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🧬 BIOCHEMISTRY - MOLECULAR (Pages 32-40)

First Aid for USMLE - Complete Lecture Notes

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PAGE 32 - CHROMATIN STRUCTURE

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What is Chromatin? (The Big Picture First)

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LINE 1: "DNA exists in the condensed, chromatin form to fit into the nucleus."

• Condensed = tightly packed, squished together
• Chromatin = the combination of DNA + special proteins (called histones) that makes it possible to pack DNA tightly
• Nucleus = the compartment inside the cell where DNA is stored

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LINE 2: "DNA loops twice around a histone octamer to form a nucleosome ('beads on a string')."

• Histone = these are special proteins that DNA wraps around. Think of them as thread spools.
• Histone octamer = a group of EIGHT histone proteins (oct = 8). Specifically: H2A, H2B, H3, H4 - two copies of each = 4 types x 2 = 8 histones total.
• Nucleosome = one unit = DNA wrapped TWICE around one histone octamer. This is the BASIC unit of DNA packing.
• "Beads on a string" = this is the classic description. Imagine a necklace with many beads (nucleosomes) connected by a string (linker DNA). That's exactly what this looks like under an electron microscope!

Non-medico analogy: Imagine you have a very long ribbon (DNA). You tie it around a spool (histone octamer) twice, then leave a bit of loose ribbon, then tie it around the next spool twice, and so on. From far away, it looks like beads on a string.

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LINE 3: "H1 binds to the nucleosome and to 'linker DNA,' thereby stabilizing the chromatin fiber."

• H1 histone = this is the LINKER histone. It is different from the H2A, H2B, H3, H4 (which are inside the bead). H1 sits on the OUTSIDE.
• Linker DNA = the stretch of DNA between two nucleosomes (the "string" between the "beads")
• Stabilizing = making the structure more tight and secure

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RIGHT SIDE OF CHROMATIN STRUCTURE:

"DNA has (-) charge from phosphate groups."

• The DNA backbone is made of phosphate groups (think of phosphate as the glue between the sugar-base units).
• Phosphate = negatively charged (-)
• This is chemically important! Opposites attract. So we need something positively charged to hold the negatively charged DNA.

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"Histones are large and have (+) charge from lysine and arginine."

• Lysine and Arginine = two amino acids (building blocks of proteins). Both carry a POSITIVE charge at normal body pH.
• The histone proteins are RICH in lysine and arginine, making them positively charged (+).
• Why? Because opposite charges attract! Negative DNA clings to positive histones. This is an electrostatic (electrical) attraction. No covalent bond needed - it's like a magnet!

Non-medico analogy: DNA is like a negatively charged wire. Histones are like positively charged cylinders. The wire naturally wraps around the cylinders due to electrical attraction.

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"DNA and histone synthesis occurs during S phase."

• S phase = the "Synthesis" phase of the cell cycle. This is when the cell is copying (duplicating) its DNA before dividing.
• Since you need MORE DNA and MORE histones together (to pack the new DNA), the cell makes both at the SAME TIME during S phase. Makes sense!

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"Mitochondria have their own DNA, which is circular and does not bind histones."

• Mitochondria = the "powerhouse" of the cell. But here's a surprise - they have their OWN separate DNA!
• Circular DNA = mitochondrial DNA is circular (like bacterial DNA), NOT linear like nuclear DNA.
• Does NOT bind histones = mitochondrial DNA is "naked" - not wrapped around histones. This is one piece of evidence that mitochondria were once ancient bacteria that got incorporated into our cells (the Endosymbiotic Theory).

Exam Tip: Mitochondrial DNA = circular + no histones + maternally inherited (passed from mother to child only through the egg).

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HETEROCHROMATIN vs EUCHROMATIN

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HETEROCHROMATIN:

"Condensed, appears darker on EM (labeled H in image; Nu = nucleolus). Sterically inaccessible, thus transcriptionally inactive. ↑ methylation, ↓ acetylation."

• Heterochromatin = "Hetero" means DIFFERENT or in this context - TIGHTLY PACKED chromatin.
• Condensed = it is very tightly coiled and packed. Dense. Compact.
• Darker on EM (Electron Microscopy) = under the electron microscope, the dark blobs are heterochromatin because the dense packing scatters electrons more.
• Sterically inaccessible = "steric" refers to physical space. The machinery that reads DNA (RNA polymerase) physically CANNOT reach the DNA because it is too tightly packed. Like trying to read a book that's rolled up into a tight cylinder.
• Transcriptionally inactive = this region CANNOT be transcribed (read) into RNA. Genes in heterochromatin are SILENCED (turned off).
• ↑ methylation = more methyl groups are added (we'll discuss this soon)
• ↓ acetylation = fewer acetyl groups (we'll discuss this soon)

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"Barr bodies (inactive X chromosomes) may be visible on the periphery of nucleus."

