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