DNA Structure and Composition

Central Dogma of Molecular Biology

Nucleotides

• Nucleotides have several functions, including building blocks of nucleic acids (DNA/RNA).
• They act as carriers of activated intermediates, structural components of coenzymes.
• They are second messengers in signal transduction pathways, principal biologic transducers of free energy, regulatory compounds in pathways and synthetic analogues as drugs.
• A nucleoside consists of a nitrogenous base and a pentose sugar.
• A nucleotide consists of a nucleoside and a phosphate group.
• Nucleotides can be nucleoside monophosphate, diphosphate, or triphosphate, depending on the number of phosphate groups attached.
• The nitrogenous bases are divided into purines and pyrimidines.
• Purines include Adenine and Guanine, which are found in both DNA and RNA.
• Pyrimidines include Cytosine, Uracil (found in RNA), and Thymine (found in DNA).
• PuGA = Purines, Guanine, and Adenosine (both DNA and RNA).
• CUTiePy = Pyrimidines, Cytosine, Uracil (RNA) and Thymine (DNA).
• Ribose is D-ribose sugar found in RNA.
• Deoxyribose is 2'-deoxy D-Ribose sugar found in DNA.

DNA Structure

• DNA consists of nucleotides held together by 3' to 5' phosphodiester bonds and is made up of 2 chains.
• DNA's two strands are complementary and antiparallel with hydrogen bonds between the bases.
• Normally, DNA twists into a right-handed double helix with 10.5 base pairs per turn.

DNA History

• The composition of DNA was known.
• Erwin Chargaff discovered Chargaff's Rule.
• Rosalind Franklin obtained X-ray crystallography images of DNA.
• James Watson and Francis Crick created the two-strand, double-helix model.

DNA Properties

• Base stacking involves hydrophobic interactions and van der Waals forces.
• A-DNA is broad and short, B-DNA is long and thin and Z-DNA is long and thin.
• A-DNA has 11 base pairs per turn, B-DNA has 10 base pairs per turn and Z-DNA has 12 base pairs per turn.
• A- and B-DNA are right-handed, while Z-DNA is left-handed.
• A-DNA is found in low humidity and high salt conditions, B-DNA is the most common form.
• Z-DNA is typically located at the 5' end of chromosomes.

Levels of DNA Organization

• 2 nm DNA double helix and is the first level of DNA organization
• 10 nm chromatin fibril where DNA wrapped 1.75x over a histone octamer (left-handed).
• Made of nucleosomes separated by linker DNA
• 30 nm chromatin fibril (solenoid).
• Supercoiled structure promotes packing DNA into compact structures.
• Chromosome results from condensation of DNA during prophase of mitosis.
• Humans have 23 pairs of chromosomes (46 total), including 22 pairs of autosomes and 1 pair of sex chromosomes.

DNA Types

• Heterochromatin is more condensed, appears darker on electron microscopy (EM).
• Heterochromatin is sterically inaccessible and transcriptionally inactive with increased Methylation and decreased Acetylation.
• Euchromatin is less condensed, lighter on EM, sterically accessible, and transcriptionally active.

RNA structure

• RNA is similar to DNA with purine and pyrimidine bases that are single stranded.
• RNA Functions can be coding or non-coding.

mRNA

• mRNA is "messenger RNA" and the most heterogenous RNA (5% of total RNA)
• mRNAs relay information from DNA to the translation machinery (ribosomes) and is a templatet for protein synthesis
• In eukaryotes, mRNA has a methylguanosine cap at the 5' end and a poly(A) tail at the 3' end.
• Primary transcript undergoes splicing prior to protein synthesis.

rRNA

• rRNA is "Ribosomal RNA", the most abundant RNA (80% of total RNA).
• rRNA Contributes to the formation and function of ribosomes.
• Molecules differ in their sedimentation coefficients and contain many loops and extensive base-pairing.
• Prokaryotes consist of 50S and 30S subunits, made up of 3 types of RNA: 16S, 23S, and 5S.
• Eukaryotes consist of 60S and 40S subunits, made up of 4 types of cytosolic rRNA: 18S, 28S, 5S, and 5.8S.

tRNA

• tRNA is "Transfer RNA", the smallest RNA (15% of total RNA).
• This molecule is an adapter that translates nucleotide sequences of mRNA into specific amino acids.
• Contains 74 to 95 nucleotides with a high percentage of unusual bases.
• There are at least 20 different species, each containing an anticodon.
• It has a 2D cloverleaf appearance.
• The acceptor arm terminates the nucleotides -CCA and receives the tRNA-appropriate amino acid.