• Barr body = a visible dark spot seen near the edge of the nucleus in female cells.
• Why? Females have TWO X chromosomes (XX). But only ONE is needed. The other X chromosome gets permanently silenced (turned into heterochromatin) in each cell - this is called X-inactivation or Lyonization (named after Mary Lyon).
• This inactivated X chromosome condenses into a Barr body - it's visible under a light microscope!
• The number of Barr bodies = number of X chromosomes - 1. So a normal female (XX) has 1 Barr body. A normal male (XY) has 0 Barr bodies.

Clinical use: Checking Barr bodies was a historical way to determine chromosomal sex. Now replaced by genetic testing.

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EUCHROMATIN:

"Less condensed, appears lighter on EM (labeled E in image). Transcriptionally active, sterically accessible."

• Euchromatin = "Eu" in Greek means TRUE or GOOD. This is the "good" chromatin that is actively working.
• Less condensed = loosely packed, open, relaxed structure.
• Appears lighter on EM = lighter under electron microscope because it's loosely packed.
• Transcriptionally active = RNA polymerase CAN access the DNA and DOES transcribe it into RNA. Genes here are ACTIVE (turned on).
• Sterically accessible = there is physical space for the transcription machinery to bind.

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DNA METHYLATION

"Reversibly changes the expression of a DNA segment without changing its sequence. Involved in aging, carcinogenesis, epigenetics, genomic imprinting, transposable element repression, and X chromosome inactivation (Lyonization)."

• DNA methylation = adding a small chemical group called a methyl group (-CH3) directly onto a base in the DNA.
• "Reversibly changes expression" = it's not a permanent mutation. The DNA sequence (the order of A, T, G, C) is NOT changed. But the GENE is turned off. This is the basis of epigenetics (changes in gene expression without changes in DNA sequence).

• Aging = methylation patterns change as we age
• Carcinogenesis = cancer cells often have abnormal methylation (wrong genes turned off)
• Genomic imprinting = in some genes, only the mom's copy or only the dad's copy is expressed - determined by methylation marks
• Transposable element repression = "jumping genes" (sequences that can move around the genome) are silenced by methylation
• X chromosome inactivation (Lyonization) = methylation is one mechanism used to silence one X chromosome in females

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"DNA is methylated in imprinting. Methylation within gene promoter (CpG islands) typically represses (silences) gene transcription."

• Imprinting = a phenomenon where a gene is expressed from ONLY ONE parent's copy (either mom's or dad's), never both. Methylation marks which copy to silence.
• CpG islands = regions of DNA where "C" (cytosine) is followed by "G" (guanine) in the sequence. These are hotspots for methylation.
• Promoter = the "start button" region of a gene - where RNA polymerase binds to begin transcription. If the promoter is methylated, the start button is jammed - the gene can't be turned on.

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"Dysregulated DNA methylation is implicated in fragile X syndrome."

• Fragile X syndrome = the most common inherited cause of intellectual disability. A region at the tip of the X chromosome gets abnormally methylated and silenced, turning off the FMR1 gene.

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HISTONE METHYLATION

"Usually causes reversible transcriptional suppression, but can also cause activation depending on location of methyl groups."

• Histone methylation = instead of methylating the DNA itself, you add methyl groups to the HISTONE proteins (specifically to the amino acids lysine or arginine in the histone "tail").
• Effect is variable - it depends on WHICH amino acid gets methylated and HOW MANY methyl groups are added:
  • Methylation of certain positions (e.g., H3K27) = SILENCING (turns gene off)
  • Methylation of other positions (e.g., H3K4) = ACTIVATION (turns gene on)

"Lysine and arginine residues of histones can be methylated."

• These are the positively charged amino acids in histones. Adding methyl groups changes their charge properties and how tightly they interact with DNA, thereby altering gene expression.

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HISTONE ACETYLATION

"Removal of histone's (+) charge → relaxed DNA coiling → ↑ transcription."

• Acetylation = adding an acetyl group (-COCH3) to lysine residues on the histone.
• Lysine is normally positively charged (+). The acetyl group NEUTRALIZES that positive charge.
• If the histone loses its (+) charge, it can no longer hold the negatively charged DNA as tightly.
• The DNA relaxes - becomes less coiled, more open.
• Open DNA = accessible DNA = RNA polymerase can get in = MORE transcription (gene turned ON)

Non-medico analogy: Imagine histone is a fist gripping a rope (DNA). Acetylation makes the fist "unclench" (neutralizes the grip). Now the rope hangs loose and you can read along it.

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HISTONE DEACETYLATION

"Removal of acetyl groups → tightened DNA coiling → ↓ transcription."

