Nucleic Acids

• What is a nucleic acid?

  A linear macromolecule who's each monomeric unit contains a sugar, base, and a phosphate
• Which OH's are involved in the formation of the nucleic acid backbone?

  3' -OH and 5' -OH
• What is the structural difference between RNA and DNA?

  RNA has a 2' OH group while DNA does not
• How are sugars linked to the backbone?

  Sugars are linked by phosphodiester bridges between the 3' OH of the sugar and 5' OH of the adjacent sugar. Known as a 3' to 5' phosphodiester linkage
• What direction is the backbone read in?

  5' to 3'
• Why does the 2' OH in RNA make unstable?

  The 2' OH can act as a nucleophile and attack the phosphodiester bond, making RNA more unstable relative to DNA
• Which nitrogen forms the glycosidic bond in purines?

  N9
• Which nitrogen forms the glycosidic bond in pyrimidines?

  N1
• Nucleotides are the monomeric units of DNA and RNA. What do nucleotides and nucleosides consist of, and how do they differ?

  Nucleotides: base, a pentose sugar, one or more phosphates

  Nucleosides: only have a base and a pentose sugar

  Nucleotides are essentially a nucleoside joined to phosphoryl group by ester linkage
• Naming conventions...

  A) glycosidic

  B) Nucleoside

  C) Base

  D) Adenylate

  
• naming conventions with deoxyribose...

  A) deoxyadenosine

  B) deoxyadenylate

  C) adenine

  D) dADP

  
• Which hydroxyl group(s) of ribose participate in formation of the phosphodiester backbone of RNA?

  5' and 3' OH groups
• Which atom forms the glycosidic linkage with a purine or pyrimidine base?

  1' Carbon
• Directionality of double helix chains?

  Two DNA chains of opposite directionality intertwine to form a right-handed double helix
• How are the bases arranged relative to the axis of the helix and how wide is the helix?

  The bases are nearly perpendicular to the axis of the helix with adjacent bases separated by 3.4 Å. The helix is approximately 20 Å wide
• What form is most DNA in under physiological conditions?

  B-DNA also known as the Watson and Crick model of DNA
• Describe the structure of the double helix in the Watson-and-Crick model?

  Adenine pairs with thymine and makes 2 hydrogen bonds. Guanine and cytosine pair to make 3 hydrogen bonds.

  • the pairing of purines and pyrimidines results in a uniform diameter
• Why are there variations in base pairing?

  • Rotation of the base in RNA or DNA ends (reverse watson crick)
  • Tautomerization (wobble base pairs)
  • Rotation around C'1-N glycosidic bond (Hoogsteen)
• How does DNA base pairs contribute to the stability of the helix?

  • hydrophobic effect: bases within the double helix are hydrophobic and hydrophobic interactions drive bases to inside the helix, while the more polar residues are on the outside
  • Base stacking: the flat, planar structure of bases allows them to be stacked which allows allows the bases to attract each other through van der waals interactions
• DNA has a major and minor groove, why do they exist?

  The major and minor grooves in DNA form because the bonds between base pairs aren't directly opposite each other. This creates spaces between the strands where proteins can bind. The major groove is larger, while the minor groove is smaller.
• Why are the major and minor grooves important?

  Each groove is lined with atoms unique to each base pair that can donate or accept hydrogen bonds, allowing proteins to interact with DNA. These interactions are crucial for processes like replication and transcription, as specific proteins bind to DNA, recognizing hydrogen bond sites on the grooves to catalyze these processes.
• A-form of DNA is a hybrid of DNA and RNA, and the base pairs are no perpendicular to the axis. While Z-form is left-handed DNA and its phosphodiester bond zigzags
• One way prokaryotic DNA can be packaged is by supercoiling. Prokaryotic DNA is circular but when unwound by two turns, will supercoil into a negative superhelix. If given two extra turns will become a positive superhelix. Both are still more condensed then the relaxed DNA
• Packaging of Eukaryotic DNA

  • Nucleosomes are histone octamers wrapped by 200 bp of DNA. The amino-terminals tails that contain positively charged residues that interact with the negatively charged DNA.
  • This step compacts linear DNA by a factor of 7, but it still must be further compacted to enter the nucleus
  • These nucleosomes form a left-handed helical stack to further compact into condensed chromatin
• RNA can fold back on itself to form complex 3D structures, what role does this allow ribosomes to take on?

