LINES = Long interspersed elements, that are sequences of 1000 to 7000 base pairs.
• SINES = Short interspersed elements, that are sequence of 100 to 400 base pairs.

Gel Electrophoresis

• Gel electrophoresis is a lab method that visualizes and separates nucleic acids.
• Southern blotting = a technique that detects and quantifies specific gene sequences using probes.
• A probe is a single-stranded small sequence of DNA (15-30 bp) that is complementary.
• Key steps in Southern blotting are gel electrophoresis to separate DNA fragments by size then transferring the separated DNA to a membrane.
• The DNA is then probed to detect specific sequences depending on experimental controls.
• Positive control = Nucleic acid to which a probe can bind.
• Negative control = Water instead of nucleic acid.