• Deacetylation = REMOVING the acetyl group from histones.
• Now the lysine's positive charge comes BACK (+).
• The histone grips the DNA tightly again.
• DNA coils up tightly → RNA polymerase cannot access → LESS transcription (gene turned OFF)

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PAGE 33 - NUCLEOTIDES

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What are Nucleotides? (The building blocks of DNA and RNA)

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"Nucleoside = base + (deoxy)ribose (sugar)"

• A nucleoside = one base + one sugar. That's it.
• Base = the nitrogen-containing ring structure (we'll see the types soon - A, T, G, C, U)
• (Deoxy)ribose = a 5-carbon sugar. If it has an OH group at the 2' position, it's ribose (used in RNA). If that OH is replaced by just H (hydrogen), it's DEOXYribose (used in DNA - hence "de-oxy" = without oxygen).

Non-medico analogy: A nucleoside = a car body without wheels (just the base + sugar, no phosphate yet).

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"Nucleotide = base + (deoxy)ribose + phosphate; linked by 3'-5' phosphodiester bond."

• A nucleotide = base + sugar + phosphate. It has all 3 components.
• The phosphate group is attached at the 5' carbon of the sugar.
• When nucleotides join together to form a DNA strand, they connect via a phosphodiester bond - the phosphate of one nucleotide connects to the 3' carbon of the previous sugar. This is why we call it a 3'-5' phosphodiester bond.

Non-medico analogy: A nucleotide = a complete car (body + wheels + engine). The 3'-5' bond is how you connect multiple cars in a train.

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"Nucleo-'tri'-des have THREE components."

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"5' end of incoming nucleotide bears the triphosphate (energy source for the bond)."

• When a new nucleotide is added during DNA synthesis, it arrives as a triphosphate (three phosphate groups attached).
• Example: dATP (deoxyadenosine triphosphate - the A used in DNA synthesis).
• When it gets incorporated, two of the three phosphate groups are RELEASED as pyrophosphate (PPi), releasing energy that drives the reaction forward.

Non-medico analogy: The three phosphates are like a compressed spring. When two are released, the energy snaps the new nucleotide into place.

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PURINES vs PYRIMIDINES

"Purines (A, G) - 2 rings"

• Purines = Adenine (A) and Guanine (G)
• They have a DOUBLE RING structure (a 6-membered ring fused to a 5-membered ring)
• Mnemonic: PURE As Gold (Purines = A and G)
• Another mnemonic: "Pure water comes from a Mountain spring" → Pure = contains A and G

Non-medico analogy: Purines are "bigger" molecules (2 rings), like a double-decker bus.

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"Pyrimidines (C, U, T) - 1 ring"

• Pyrimidines = Cytosine (C), Uracil (U), Thymine (T)
• They have a SINGLE RING structure (just one 6-membered ring)
• Mnemonic: CUT the PYRamid (CUT = C, U, T are pyrimidines; PYRamid = PYRimidine)
• Smaller molecules.

Non-medico analogy: Pyrimidines are like single-decker buses (1 ring).

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"Thymine has a methyl."

• Thymine is unique among pyrimidines because it has a methyl group (-CH3) attached to its ring.
• Uracil is basically thymine WITHOUT this methyl group.
• Thymine is only in DNA. Uracil is only in RNA. Why? Uracil can appear in DNA by accident (through deamination of cytosine). The cell recognizes it as wrong and repairs it. If uracil were normal in DNA, the repair system wouldn't know which ones are mistakes. By using THYMINE (methylated uracil) in DNA, the cell can tell: "Uracil in DNA = always a mistake, always repair it."

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DEAMINATION REACTIONS:

• Deamination = removing an amino group (-NH2) from a base.
• This happens spontaneously in cells, especially in the presence of heat, acid, or certain chemicals.

• Cytosine → Uracil: Cytosine loses its amino group and becomes uracil. This is the most common deamination. Since uracil doesn't belong in DNA, the cell repairs it. (If unrepaired → C replaced by U → next replication puts A opposite U → permanent C:G to T:A mutation)
• Adenine → Hypoxanthine: Adenine deaminates to a base called hypoxanthine (which pairs with C instead of T, causing mutations)
• Guanine → Xanthine: Guanine deaminates to xanthine (xanthine pairs correctly with C, so this is less mutagenic)
• 5-methylcytosine → Thymine: This one is tricky! Methylated cytosine (5-methylcytosine) when deaminated produces THYMINE (a normal DNA base). The repair system can't easily recognize thymine as wrong in a C:G context. This is why CpG sites are mutation hotspots - the methylated C frequently deaminates to T.

"Uracil found in RNA; Thymine in DNA. Methylation of uracil makes thymine."

• RNA uses URACIL instead of thymine.
• DNA uses THYMINE instead of uracil.
• If you add a methyl group to uracil, you get thymine. So thymine = methylated uracil.

