Nucleotides Study Notes

Unit 6: Nucleotides

The Building Blocks of Energy and Life

• Nucleotides play a critical role as:

  • Base of Energy Currency:

  • ATP (Adenosine Triphosphate)

  • GTP (Guanosine Triphosphate)

  • Structural Components:

  • Coenzymes

    • NAD (Nicotinamide Adenine Dinucleotide)

    • FAD (Flavin Adenine Dinucleotide)

  • Building Blocks of Genetic Code:

  • DNA (Deoxyribonucleic Acid)

  • RNA (Ribonucleic Acid)

Structural Characteristics of Nucleotides

• Nucleotides consist of three main components:

  1. Nitrogenous Base:

  • Can be either a purine or a pyrimidine

  1. Five-Carbon Sugar:

  • Ribose (in RNA)

  1. Phosphate Tail:

  • Contains one, two, or three phosphate groups

• The structure without the phosphate is referred to as a nucleoside.

Common Structural Moieties

• Phosphate Types:

  • Monophosphate

  • Diphosphate

  • Triphosphate

• Sugars:

  • Ribose

  • 2’-Deoxyribose

• Purines:

  • Adenine

  • Guanine

• Pyrimidines:

  • Cytosine

  • Thymine (DNA)

  • Uracil (RNA)

Naming Conventions

• As Nucleosides:

  • Purines: –ine is replaced with –osine

  • Pyrimidines: End with –idine; specific suffixes differ based on the base

  • Bases attached to a deoxy sugar include deoxy- prefix

• As Nucleotides:

  • Purines: –ine replaced with –ylate

  • Pyrimidines: End with –idylate

  • Expressed by the nucleoside name followed by 5’-phosphate (indicating the number of phosphate groups)

  • One-letter codes: A, G, C, T, or U (with “d” to indicate deoxy)

  • Phosphate indicators: MP (monophosphate), DP (diphosphate), TP (triphosphate)

Examples of Nucleosides and Nucleotides

• Nucleoside: Deoxycytidine

  • Letter: C or dC

  • Nucleotide: Deoxycytidylate or Deoxycytidine Diphosphate

• Nucleoside: Deoxyadenosine

  • Letter: A or dA

  • Nucleotide: Deoxyadenylate or Deoxyadenosine Triphosphate

• Nucleoside: Uridine

  • Letter: U or UMP

  • Nucleotide: Uridylate or Uridine Monophosphate

Atomic Numbering of Altered Bases

• Chemical alterations designate the number of the atom involved.

  • If a substituent is covalently bound to the purine/pyrimidine ring, it is assigned the corresponding number.

  • If bound to an exocyclic hetero atom, the designation changes to the hetero atom and the attachment point on the ring.

• Nitrogenous bases are numbered normally, while sugars use a prime designation:

  • Examples:

  • 5-Methylcytosine (methyl at position 5)

  • 7-Methylguanine (methyl at position 7)

  • N6-Methyladenine (methyl at position 6)

Phosphate Bonds and Phosphoester Linkages

• The 5’ monophosphate is the most prevalent form; however, phosphates can bind to any available hydroxyl group.

• Cyclic monophosphates are isolatable intermediates and influence RNA instability.

• 3’,5’-Cyclic Monophosphates:

  • Regulatory molecules, abbreviated as cAMP or cGMP

• During DNA and RNA synthesis, the 5’-phosphate of one nucleotide binds to the 3’ hydroxyl group of another, forming a phospho-ribo backbone.

  • Variants:

  • 5’-Monophosphate

  • 3’-Monophosphate

  • 2’-Monophosphate

  • 2’,3’-Cyclic Monophosphate

DNA Ligation

• Occurs in a 5’ to 3’ orientation via pyrophosphate transfer in phosphodiester linkages.

• Nucleotides covalently link through phosphate bridges.

  • The 5’ phosphate of one nucleotide binds to the 3’ hydroxyl of the next.

• Phosphate groups exhibit a pKa near 0, hence they are completely deprotonated.

• Single strands of DNA and RNA possess specific 5’ to 3’ polarity.

