MCB 150 90-120 Lecture Notes: Energy, Metabolism, and DNA Replication

Energy Production and ATP

  • Proton gradient is unstable, protons want to go back in.
  • Energy from glucose breakdown is used to make ATP:
    Glucose + 6O2 \rightarrow 6CO2 + 6H_2O
  • Theoretical ATP production from glucose oxidation:
    • 2 ATP from Glycolysis = 2 ATP
    • 2 NADH from Glycolysis (x 2 ATP each) = 4 ATP
    • 2 GTP (≡ ATP) from Krebs cycle = 2 ATP
    • 8 NADH from Krebs cycle (x 3 ATP each) = 24 ATP
    • 2 FADH2 from Krebs cycle (x 2 ATP each) = 4 ATP
    • Total (theoretical) ATP per Glucose molecule = 36 ATP

Announcements

  • Student hours are available for questions about performance and preparation.
  • One-on-one conversations available for checking in.
  • No lecture or student hours on Friday; lecture video from FA24 is available on Canvas.

Cellular Respiration

  • The path of carbon and hydrogen are followed in glucose to complete oxidation in the presence of O_2:
    • Glycolysis: 1 glucose to 2 pyruvate.
    • Pyruvate oxidation & Krebs Cycle: Carbons released as CO_2.
    • ETC and Chemiosmosis: Hydrogens combine with oxygen to form water.
  • The path of energy in glucose (as electrons) is followed to make ATP:
    • Some ATP generated by SLP in glycolysis and Krebs.
    • Most electrons transferred to cofactors to the ETC, creating a proton gradient that powers ATP synthase.

Anaerobic Conditions and Fermentation

  • Under anaerobic conditions:
    • Cells perform glycolysis, but no Krebs cycle or oxidative phosphorylation; ATP comes from glycolysis.
    • Pyruvate undergoes fermentation to regenerate NAD^+, which was reduced to NADH in glycolysis.
    • No additional energy is released per molecule of glucose.
    • The rate of glycolysis is increased to compensate for the loss of oxidative phosphorylation.
    • Fermentation products accumulate.

Fermentation Examples

  • Fermentation in yeast:
    • Pyruvate is converted to Acetaldehyde, then to Ethanol.
    • NADH is converted to NAD^+ + H^+.
    • CO_2 is released.
    • Enzymes involved: Pyruvate Decarboxylase and Alcohol Dehydrogenase.
  • Fermentation in muscle cells and many bacteria:
    • Pyruvate is converted to Lactic Acid (Lactate).
    • NADH is converted to NAD^+ + H^+.
    • Enzyme involved: Lactate Dehydrogenase.

Regulation of Metabolism

  • Catabolic and biosynthetic pathways are regulated and coordinated to avoid using more energy than necessary.
  • Coordination comes from:
    • Amount of enzyme
    • Activity of allosterically-regulated enzyme
  • Allosteric regulation:
    • Some enzymes have binding sites other than the active site for regulatory molecules (allosteric regulators).
    • Regulators change the conformation of the active site.
    • Increase or decrease enzyme activity.
      • Positive Regulator: increases activity
      • Negative Regulator: decreases activity
  • Feedback inhibition:
    • Allosteric regulator is a product of a later reaction in that pathway.
    • Example: Feedback inhibition of glycolysis by ATP:
      • If ATP binds to the regulatory site, the activity of PFK is decreased (negative regulation).
      • If ADP (or AMP) binds to the regulatory site, the activity of PFK is increased (positive regulation).
      • Phosphofructokinase (PFK) has allosteric binding sites.
  • Some allosteric regulators can up-regulate one reaction and down-regulate a different reaction.

Central Dogma of Molecular Biology

  • DNA encodes genetic information that directs the cell to make proteins and RNAs.
  • Information in genes does not pass directly from DNA to proteins.
  • Information in the nucleotide sequence of genes is copied into an RNA intermediate (transcription).
  • The nucleotide sequence information in RNA is used to build proteins (translation).
  • The flow of genetic information from DNA to RNA to Protein is the Central Dogma of Molecular Biology.

Discovering the Function and Structure of DNA

  • By the 1940s, hereditary material was known to reside on chromosomes.
  • Chromosomes are composed of chromatin, which is a complex of DNA and protein.
  • Proteins were known to be made of 20 amino acids, and DNA was made of 4 nucleotides.
  • It seemed logical that protein was the genetic material because of its diversity.
  • The next step was to deduce the 3D structure of DNA and figure out how it is copied and turned into protein.
  • Chargaff’s Rules (1949):
    • The amount of each dNTP varies between organisms.
    • [dA] = [dT] and [dC] = [dG] in all organisms.
  • Rosalind Franklin & Maurice Wilkins:
    • X-ray diffraction suggested a helix of two strands with a uniform width that stacks bases, with sugar-phosphate on the outside.
  • James Watson & Francis Crick:
    • Created a scale model that fit all the available data.

Forces Holding DNA Strands Together

  • Hydrogen bonds form between a purine on one strand and a pyrimidine on the other.
    • Explains uniform width and Chargaff’s rules.
    • Purine-Purine: > 2nm
    • Pyrimidine-Pyrimidine: < 2nm
    • Purine-Pyrimidine: = 2nm
  • Only G can “fit” opposite C and A opposite T for groups to be precisely positioned for H-bonds.
    • Called Watson-Crick or complementary base pairing.