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HYDROGEN BONDS BETWEEN BASE PAIRS:

"C-G bond (3 H bonds) stronger than A-T bond (2 H bonds). ↑ C-G content → ↑ melting temperature of DNA."

• Bases pair across the two DNA strands via hydrogen bonds (weak individual bonds, but powerful in large numbers).
• A-T pair = connected by 2 hydrogen bonds
• C-G pair = connected by 3 hydrogen bonds
• More C-G = more hydrogen bonds = you need MORE heat to separate the strands = higher melting temperature

Non-medico analogy: A-T is like velcro with 2 hooks. C-G is like velcro with 3 hooks. Obviously 3 hooks hold more tightly. High C-G content = the DNA is harder to "unzip."

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Amino acids necessary for PURINE synthesis:

"cats purr until they GAC: Glycine, Aspartate, Glutamine"

• Purines are built de novo (from scratch) using several amino acids as raw materials.
• Glycine = donates carbon and nitrogen to the purine ring
• Aspartate = donates nitrogen
• Glutamine = donates nitrogen
• Also used: CO2, Formyl-THF (from folate), PRPP (a ribose phosphate)

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PAGE 34 - DE NOVO PYRIMIDINE AND PURINE SYNTHESIS

"Various immunosuppressive, antineoplastic, and antibiotic drugs function by interfering with nucleotide synthesis."

• Immunosuppressive = suppresses the immune system (e.g., for organ transplant patients)
• Antineoplastic = anti-cancer (neoplastic = tumor)
• Antibiotic = kills bacteria
• All these drugs work by blocking the MAKING of nucleotides, thereby preventing DNA synthesis, thereby stopping rapidly dividing cells (cancer cells, bacteria, immune cells).

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PYRIMIDINE SYNTHESIS DRUGS:

"Leflunomide: inhibits dihydroorotate dehydrogenase"

• Leflunomide = a drug used in Rheumatoid Arthritis (an autoimmune joint disease)
• It blocks dihydroorotate dehydrogenase - an enzyme in the de novo pyrimidine synthesis pathway
• By blocking pyrimidine synthesis → lymphocytes (immune cells) can't proliferate → immune suppression

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"5-fluorouracil (5-FU) and its prodrug capecitabine: form 5-F-dUMP, which inhibits thymidylate synthase (↓ dTMP)"

• 5-fluorouracil (5-FU) = a chemotherapy (anti-cancer) drug
• Capecitabine = the "prodrug" - taken by mouth, converted to 5-FU inside the body (prodrug = inactive form that becomes active after metabolism)
• Once activated, they form 5-F-dUMP which inhibits thymidylate synthase
• Thymidylate synthase = the enzyme that makes dTMP (deoxythymidine monophosphate - the T used in DNA)
• No dTMP = no thymine = DNA synthesis stops = cancer cell dies

Think of it as: These drugs jam the factory that makes the "T" building block of DNA.

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PURINE SYNTHESIS DRUGS:

"6-mercaptopurine (6-MP) and its prodrug azathioprine: inhibit de novo purine synthesis (guanine phosphoribosyltransferase); azathioprine is metabolized via purine degradation pathway and can lead to immunosuppression when administered with xanthine oxidase inhibitor"

• 6-MP = used in leukemia (blood cancer) and inflammatory bowel disease
• Azathioprine = used as an immunosuppressant (transplant rejection, autoimmune diseases)
• Both block HGPRT (hypoxanthine-guanine phosphoribosyltransferase) - an enzyme that recycles purines
• Important drug interaction: Azathioprine is broken down by xanthine oxidase. If you give allopurinol (a xanthine oxidase inhibitor used for gout), azathioprine levels rise dangerously high!

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"Mycophenolate and ribavirin: inhibit inosine monophosphate dehydrogenase"

• Mycophenolate = powerful immunosuppressant used in organ transplantation
• Ribavirin = antiviral drug
• Both inhibit IMP dehydrogenase - an enzyme needed to make GMP (guanosine monophosphate) from IMP

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PURINE AND PYRIMIDINE SYNTHESIS (Both):

"Hydroxyurea: inhibits ribonucleotide reductase"

• Hydroxyurea = used in sickle cell disease AND some cancers
• Ribonucleotide reductase = converts ribonucleotides (used in RNA) to DEOXYribonucleotides (used in DNA)
• Without this enzyme, cells can't make DNA building blocks = DNA synthesis stops

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"Methotrexate (MTX), trimethoprim (TMP), and pyrimethamine: inhibit dihydrofolate reductase (↓ dTMP) in humans (methotrexate), bacteria (trimethoprim), and protozoa (pyrimethamine)"

• Dihydrofolate reductase (DHFR) = the enzyme that converts dihydrofolate → tetrahydrofolate (the active form of folate/folic acid)
• Tetrahydrofolate is needed to donate carbons in nucleotide synthesis (specifically for making dTMP and purines)
• Without active folate → can't make nucleotides → can't make DNA → cells die

Non-medico analogy: Same lock (DHFR), different keys for different organisms. Each drug is a "key" shaped to fit the specific organism's version of the enzyme.