  • These strands are referenced from 5’ to 3’, similar to proteins referenced from N to C.

RNA Hydrolysis in Alkaline Conditions

• Phosphodiester linkages hydrolyze in alkaline environments, leading to RNA instability.

• The 2’ hydroxyl directly engages in hydrolysis, creating cyclic 2’-3’ monophosphate intermediates.

  • This intermediate can further hydrolyze into 2’ or 3’-monophosphate species.

• DNA, lacking the 2’ hydroxyl, demonstrates greater stability under alkaline conditions.

Structural Characteristics of DNA and RNA

• Nitrogenous Bases:

  • Planar and conjugated, strong UV light absorbers

  • Their planar nature allows hydrophobic pi-stacking, stabilizing adjacent bases

  • Chemical groups on bases form hydrogen bonds across strands in specific orientations, leading to base pair complementarity.

Structural Characteristics of DNA Double Helix

• The initial 3D structure was established in 1953 by Rosalind Franklin and Maurice Wilkins.

• Key observations:

  • Species-specific differences in G, C, A, and T counts, with G = C and A = T.

  • Base pairs are separated by a distance of 3.4 Å and one helical rotation demonstrates 10.5 base pairs, equating to a periodicity of 36 Å.

• Non-polar nitrogenous bases are buried internally by the highly negative, solvent-facing sugar-phosphate backbone.

Structural Characteristics of DNA Helix

• The furanose ring of deoxyribose is in the C-2’ endo position, folding alongside the C-5’ methyl hydroxyl.

• Base pairing offsets the backbone creating major and minor grooves.

• Complementary strands run anti-parallel (5’ end of one aligns with the 3’ end of another).

Ribose Conformation and Helical Forms

• The furanose can adopt various puckered orientations impacting helix geometry:

  • C-2’ and C-3’ on the same side as C-5’ (endo) or opposite side (exo)

  • Rotation about C-1’ and nitrogenous bases can either be on the same side (syn) or opposite sides (anti)

• Varieties leading to different helical forms:

  • A form: Right-handed, C-3’ endo sugars, anti bases, bases tilted at 20° to the helical axis.

  • B form: Right-handed, C-2’ endo sugars, anti bases, bases tilted at 6° to the helical axis (most commonly observed).

  • Z form: Left-handed, C-2’ endo sugars for pyrimidines, C-3’ endo sugars for purines, bases anti for pyrimidines, syn for purines, with bases positioned at 7° to the helical axis.

DNA Sequence and Secondary Structure

• Palindromes:

  • DNA sequences showing inverted repeats creating two-fold symmetry, self-complementary, potentially forming hairpin or cruciform structures.

• Mirror Repeats: Inverted repeats on the same strand not forming hairpins or cruciform structures.

• Self-complementarity results in formation of complex structures in solution irrespective of length.

Triplex and Tetraplexed DNA

• Watson-Crick Base Pairing: Regular A=T and G≡C pairings.

  • Third bases can interact with Watson-Crick pairs in the major groove.

  • Protonated cytidine pairs with G≡C, thymidine pairs with A=T.

• This triplex pairing leads to Hoogsteen Pairing, particularly stable at lower pH, as G≡C pairing requires C+.

• G-Tetraplex or G-Quadruplex: Guanosine-rich strands can also aggregate into tetraplexes.

Structure and Pairing in RNA

• RNA, transcribed from DNA, exists as a single strand.

• Tends to adopt a right-handed helical conformation, dominated by base stacking.

  • Purine-purine interactions are particularly strong, often displacing pyrimidines.

• RNA is known to form self-complementary interactions and can hybridize with RNA and DNA strands.

  • Common pairings include G≡C, A=U, and sometimes G=U.

• Primarily adopts A-form helix with possible instances of Z-form.

RNA Secondary Structure

• Self-complementarity of single-stranded RNA results in complex secondary structures.

• Types of RNA synthesized:

  • mRNA (Messenger RNA): Carries genetic instructions for protein synthesis.

  • tRNA (Transfer RNA): Acts as adapter molecules linked to amino acids for recognizing mRNA during translation.

  • rRNA (Ribosomal RNA): Integral structural and catalytic component of ribosomes in protein synthesis.