snRNA

• snRNA is "Small nuclear RNA".
• Functions in mRNA processing and rRNA processing.
• Splices together the exons to form, making the mature mRNA.

miRNA

• miRNA is "micro-RNA".
• Interacts with the 3' untranslated region of mRNA to induce mRNA degradation and translational repression.

siRNA

• siRNA, or "silencing RNA”
• Double-stranded RNA (20-24 bp).
• Interferes with the expression of genes that have a complementary nucleotide sequence to that of siRNA, inducing mRNA degradation.

lncRNA

• IncRNA or"long non-coding RNA"
• Non-coding transcripts of >200 nt.
• Involved in regulation of cell differentiation & development and maintenance of telomere length (TERC and TERRA).

DNA vs RNA

• DNA contains Deoxyribose, while RNA contains Ribose.
• DNA contains Adenine & Guanine, while RNA also contains them.
• DNA contains Cytosine & Thymine, while RNA contains Cytosine & Uracil.
• DNA is double stranded, while RNA is single stranded.
• Chargaff's rule applies to DNA, but does not apply to RNA.
• DNA is stable and not hydrolyzed by alkali due to the absence of a 2' hydroxyl group.
• RNA is unstable, and can be hydrolyzed by alkali to 2', 3'-cyclic diesters of the mononucleotides.

Proteins

• Proteins are the most abundant and functionally diverse molecules in living systems.
• Proteins are linear polymers of amino acids that are linked together by peptide bonds.
• The general formula for amino acids is N-CX-COOH (X: variable R group)
• Amino acids can have basic, uncharged polar or acidic side chains.
• Proteins range from 100-1000 amino acids (AAs) in length.
• The protein sequence can be determined by removing one AA at a time via Edman degradation.
• Proteins regulate metabolism and facilitate muscle contraction.
• They provide structural frameworks, shuttle molecules in the bloodstream, and are components of the immune system.

Structural Organization of Proteins

• Primary structure is the sequence of a chain of amino acids.
• Secondary structure is the hydrogen bonding of the peptide backbone, causing the amino acids to fold into a repeating pattern.
• Tertiary structure is the three-dimensional folding pattern of a protein due to side chain interactions.
• Quaternary protein structure consisting of more than one amino acid chain.

Protein Structures

• Primary structure is determined by the AA sequence with an N (NH3) and a C (COOH) terminus.
• Peptide bonds attach the α-amino group of one to the α-carbonyl group of another.
• Peptide bonds exibit partial double-bond character, a Trans-configuration and can be disrupted by hydrolysis.
• Secondary structure that causes folding of short (3-30 residues) segments of polypeptide into geometrically ordered units, stabilized by hydrogen bonds.
• Motifs are supersecondary structures produced by packing of sidechains from adjacent secondary structural elements.
• Alpha helix is the most common secondary structure; it is a spiral with polypeptide backbone core and side chains extending outward with ~3.6 AAs per turn.
• Beta sheets consist of AA residues folded into zigzags or with a pleated pattern.
• The "r" groups of adjacent residues project in opposite directions and can be structured in parallel or antiparallel.
• Tertiary Structure is overall 3D shape of the protein, stabilized by hydrophobic clustering force, disulfide bridges (Cysteine), hydrogen bonds, ionic interactions, and van der Waals forces.
• Proteins fold into a conformation of lowest energy with folding assisted by molecular chaperones.
• Quaternary Structure consists of 2 or more polypeptide chains forming one macromolecule, but not all proteins have a corresponding quaternary structure.

Clinical Correlate: Sickle Cell Disease

• Sickle Cell Disease is due to a point mutation (missense) in both genes coding for the β-chain.
• It involves a change from Glu to Val at position 6 and is a homozygous recessive disorder.
• Polymerization and decreased solubility of the deoxy form of Hb in low oxygen tension causes distortion of the RBC,
• This leads to sickling of RBCs and occlusion of capillaries.
• Clinical manifestations include Anemia, Tissue anoxia and Painful crises.
• Treatments include hydration, analgesics, *antibiotics, transfusions, hydroxyurea, L-glutamine, Crizanlizumab and Voxcelotor.