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"CPS1 = mItochondria, urea cycle, found in liver. CPS2 = cYtosol, pyrimidine synthesis, found in most cells."

• CPS = Carbamoyl Phosphate Synthetase - an enzyme that makes carbamoyl phosphate (an important molecule)
• CPS1 = works in the MItochondria, part of the UREA CYCLE (which detoxifies ammonia), found mainly in LIVER
  • Mnemonic: CPS1 = mItochondria
• CPS2 = works in the CYtosol (the fluid part of the cell), part of PYRIMIDINE synthesis, found in MOST cells
  • Mnemonic: CPS2 = cYtosol

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PAGE 35 - PURINE SALVAGE DEFICIENCIES

What is Purine Salvage?

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ADENOSINE DEAMINASE (ADA) DEFICIENCY

"ADA is required for degradation of adenosine and deoxyadenosine. ↓ ADA → ↑ dATP → ↓ ribonucleotide reductase activity → ↓ DNA precursors in cells → ↓ lymphocytes."

1. ADA = the enzyme that breaks down adenosine and deoxyadenosine
2. When ADA is missing (deficiency), adenosine and deoxyadenosine ACCUMULATE
3. These accumulate and get converted to dATP (deoxyadenosine triphosphate)
4. High dATP inhibits ribonucleotide reductase (the enzyme that makes ALL DNA building blocks)
5. Without ribonucleotide reductase activity, cells can't make DNA precursors
6. No DNA precursors = cells can't divide = lymphocytes (immune cells) die
7. No lymphocytes = no immune system

"One of the major causes of autosomal recessive SCID."

• SCID = Severe Combined Immunodeficiency Disease
• "Severe" = very serious
• "Combined" = both T cells and B cells (both arms of immunity) are affected
• "Immunodeficiency" = the immune system doesn't work
• SCID is the "Bubble Boy" disease - children with SCID must live in completely sterile environments because even minor infections are fatal

ADA deficiency is the most famous genetic cause of SCID. These children have no functional immune system because their lymphocytes self-destruct due to toxic buildup of dATP.

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LESCH-NYHAN SYNDROME

"Defective purine salvage. Deficient or mutated HGPRT → ↓ GMP (from guanine) and ↓ IMP (from hypoxanthine) formation. Compensatory ↑ in purine synthesis (↑ PRPP amidotransferase activity) → excess uric acid production. X-linked recessive."

1. HGPRT is absent or mutated
2. Can't salvage (recycle) hypoxanthine or guanine
3. These free bases can't be reused → they get DEGRADED all the way to uric acid
4. Also, because the salvage pathway is blocked, PRPP (the raw material for de novo synthesis) ACCUMULATES
5. More PRPP → more de novo purine synthesis → even MORE uric acid production
6. Result: MASSIVELY elevated uric acid (hyperuricemia)

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Clinical Features: "Intellectual disability, self-mutilation, aggression, hyperuricemia (red/orange 'sand' in diaper), gout, dystonia, macrocytosis."

• Intellectual disability = brain development is impaired because uric acid is neurotoxic (toxic to brain cells)
• Self-mutilation = these children bite their own fingers, lips, shoulders. This is pathognomonic (specific to this disease). Nobody fully understands why, but it's characteristic.
• Aggression = behavioral/psychiatric manifestation
• Hyperuricemia = high uric acid in blood
• Red/orange "sand" in diaper = uric acid crystals in urine that look like orange-red sand in the baby's diaper. Classically described.
• Gout = uric acid deposits in joints causing painful inflammation (because uric acid forms crystals in joints)
• Dystonia = involuntary muscle contractions causing twisting movements (neurological)
• Macrocytosis = enlarged red blood cells (seen in some cases)

• Both drugs reduce uric acid production (allopurinol and febuxostat both inhibit xanthine oxidase, the enzyme that makes uric acid)

• Hyperuricemia
• Gout
• Pissed off (aggression, self-mutilation)
• Red/orange crystals in urine
• Tense muscles (dystonia)

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GENETIC CODE FEATURES

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"Unambiguous - Each codon specifies only 1 amino acid."

• Codon = a set of 3 nucleotide bases in mRNA that codes for ONE specific amino acid
• Unambiguous = there is NO ambiguity. Each codon has one and only ONE meaning. For example, the codon AUG always means Methionine (and also "start here"). Never two different amino acids.

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"Degenerate/Redundant - Most amino acids are coded by multiple codons."