DNA Structure and Information

  • Double-stranded DNA is antiparallel & complementary.
  • The information content of DNA resides in the sequence of its bases.
    • There are only 4 bases to choose from.
    • Potential for different combinations is staggering when considering the size of chromosomes.
      • If chain has 2 nucleotides, 4x4 = 16 possibilities (4^2).
      • If chain has 3 nucleotides, 4x4x4 = 64 (4^3).
      • If chain has n nucleotides, 4^n possibilities.
    • Some human chromosomes have 250 million bases - 4^{250,000,000} possibilities!

DNA Replication: Watson & Crick's Model

  • Watson & Crick’s model placed importance on complementary bases.
  • Suggested a copying mechanism.
  • Each DNA strand contains the information needed to make a new identical double helix.
  • If the parental double helix is unwound, adding complementary bases to the now single-stranded DNA chains (templates) builds two identical daughter helices.

DNA Replication: Order of Events

  1. Determine where to start
  2. Separate the strands
  3. “Prime the pump”
  4. Synthesize DNA
  5. Clean up

DNA Replication: Start Signal

  • The start signal for DNA Replication is the Origin of Replication (ori).
  • The ori is a specific sequence of bases in the DNA
  • Strands will be separated at the ori, and synthesis of new DNA will occur from both parent strands in both directions away from the ori.
  • Circular bacterial chromosomes typically have a few million base pairs and a single ori.
  • Eukaryotes must initiate replication in multiple locations to finish prior to cell division.

DNA Replication: Strand Separation and Synthesis

  • Strands are separated by Helicase enzymes, and are kept single-stranded by Single-Stranded DNA Binding Protein.
  • DNA strand synthesis:
    1. Incoming dNTP is hybridized to parental template
    2. Phosphodiester bond formed with 3’ end of chain by Polymerases
  • DNA Replication is Bidirectional:
    • New DNA needs to be synthesized on both strands on both sides of the ori
    • Synthesis only occurs in the 5’ to 3’ direction, and the new strands have to be ANTIPARALLEL to the template!
      • To solve this problem, DNA synthesis on one side of the ori begins at the ori and proceeds normally =
        • Leading strand
      • But DNA synthesis on the other side of the ori starts a short distance away from the ori and works back toward the ori
        • Lagging strand
      • Small fragments of DNA are called Okazaki fragments
      • This way, all synthesis occurs 5’→3’

DNA Replication: Priming

  • DNA polymerases cannot start a new DNA strand from scratch!
    • They absolutely require a free 3'-OH group to which to add the incoming dNTPs
  • Solution: RNA synthesizing enzymes can use a single-stranded DNA template to make an RNA strand from scratch
    • The special DNA-dependent, RNA-synthesizing enzyme used in DNA Replication is called Primase
    • Primase creates a short (5–15 nucleotide) strand of RNA opposite an ss-DNA template called a primer
    • This gives the major DNA-dependent, DNA-synthesizing enzyme (DNA polymerase III in E. coli) what it needs—a free 3'-OH group
  • Every fragment is primed
  • RNA primers must now be removed, or the genome would be littered with RNA bases:
    • RNA nucleotides removed and replaced with DNA nucleotides by DNA polymerase I
  • One last problem: Backbone of new chain has "nicks" in it where no covalent linkage exists between nucleotides
    • These "nicks" are sealed by DNA Ligase (or just Ligase)

DNA Replication: Terminology

  • "X"-dependent "Y"-synthesizing enzyme
    • "X" = what it uses as a template
    • "Y" = what it is making
  • An enzyme that degrades (hydrolyzes) a phosphodiester linkage = a nuclease
  • A nuclease that hydrolyzes nucleic acid from the end of a chain = an exonuclease
  • A nuclease that hydrolyzes nucleic acid internally (i.e., not at one end or the other) = an endonuclease
  • If an exonuclease starts at the 5' end, working toward the 3' end, it is called a 5'–3' exonuclease
  • If an exonuclease starts at the 3' end, working toward the 5' end, it is called a 3'–5' exonuclease
  • DNA polymerase I's ability to remove primers is due to its 5'–3' exonuclease activity, which is a separate enzymatic activity from its DNA synthesizing ability
    Proofreading: an example of 3'–5' exonuclease activity

DNA Organization

  • Nucleotides make up nucleic acid chain.
  • Bases pair with each other to make a double helix.
  • A very long double helix of DNA (+associated proteins) is a chromosome.
  • Chromosomes can be linear or circular.
  • How do we pack millions or billions of bp (base pairs) of DNA into such a small space?
    • In bacteria like E. coli, extended lengths of DNA can be 1,000 times the width of the cell.
    • In humans, all of the chromosomes stacked end to end would be 2 meters in length!
    • Either way, we need to compact all that chromatin into a space that's only 1–10 μm.
  • Bacterial chromosomes are supercoiled:
    • Done by topoisomerases, which nick DNA, wind or unwind, then reseal DNA
  • Relaxed versus supercoiled DNA
  • What about Eukaryotes?
    • How do we get 2 meters of DNA into a nucleus that is 5–8 μm in diameter?
  • Organization of chromatin in the nucleus:
    • Chromatin first described by R. Kornberg in 1974