Proteins

• Nucleotides


  The building blocks of nucleic acids (synonymous to amino acids being the building blocks of proteins)

  
• Nucleotide


  A nitrogenous base linked to a sugar (will always be ribose in our case) to which at least one phosphate group (mono, di or tri phosphate) is attached. Bases are derivatives of either purines or pyrimidines (heterocycles)

  
• Know how to draw nucleotides
• Nucleoside


  Take nitrogenous base and add a sugar to it (ribose)
• Nucleotide


  Take nucleoside and add a phosphate to it (phosphate is being covalently linked to the ribose)

  
• The pentose sugar is ribose
• DNA


  Contains deoxyribonucleotides: lacking the –OH group at the 2' position, just has a hydrogen it is dehydrogenated

  
• RNA


  Contains ribonucleotides: has a –OH group at the 2' position

  
• Purines


  A and G conjugated nitrogenous base with an imidazole group that pyrimidines don't have

  
• Pyrimidines


  T, C and U

  
• Purines form bonds to a pentose via N9 atoms; pyrimidines do so through their N1 atoms

  
• Primed numbers refer to the atoms of the pentose; unprimed refer to the atoms of the nitrogenous base
• Difference between T and U

  A methyl group
• Difference between C and T

  Amine vs carbonyl
• Each base is being conjugated
• Know structures of the nucleotides
• Ribose


  A 5 membered ring
• Pentose phosphate group


  Predominantly mono-, di- or tri- phosphate. Bonded at the 3' or 5' position

  
• Phosphates of polynucleotides


  Acidic so at physiological pH, nucleic acids are polyanions

  
• Bacterial can have penta – used as energy stores, or toxins
• Phosphodiester bond


  When building blocks come together it is a subsequential link from 5' to 3' that form a phosphodiester backbone (careful of where you're adding functional groups, because phosphate doesn't exist on 1', 2', or 4')

  
• Can be a single phosphate or many substituent phosphates
• Ribose is 1' to 5' ;1' (linkage site)

  
• Chargaffs rules


  Described the base composition of DNA – equal numbers of A and T; G and C
• Structure of the nucleotide ATP (adenosine 5' triphosphate)

  • Nitrogenous base, sugar linked to it at the 1' position, triphosphate substitution at the 5' position
  • ATP is a nucleotide containing adenine, ribose and a triphosphate group. It is an energy carrier or energy transfer agent

  
• Structure of dADP


  • 5' substitution of a diphosphate
• Can see many forms but all make of same base components
• Anti and syn conformations

  • The orientation of the base relative to the ribose (glycosidic bond) is defined by anti and syn notation – the rotation of nitrogenous base relative to the ribose is either going to be anti or syn
  • Syn – base is turned in a position that is directly opposite to the remainder of the pentose ring. Little bit of steric hinderance going on
  • Anti – completely opposite direction

  
• Naturally, with pyrimidines they strongly favor anti conformation because the oxygen has a lot steric clashes with 5' position of the ribose
• Purines can be either conformation but are mostly anti as well
• Chi rotational axis – one of 7 degrees of freedom of nucleic acids
• Predominant nucleotides found in nature

  • DNA: deoxyribose sugar with A, T, C and G
  • RNA: ribonucleotides with A, U, C and G

  
• Nucleic acids


  • Biopolymers of nucleotides
  • Individual units form nucleic acids. Linked extended chains get interpreted 5' to 3' because as they are being linked together because a phosphodiester backbone is being made between a 3' of an upstream ribose and the 5' of an upstream ribose
  • 5' free phosphate and a 3' free hydroxyl
  • The 3' and 5' positions of the ribose sugar of nucleotides are joined by a phosphodiester bond
  • The chain is conventionally read 5' to 3' (synonymous to reading a peptide chain N to C)
  • Instances where you can interpret 3' to 5'

  
• DNA structure


  • Dictated by sequence
  • Compromised of deoxyribonucleotides: A, G, T and C
  • DNA unlike RNA, has an equal number of A and T (A=T) and G and C (G=C)… why? Complementary base pairing between purines and pyrimidines
  • Chargaff's rule: purines and pyrimidines are 1:1 stoichiometric ratio because base pairing between purines and pyrimidines
  • DNA is a double stranded antiparallel helix of polynucleotides, known as a double helix
  • Each helix is right handed
  • Strict conformation unlike proteins
  • 20A wide, 3.4A between nucleotides, 34A rise per turn
  • Bases occupy the core of the helix; sugar-phosphates are on the periphery
  • Base pairs are planar H-bonded groups from complementary bases
  • DNA is large: ~5kb to millions of kb
  • Diffraction pattern of DNA, you get fiber diffraction (DNA is long polymers of fibers). This allows us to solve the structure of DNA
  • A-T two primary hydrogen bonds, G-C three hydrogen bonds. G-C base pair is more thermodynamically stable than A-T
  • Analyzing DNA, you have to consider that DNA is way bigger than proteins. The individual units that make it up are far larger building blocks and many more. Oligomeric state is much larger. Have to sometimes change methods of analysis – with DNA you tend to run an agarose gel, whereas with proteins you would use acrylamide. With agarose, the pore sizes are far bigger, even an high percent will have larger pore sizes than smaller pore sizes in acrylamide