• There are 64 possible codons (4 bases x 4 bases x 4 bases = 4³ = 64 combinations)
• But there are only 20 amino acids
• So mathematically, most amino acids must be coded by MORE THAN ONE codon
• Example: Leucine is coded by 6 different codons. Alanine by 4. Methionine by only 1.
• This is called degeneracy or redundancy

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"Wobble hypothesis - first 2 nucleotides of codon are essential for anticodon recognition while the 3rd nucleotide can differ ('wobble')."

• When the tRNA (transfer RNA, the adaptor molecule) recognizes an mRNA codon, it's the FIRST TWO positions that must match perfectly.
• The 3rd position is flexible - it can "wobble" (mismatch slightly) and still work.
• This is why many synonymous codons differ only in the 3rd position (e.g., GCU, GCC, GCA, GCG all code for Alanine - the first two bases GC are fixed, but the 3rd wobbles).

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"Commaless, nonoverlapping - Read from a fixed starting point as a continuous sequence of bases."

• Commaless = no punctuation marks between codons. The ribosome reads AUG GUU CAA CGU... continuously without stopping between codons.
• Nonoverlapping = each nucleotide belongs to only ONE codon. (In contrast, in an overlapping code, one base could be in two codons - the genetic code is NOT like that.)
• Fixed starting point = reading always starts from the AUG start codon and reads in one direction (5' to 3') without jumping.
• Exceptions: some viruses use overlapping reading frames as a way to pack more genetic information into a small genome.

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"Universal - Genetic code is conserved throughout evolution."

• The SAME codons code for the SAME amino acids in bacteria, fungi, plants, and humans. The code is essentially IDENTICAL across all life forms.
• This is powerful evidence that all life shares a common ancestor.

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PAGE 36 - DNA REPLICATION

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"Occurs in 5' → 3' direction ('5ynth3sis') in continuous and discontinuous (Okazaki fragment) fashion. Semiconservative."

• 5' → 3' direction = DNA polymerase can only ADD new nucleotides to the 3' end of a growing chain. It reads the template strand 3' to 5', but builds the new strand 5' to 3'. Always.
  • Mnemonic: "5ynth3sis" - the 5 and 3 tell you the direction of synthesis.
• Continuous vs Discontinuous (Okazaki fragments):
  • The two strands of DNA run in OPPOSITE directions (antiparallel).
  • When the replication fork moves, one template strand runs 3' to 5' (so the new strand can be made 5' to 3' continuously) - this is the LEADING STRAND.
  • The other template strand runs 5' to 3' in the direction of fork movement. DNA polymerase can only go 5' to 3', so it has to synthesize in the OPPOSITE direction of fork movement, in short chunks - these are Okazaki fragments - this is the LAGGING STRAND.
• Semiconservative = when DNA replicates, each new double helix contains ONE original (parental) strand and ONE newly synthesized strand. Half of the original DNA is "conserved" in each daughter molecule.

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THE REPLICATION MACHINERY (Each enzyme explained):

ORIGIN OF REPLICATION:

"Particular consensus sequence in genome where DNA replication begins. May be single (prokaryotes) or multiple (eukaryotes)."

• Origin of replication = the specific spot on the DNA where replication STARTS
• Prokaryotes (bacteria) = have ONE origin of replication (simple circular chromosome)
• Eukaryotes (humans, animals) = have MULTIPLE origins of replication (because the genome is much larger - you need many starting points to replicate it in a reasonable time)
• AT-rich sequences (TATA box regions) are commonly found at origins of replication because A-T pairs (only 2 H-bonds) are easier to separate/unwind than C-G pairs.

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REPLICATION FORK:

"Y-shaped region along DNA template where leading and lagging strands are synthesized."

• As replication proceeds, the two strands of DNA are pulled apart at a specific point, creating a Y-shaped structure visible under electron microscopy.
• The "arms" of the Y = the two template strands being read
• The "stem" of the Y = the double-stranded DNA that hasn't been replicated yet

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HELICASE:

"Unwinds DNA template at replication fork."

• Helicase = the enzyme that UNZIPS the double helix. It breaks the hydrogen bonds between the two strands, separating them so each can be used as a template.
• Mnemonic: Helicase Halves DNA (it splits the double helix in two)
• Bloom syndrome = a genetic disease caused by mutation in the BLM gene (encodes a DNA helicase). Patients have short stature, sun-sensitive rashes, and increased cancer risk.

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SINGLE-STRANDED BINDING PROTEINS (SSBPs):

"Prevent strands from reannealing or degradation by nucleases."

• After helicase unwinds the DNA, the separated single strands want to come back together (re-anneal) or get chewed up by enzymes (nucleases).
• SSBPs = proteins that coat the single-stranded DNA and keep it stretched out and protected.
• They don't catalyze anything - they just stabilize.

