nucleic lec 11

Nucleic Acid Structure

• Nucleic acids are composed of fundamental building blocks called nucleotides
• Nucleotides consist of a phosphate group, a sugar (deoxyribose in DNA, ribose in RNA), and a nitrogenous base
• Purines (adenine and guanine) and pyrimidines (cytosine, thymine in DNA, uracil in RNA) are the nitrogenous bases
• Nucleotides are linked together through phosphodiester bonds, forming a sugar-phosphate backbone
• Bases are attached to the sugar at the 1' position.
• The sugar is phosphorylated at the 5' position.

Nucleotide Organization

• In DNA, the sugar is 2' deoxyribose; in RNA, the sugar is ribose
• The bases are attached at the 1' position of the sugar.
• The phosphate group is attached at the 5' position of the sugar.

Purines vs. Pyrimidines

• Purines and pyrimidines are heterocyclic aromatic compounds.
• Purines have two rings, while pyrimidines have one ring.
• Biological bases are derivatives of these structures with additional keto, amine, and methyl groups.

Standard Bases

• Adenine (A) and guanine (G) are purines
• Cytosine (C), thymine (T) (in DNA), and uracil (U) (in RNA) are pyrimidines
• Bases can be methylated on heteroatoms
• In DNA, methylation is typically regulatory
• In RNA, modifications (e.g., methylation in ribosomes) often play a structural role.

Deoxynucleotides

• Deoxynucleotides are the building blocks of DNA
• Nomenclature: Deoxyadenosine 5'-monophosphate (dAMP), deoxyguanosine 5'-monophosphate (dGMP), deoxythymidine 5'-monophosphate (dTMP), deoxycytidine 5'-monophosphate (dCMP)
• Note a nucleoside refers only to the base.

Nucleic Acid Structural Hierarchy

• Nucleic acids have primary, secondary, and tertiary structures
  • Primary structure is the sequence of nucleotides
  • Secondary structure is the base pairing interactions (e.g., double helix)
  • Tertiary structure is the three-dimensional arrangement of secondary structures.

DNA Backbone and Interactions

• DNA backbone is comprised of 5'OH of one ribose group linked to 3'OH of the next through a phosphate group
• DNA backbone has no H-bonding donating groups, and no positively charged groups
• Phosphate groups interact well with water
• Deoxyribose is nonpolar
• Unlike peptides, the backbone cannot make strong interactions with itself

Ribos-Phosphate Backbone

• Six single bonds (alpha, beta, gamma, delta, epsilon, and zeta) exist between the 3' of one ribose and the next, allowing free rotation
• Angles are highly limited by ribose ring constraints
• Nucleic acid backbones have greater flexibility per residue compared to peptides.

Sugar Pucker

• Deoxyribose has limited flexibility
• Four atoms in the ring are planar while the other is either endo (toward C5') or exo (away from C5') for C-2' and C-3'

Base Interactions

• Bases have H-bond donor and acceptor properties
• Bases have a big flat, hydrophobic surface facing away from the minor groove.
• Hetero-cyclic bases develop complex electrostatic fields
• These fields dictate base stacking behavior
• Base stacking is a crucial force in DNA stability.

Base Pairs

• Watson-Crick pairs include A-T with two hydrogen bonds and G-C with three hydrogen bonds
• C1-C1 distances are essentially identical at 10.9 Å in WC pairs
• GC base pairs have stronger stacking than AT
• Non-Watson-Crick base pairs can exist and are important for RNA structures
• These non-canonical interactions distort the backbone.

DNA Double Helix

• DNA exists in an antiparallel fashion
• Bases interact through hydrogen bonds inside
• Phosphate groups are on the outside to minimize electrostatic repulsion
• The helix is right-handed

Major and Minor Grooves

• The major groove is longer while the minor groove is shorter
• The major groove has more room for interactions with proteins
• The minor groove offers potential for sequence specific interactions to recognize the DNA backbone.

DNA Geometry

• B-DNA: right-handed helix, base pairs perpendicular to the helix axis, ~23 Å diameter
• A-DNA: right-handed helix, base pairs tilted, ~27 Å diameter
• Z-DNA: left-handed helix, zig-zag backbone.

DNA Hydration

• DNA forms hydrogen bonds with surrounding water molecules
• Specific positions bind water molecules via standard interactions within the major groove or the minor groove/backbone
• Water significantly contributes to B-DNA stability

Metal Ions in Nucleic Acid Structure

• The large negative charge on the phosphate backbone of DNA needs counter ions (like Mg2+) to stabilize the structure
• These metal ions facilitate the formation of the DNA double helix in solutions with predominantly water molecules

Forces Stabilizing Nucleic Acid Structure

• Base Stacking (hydrophobic interactions, van der Waals and electrostatic).
• Hydrogen bonds between bases
• Entropic gain from releasing waters associated with single-stranded nucleotides into the bulk solution.
• Metal ion binding

Energetics of DNA Duplex Formation

• DNA duplex formation is favored under physiological conditions due to the substantial enthalpic gain from hydrogen bonding and van der Waals forces in base stacking.
• Entropy of duplex formation (TDS) is unfavorable, reflecting the cost of restricting conformational freedom
• DNA is readily melted by increasing temperature.

DNA Tetraplex

• DNA tetraplex forms in guanine rich sequences.
• Stable over a wide range of conditions.
• Can be parallel or antiparallel.
• Influences transcription, replication and recombination.

DNA Holliday Junction

• Formed during DNA repair or meiotic crossover
• Adjacent helices have complementary DNA strands exchanged
• DNA flexibility allows for this configuration without significantly distorting the DNA duplex.

DNA Structure and Protein Binding

• Most cellular DNA is in B-DNA conformation.
• Complementary base pairing provides built-in ability for DNA to recognize complementary sequences.