  
• DNA structure


  • The DNA helix can be in the A, B or Z conformation....B-DNA is most common
  • Note the bases in A-DNA are no longer planar, they are tilted: formed by desiccation and found in the DNA pol active site
  • Z-DNA forms under high salt conditions due to stabilized phosphate groups
  • Transition from B to A or Z is not spontaneous. Very few instances we have A or Z
  • In A-DNA, see a slightly larger helix. Hydrogen bond network is happening on the axis of the helix – same direction as the backbone is moving
  • Take DNA and dry it out it will take on A-DNA structure
  • In active site of DNA polymerase there is a small stretch of B DNA that goes into a conformation of A DNA has catalysis is happening
  • Enzymes can alter structure
  • Z-DNA tends to occur under high salt because extremely tight conformation of helices brings the phosphate backbones in close proximity about 8A – this has a lot of repulsion, only way to stabilize that is to have lots of salt. Counter ions will eliminate that repulsive force. Only happens in niche instances
  • In B DNA phosphates are about 12A
  • Know what's highlighted
  • Major and minor groove that drive interactions with proteins. Provide features. Landscapes that protein can interact with. Difference between forms of DNA and influence how proteins interact, Z-DNA has an vulnafied interacting protein
  • With A and B DNA since they are so similar you have the prevalence of anti DNA and in Z DNA you have both anti and syn depending on nitrogenous base pyrimidines can take on the anti happens exclusively because carbonyl group has a lot of steric interaction with the ribose and purines can take on either anti or syn (Z-DNA purines usually take on the syn conformation – usually isn't as sterically favored when looking at A or B)
  • DNA has inherent flexibility important for biological function, such as condensation (supercoiling) or binding to proteins
  • 7 degrees of freedom
  • 6 backbone rotations and 1 chi rotation of the nitrogenous base
  • Even though there's 7 degrees of freedom, their conformational landscape is limited especially for DNA. Double stranded, anti-parallel double helix that for the most part is linear and only under the influence of enzymes will lead to something like super coiling

  
• RNA structure


  • Comprised of ribonucleotides: A, G, U and C
  • Usually single-stranded, forming complex structures of intramolecular base-pairs
  • In the single stranded form it can form on itself, form hairpins, form loop structures. The structure dictates its function.
  • RNA as itself can act as an enzyme. Primarily due to the fact they can take on such interesting conformations
  • Far more conformationally complex compared to DNA
  • RNA holds a lot more potential for activity in the cell
  • DNA is primarily an information storage
  • More sensitive to degradation – hydrolysis. Presence of 2'OH makes it susceptible to that. In a nucleic acid strand of ribonucleotides, the 2'OH can be hydrolyzed and can lead to cleavage of phosphodiester backbone
  • An RNA helix is possible and resembles A-DNA, not B-DNA due to steric clashes between 2'-OH groups
  • Inherently less stable than DNA; susceptible to hydrolysis due to the 2'-OH group

  
• Nucleic acids


  • Mainly held together by Watson-Crick base pairs, but non-canonical base pairs exist, such as Hogsten pairs
  • Watson-crick base pairs are the most stable in a double helix
  • Even though they're rigid, there is some exceptions to these rules that can lead to non-Watson and crick base pairing
  • With Hoogsteen, the anomeric conformation of the nucleotides, as the nitrogenous bases are being rotated - A-T in WC everything is anti, Hoogsteen we have a syn A and an anti T which changes the base pairing pattern
  • Structures are skewed from each other
  • In G-C we lose one of the hydrogen bonds in Hoogsteen, and the geometry is changing its elongating and being thrown out of alignment which can lead to weaker hydrogen bonding
  • H-bond drive base pair complementarity, but add little to the stability of nucleic acids
  • Stacking interactions (hydrophobic interactions) are primary stabilizing force
  • Hydrogen bonds aren't that strong of a force, it's the base pair stacking interactions that allows for the stability
  • Looking at a simple sequence, can expect one to be more stable than another
  • Note, nucleic acids with high GC content are more stable duplex than one with a low GC content; stability is sequence-dependent
  • Therefore, CCGAGCTTGG is more stable than TTACATGGAA
  • Further stability is achieved by counterions, namely cations
  • Divalent cations like Mg2+ are most effective shielding agents
  • General trend, the stacking between G and C gives greater energy – therefore greater stability
  • Pyrimidines and purines if they are stacking over one another they are not as strong as purines and purines would otherwise be

  
• Modern method analyzing base pair energies (stability) in nucleic acids

  • Made synthetic nucleic acids that one end of it is immobilized to a solid support and one end was immobilized to an agarose bead
  • Strands that come together are complementary, and being held together at their ends by base stacking
  • Using centrifugal force microscopy to look at stacking energies
  • Spun in centrifuge, and will separate after time. What's holding it together is a single stacked base, so the force that is used to separate them must be equal to the force that is holding the single stacked base together
  • A stacked base between two purines is a greater stability than a purine and pyrimidine which is greater strength than two pyrimidines
  • The reason this trend exists is because purines have more heavy atoms and looking at geometry of these two groups stacking together there is a large contribution of Lennard-Jones – plays into strength of intermolecular interaction. Greater the heavy atoms, greater that strength

  
• Thermal denaturation and renaturation (annealing of DNA)

  • Heat DNA – denatures it – two individual strands that come apart. Breaking hydrogen bonds between complementary bases and stacked bonds between neighboring bases
  • When you unfold DNA, one characteristic of DNA when you do so is an increase in its absorbance

  
• Absorbance maximum of DNA is at 260nm (protein is 280nm)