Non-medico analogy: Imagine unzipping a zipper, then putting little clips along each side so the zipper teeth can't zip back together. SSBPs are those clips.

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DNA TOPOISOMERASES:

"Creates a single- (topoisomerase I) or double- (topoisomerase II) stranded break in the helix to add or remove supercoils."

• The problem: As helicase unwinds DNA at the fork, the DNA ahead of the fork gets OVERWOUND (like twisting a rope). These tangles are called supercoils. They must be removed or replication stops.
• Topoisomerases = enzymes that manage the topology (3D shape/coiling) of DNA.
  • Topo I = cuts ONE strand, lets it swivel, then re-seals. Relieves supercoils without using ATP.
  • Topo II = cuts BOTH strands, passes another segment through, re-seals. Uses ATP. Can change the linking number of DNA.

• In Eukaryotes/Humans:
  • Irinotecan/topotecan → inhibit Topo I (cancer drugs)
  • Etoposide/teniposide → inhibit Topo II (cancer drugs)
• In Prokaryotes/Bacteria:
  • Fluoroquinolones (ciprofloxacin, levofloxacin) → inhibit Topo II (called DNA gyrase in bacteria) and Topo IV (antibiotic)

Why do fluoroquinolones only kill bacteria? Because bacterial topo II (DNA gyrase) is structurally different from human topo II. The drug fits the bacterial enzyme but NOT the human one - selective toxicity!

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PRIMASE:

"Makes RNA primer for DNA polymerase III to initiate replication."

• The problem: DNA polymerase CANNOT start a new DNA chain from scratch. It can only ADD to an existing chain. So how does it start?
• Primase = an RNA polymerase that makes a short RNA strand (RNA primer, about 10 nucleotides) to give DNA polymerase a starting point.
• RNA primer = a short "landing pad" with a free 3'-OH end that DNA polymerase can extend from.

Non-medico analogy: DNA polymerase is like a copy machine that needs a page already in the tray to start. The primase puts that starting page (RNA primer) in the tray.

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DNA POLYMERASE III (Prokaryotes only):

"Prokaryotes only. Elongates leading strand by adding deoxynucleotides to the 3' end. Elongates lagging strand until it reaches primer of preceding fragment."

• DNA Pol III = the main workhorse DNA polymerase in bacteria.
• It extends the chain by adding new dNTPs (deoxynucleotide triphosphates) to the 3' end of the growing strand.
• It does the heavy lifting - synthesizes most of the new DNA.

"DNA polymerase III has 5'→3' synthesis and proofreads with 3'→5' exonuclease."

• 5'→3' synthesis = builds DNA in the 5' to 3' direction
• 3'→5' exonuclease = proofreading activity! If the wrong base is added, the enzyme backs up (3' to 5') and removes it, then continues. This is how DNA replication has such high accuracy.

"Drugs blocking DNA replication often have a modified 3' OH, thereby preventing addition of the next nucleotide ('chain termination')."

• Many antiviral and anticancer drugs are nucleoside analogs (fake building blocks).
• They get incorporated into the growing DNA chain.
• But they have a MODIFIED (or missing) 3'-OH group.
• Without a free 3'-OH, no more nucleotides can be added = chain termination = DNA synthesis stops = virus can't replicate.
• Examples: AZT (used in HIV treatment), acyclovir (used in herpes).

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DNA POLYMERASE I (Prokaryotes only):

"Prokaryotes only. Degrades RNA primer; replaces it with DNA."

• After DNA Pol III has extended all the Okazaki fragments, each fragment is separated by an RNA primer at the 5' end.
• DNA Pol I comes along with its 5'→3' exonuclease activity - it chews away the RNA primer while simultaneously replacing it with proper DNA.

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DNA LIGASE:

"Catalyzes the formation of a phosphodiester bond within a strand of double-stranded DNA."

• After DNA Pol I fills in the gaps left by RNA primers, there are still nicks (breaks in the sugar-phosphate backbone) between adjacent DNA segments.
• DNA Ligase = the enzyme that seals these nicks by forming the final phosphodiester bond.
• It joins the Okazaki fragments together on the lagging strand.

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TELOMERASE:

"Eukaryotes only. A reverse transcriptase (RNA-dependent DNA polymerase) that adds DNA (TTAGGG) to 3' ends of chromosomes to avoid loss of genetic material with every duplication."