Ribonucleotides and RNA

• RNA is made from Ribonucleotides
• Contains ribose sugar instead of deoxyribose in DNA
• Uracil (U) replaces thymine in RNA
• RNA generally forms A-form helical structure.

RNA Topology Diagrams

• Depict the relative locations of secondary structural elements like bulges, internal loops, and hairpins

RNA Function

• Function of many RNA molecules depends on folding into complex 3D structures
• Including rRNA, tRNA, and mRNA in protein synthesis, as well as involved in splicing machinery.

Riboswitches

• Occur in 5' untranslated regions of mRNAs in gram-positive bacteria
• Control protein levels post-transcriptionally
• Regulate transcription and translation depending on the presence of specific metabolites in the cell.

SAM Riboswitch

• A highly complex three-dimensional RNA structure.
• Contains regions that resembles standard A-RNA structures, and others with more random appearances
• Many odd pairings (non-canonical interactions) in this structure.

Ligand Recognition by RNA

• S-Adenosyl methionine (SAM) binding pocket involves 11 nt from 5 strands.
• Interactions involving base stacking, van der Waals interactions, and multiple hydrogen bonds using both base edges and ribose atoms.
• Clefts in the structure are used to stabilize SAM binding.

Hammerhead Ribozyme

• Enzyme RNA strand binds and cleaves a substrate RNA strand.
• Multiple Na+ ions involved in stabilizing structure.
• Regions of the enzyme interact successively with the substrate RNA

Protein-Nucleic Acid Interactions

• Essential for DNA maintenance, replication, and transcription.
• Specific protein interactions with DNA are essential for biological processes
• This include enzymatic reactions, regulation and transcription, replication.

DNAse I-DNA Interactions

• DNase I is a relatively non-specific double-stranded nuclease from bovine pancreas
• Binds in the minor groove of DNA, opening it up slightly
• Interacts mainly with phosphate groups of the backbone through hydrogen bonds and electrostatic interactions.
• Arginine residues are favored during the interactions.

Protein Targeting

• There are 4 bases in DNA (A, G, C, and T).
• A set of n nucleotides has 4n possible sequences.
• In the human genome, this is a very large number.

"Unwind and Read" Strategy

• Proteins may possibly unzip DNA to "read" the base pairing edges.
• This strategy is energetically expensive.
• However, some proteins use modified versions of this strategy exploiting chemical modifications of bases to flip a base out of the DNA

Recognising Base Edges

• This strategy is plausible because each base pair has a unique pattern of potential interactions in the major groove.
• The minor groove is more ambiguous (difficult to interpret).

Helix-Turn-Helix Transcription Factors

• Recognition helices in these types of proteins often insert into major groove of DNA.

CRO Protein and DNA Binding

• Recognizes a 17 residue pseudo-palindromic sequence.
• Forms a dimer.
• Helix 3 for both protomers sticks out on the same surface interacting with DNA half site.

Cro Interactions

• helix 3 inserted into DNA major groove
• Hydrogen bonds mediated with individual bases
• Non-specific interactions with backbone, dominated by basic residues (Lys/Arg) and electrostatic interactions.
• DNA is distorted upon binding to form a specific interaction site between the protein and the DNA

HTH Motif in DNA Binding Proteins

• Recognition helix in DNA-binding proteins inserts into major groove and interacts specifically with bases
• Helix near the N-terminus position is responsible for recognition helix position and orientation

Other Non-HTH Proteins

• Some proteins also use a-helices to recognize DNA (e.g. coiled-coil, zinc fingers or leucine zipper)

TATA Binding Protein (TBP)

• Ubiquitous eukaryotic transcription factor.
• Directly binds to DNA TATA box, assisting in recruitment of RNA polymerase.

TBP Structure

• Monomeric protein with structurally similar halves.
• Shaped like a saddle.

TBP DNA-Binding

• Binds in the shallow minor groove.
• Contacting 8 consecutive base pairs.
• Extensive interactions with the DNA backbone using basic amino acid residues.

TBP DNA Contacting Bases, Additional interactions

• TBP forms specific hydrogen bonds with AT edges using basic residues.
• Extensive interactions with the DNA backbone due to interactions with the PO4 groups
• Surface contacting DNA bases is primarily composed of aliphatic and aromatic residues, contributing to the overall hydrophobic nature of the protein-DNA interface

Modular DNA Recognition by TAL Effector Proteins

• TAL proteins are built from sequential repeats.
• Each repeat comprises two helices.
• This modular design makes them highly customizable for protein engineering.

K16 and Q17 of Each Repeats Contact PO4

• K16 and Q17 amino acids contact the phosphate group of the DNA strand.
• These contacts provide high binding energy without sequence specificity

Positions 12 and 13 Specifying the Base

• Position 12 (Asn or His) and 13 (Gly, Asp, Asn, or Ser) residues together determine nucleotide specificity.

RNA Topology diagrams(additional details)

• Depicts secondary structural elements like bulges, internal loops, and hairpins.

Disorder-Order Transition in NA Binding

• DNA binding proteins often have disorder.
• Binding to DNA partner forces the protein into ordered conformation.
• Large number of basic amino acids and non-polar base stacking observed.

General Conclusions

• DNA binding proteins primarily interact with phosphate backbone to derive their binding energy.
• Almost all sequence-specific DNA-binding proteins distort DNA.
• Specificity is heavily influenced by the DNA's ease of twisting/distortion into the specific conformation for binding
• Hydrogen bonds with exposed bases are essential for sequence recognition.

This quiz explores essential concepts related to DNA binding proteins, including their structural features, interactions with DNA, and the significance of hydrophobic interactions and ordering transitions. Test your knowledge on topics like the major groove's role in recognition, the phosphate backbone, and the mechanics of DNA distortion.