• RNA structure is essential for driving stability, regulation, and catalytic activity.

Chemical Modifications to DNA

• Slow spontaneous processes can cause modifications in DNA bases.

  • Proofreading mechanisms can generally correct these modifications.

• Mutation: A permanent change in the genetic code resulting from chemical modifications.

• Deamination: Base loses an exocyclic amine group.

  • Frequencies:

  • Cytidines: 1/10^7 per 24 hours.

  • Adenines/Guanines: 1/10^9 per 24 hours.

• Hydrolysis: Leads to abasic sites, more common in purines at 1/10^5 per day.

Environmental DNA Damage

• Ionizing Radiation: UV or gamma radiation causes photoproducts in neighboring bases.

• Nitrosamines: Food preservatives form nitrous acid, which accelerates deamination.

• Alkylating Agents: Compounds like dimethyl sulfate disrupt base pairing.

• Oxidative Stress: Forms radical oxygen species leading to base and deoxyribose oxidation, causing strand breaks.

DNA Denaturation

• DNA strands separate with increased temperature or altered pH, disrupting hydrogen bonds.

• Denaturation can be complete or partial, resulting in the unwinding of the helix.

• Renaturation (or Annealing): Occurs when conditions revert to favorable levels, proceeds as follows:

  • Initial slow step: finding complementary regions via random collisions.

  • Once found, remaining unpaired bases quickly pair up and zip together.

DNA TM of Unwinding

• Stacked base pairs initially lower absorption of UV light.

• Pairing base pairs further diminishes absorption, demonstrating the hypochromic effect.

• As DNA denatures, increased UV absorption is noted, termed the hyperchromic effect.

• The Melting Temperature (TM): Determined when half the DNA sample is unwound, measured by UV absorption at varying temperatures.

  • G≡C pairs have more hydrogen bonds than A=T pairs, affecting relative TM.

• Higher GC content correlates with a higher TM.

• TM values observable for DNA-DNA, DNA-RNA, and RNA-RNA duplexes, with RNA-RNA being the most stable and exhibiting TM values 20°C higher than DNA-DNA.

Central Dogma of Biology

• DNA:

  • Replication

  • Converts into RNA:

  • Transcription

  • Converts into Protein:

  • Translation

Replication

• Begins at an origin and proceeds bidirectionally.

• A replication bubble forms, generating two forks moving in opposite directions.

• Origins generally contain high A=T content.

• Replication only occurs in a 5’-3’ direction, dependent on a free 3’-OH at the elongation starting point.

• Two product forms arise:

  • Leading Strand: Continuous

  • Lagging Strand: Discontinuous, forming Okazaki fragments.

DNA Polymerase Template

• Replication occurs within the active site of DNA Polymerase I, catalyzing the following reaction:

• DNA polymerization necessitates two elements:

  1. Template: Guides specific base additions.

  2. Primer: Pre-existing complementary strand attached for polymerase to utilize.

DNA Polymerase - Primer

• The primer serves as a complementary strand segment to the template; polymerase cannot initiate without an existing strand.

Polymerase Chain Reaction (PCR)

• Developed by Kary Mullis during a life-altering car ride, PCR is an essential technique combining replication and denaturation.

• Utilized for cloning, gene amplification, and quantifying expression.

• Primers flank the target sequence, combined with DNA, nucleotides, and polymerase.

• The process involves:

  • Heating: Denatures the DNA

  • Cooling: Allows primer annealing

  • Polymerase replicates along each strand in the 5’ to 3’ direction.

Polymerase Chain Reaction (PCR) Continued

• Each cycle leads to production of the original template and two truncated products extending to both ends of the template.

• The process is repeated, and in the second run, primers bind to the truncated ends from the first round.

• Post-process, the original template remains, alongside extended truncated products and two short segments flanked by primer binding regions.

Polymerase Chain Reaction (PCR) Final Notes

• This denaturing, annealing, and extending cycle generally repeats for 20 to 40 cycles.

• After completion, the target sequence amplification can be calculated by the formula $2^{(n-1)}$, where $n$ represents the number of cycles.