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
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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
Determine where to start
Separate the strands
“Prime the pump”
Synthesize DNA
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:
Incoming dNTP is hybridized to parental template
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?