  They can act as catalysts known as ribozymes that can help peptide bond synthesis or in splicing
• What does the polymerases do in E. coli?

  DNA polymerase catalyzes the addition of deoxyribonucleotides to a DNA strand.

  • Polymerase I: removes primers, and has 5' to 3' exonuclease activity and 3' to 5' exonuclease activity
  • Polymerase III: replicates DNA, and has 3' to 5' exonuclease activity
• Exonuclease component of polymerases hydrolyzes phosphodiester bonds to remove incorrect nucleotides from the 5' or 3' end
• How does the polymerase help ensure the right dNTP is being incorporated?

  The binding of the correct dNTP triggers a conformational change in the polymerase that causes the helical region (fingers domain) to clamp down on the correct nucleotide once the correct hydrogen bond interactions is formed with the template strand.
• What are the basic requirements for DNA strand-elongation reaction?

  Requires: dNTPs, Mg2+, and a primer to start
• Which strand is the new DNA strand assembled on?

  On the template strand.
• Which direction is DNA elongation in?

  5' to 3' direction
• How exactly are mistakes corrected by polymerase?

  a mismatch results in a stall, this pause in replication gives time for the incorrect nucleotide to flop into the active site of the exonuclease where it can be hydrolyzed and removed from the 3' growing strand.
• How is DNA separated into single strands?

  Helicases are ring-like hexamers with a channel that can fit a strand of DNA and can hydrolyze ATP. When ATP is hydrolyzed, loops on the subunits move and down which help force the double helix apart and ratchet the strand through the channel.
• What is the start site of DNA replication?

  Origin of replication (oriC) is a region of DNA with special sequences such as:

  • 4 binding sites for protein DnaA
  • AT rich tandem repeats
• Step by step process of DNA replication (part 1, separating the strands and making primers)

  • DnaA binds to the origin to initiate the pre-priming complex. DNA wraps around the DnaA proteins.
  • AT rich tandem array are pulled apart
  • SSB (single-stranded binding protein) binds to now SS AT rich region to prevent the duplex (DS structure of DNA) from reannealing.
  • DnaB is a helicase that uses ATP to unwind the rest of the DNA
  • Primase (RNA polymerase) synthesizes an RNA primer (approximately 10 nt long) in 5' to 3' direction on template
• Step by step process of DNA replication (part 2, lagging and leading strand)

  • In the replication fork one strand is synthesized continuously while the other is made in fragments that must be joined together by DNA ligase which hydrolyzes ATP to join the fragments together.
  • DNA pol III synthesizes the leading and lagging strand simultaneously
• Step by step process of DNA replication (lagging strand)

  • Lagging strand is looped out so it can still be synthesized in the same direction as the leading strand
  • As the polymerase core follows the template strand it will eventually encounter another RNA primer from a previous Okazaki fragment. This causes the sliding clamp to be released.
  • A new loop is formed and the sliding clamp is added
  • Primase adds RNA primer
  • DNA pol III makes new Okazaki fragment
  • DNA pol I uses its 5' to 3' exonuclease to remove the primer between the Okazaki fragments
  • Ligase fills gaps
• How does Ligase seal Okazaki fragments?

  Ligase joins the 3' hydroxyl group and 5' phosphate group of two fragments using ATP hydrolysis in 3 steps:

  1. Initial charging: Lysine residue of enzyme is deprotonated and attacks the alpha phosphate of ATP, releasing pyrophosphate and generating lysyl-AMP.
  2. Transfer: Adenylated ligase transfers AMP to the 5' phosphate of the nicked backbone as the phosphate attacks AMP
  3. Phosphodiester bond formation: 3' OH from nick site attacks 5' phosphate, making the phosphodiester bond and releasing AMP
• Does DNA pol III have high processivity in the lagging strand?

  No, it must release the substrate over and over and makes short Okazaki fragments
• Mutagens are chemical agents that alter DNA bases