• The end-replication problem: Every time DNA replicates, the very END of the chromosome (the 3' overhang) cannot be fully replicated because there is no place to put a primer. So chromosomes shorten a little with each division.
• Telomeres = repetitive DNA sequences (TTAGGG repeated many times) at the ends of chromosomes. They serve as protective "buffer zones" - it's okay if some of these repetitive sequences are lost, rather than losing actual genes.
• Telomerase = the enzyme that ADDS BACK these TTAGGG repeats to the chromosome ends, preventing shortening.
• Telomerase is a reverse transcriptase = it carries its own RNA template (containing the sequence AAUCCC) and uses it to build the DNA sequence TTAGGG. Normally, RNA → DNA is reverse transcription (used by viruses like HIV). Telomerase uses the same trick.
• Telomerase is active in:
  • Progenitor/stem cells (needed because they divide a lot)
  • Cancer cells (highly upregulated - allows cancer cells to divide indefinitely = immortality)
  • Downregulated in aging/normal somatic cells

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PAGE 37 - DNA REPAIR

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DOUBLE-STRAND BREAKS:

NONHOMOLOGOUS END JOINING:

"Brings together 2 ends of DNA fragments to repair double-stranded breaks. Homology not required. Part of the DNA may be lost or translocated."

• Double-strand break (DSB) = BOTH strands of the DNA double helix are cut. Very dangerous.
• Nonhomologous end joining (NHEJ) = the cell just grabs the two broken ends and glues them back together - no template needed.
• Downside: It's error-prone. DNA may be lost at the junction, or pieces from different chromosomes may be incorrectly joined (translocations).
• Occurs throughout the cell cycle (no need for a sister chromatid to be available).

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HOMOLOGOUS RECOMBINATION:

"Requires 2 homologous DNA duplexes. A strand from damaged dsDNA is repaired using a complementary strand from intact homologous dsDNA as a template. Defective in breast/ovarian cancers with BRCA1 or BRCA2 mutations and in certain types of Fanconi anemia. Restores duplexes accurately without loss of nucleotides."

• Homologous recombination (HR) = uses an IDENTICAL (or nearly identical) sequence from the sister chromatid or homologous chromosome as a template to repair the break accurately.
• Much more accurate than NHEJ - no information is lost.
• Requires the cell to be in S or G2 phase (when a sister chromatid is present to use as template).

• BRCA1 and BRCA2 = genes that encode proteins essential for homologous recombination.
• When these genes are mutated → HR is defective → DSBs are repaired inaccurately by NHEJ → genome instability → mutations accumulate → cancer.
• BRCA1 or BRCA2 mutation = dramatically increased risk of breast cancer and ovarian cancer.
• This is why Angelina Jolie (who has BRCA1 mutation) had prophylactic mastectomy.

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SINGLE-STRAND DNA REPAIR:

NUCLEOTIDE EXCISION REPAIR (NER):

"Specific endonucleases remove the oligonucleotides containing damaged bases; DNA polymerase and ligase fill and reseal the gap, respectively. Repairs bulky helix-distorting lesions (e.g., pyrimidine dimers)."

• This is used for BULKY DNA damage - damage that physically distorts the double helix shape.
• The classic example: Pyrimidine dimers = UV radiation from sunlight causes two adjacent thymine (or cytosine) bases on the SAME strand to be covalently linked together, forming a "dimer." This distorts the helix.

1. Specific enzymes recognize the distorted helix
2. Endonucleases cut out a ~12-27 nucleotide segment containing the damage
3. DNA polymerase fills in the gap using the other strand as template
4. DNA Ligase seals the nick

"Occurs in G1 phase of cell cycle."

"Defective in Xeroderma Pigmentosum."

• Xeroderma Pigmentosum (XP) = a genetic disease where NER is defective.
• These patients CANNOT repair UV damage.
• Clinical features: Extreme sun sensitivity, dry skin (xeroderma = dry skin), photosensitivity, and very high rates of skin cancer even in childhood from minimal sun exposure.

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BASE EXCISION REPAIR (BER):

"Base-specific Glycosylase removes altered base and creates AP (apurinic/apyrimidinic) site. AP-Endonuclease cleaves 5' end, removing one or more nucleotides. AP-Lyase cleaves 3' end. DNA Polymerase-β fills the gap. DNA Ligase seals it."

• BER is used for SMALL, chemically altered bases (not big distortions, just chemically changed single bases).
• Classic example: Deamination of cytosine → uracil (as we discussed on page 33).

1. DNA Glycosylase - recognizes the abnormal base and snips it out, leaving an "AP site" (a spot with no base - like a gap in a word where a letter was removed)
2. AP-Endonuclease - cuts the backbone at the 5' side of the AP site
3. AP-Lyase - cuts the backbone at the 3' side
4. DNA Polymerase-β - fills in the gap with the correct nucleotide
5. DNA Ligase - seals the nick

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MISMATCH REPAIR:

"Mismatched nucleotides in newly synthesized strand are removed and gap is filled and resealed."

• During DNA replication, DNA polymerase occasionally incorporates the WRONG base (e.g., puts A opposite C).
• Mismatch repair = finds and fixes these errors in the newly synthesized strand.
• The system identifies WHICH strand is new (because the new strand is not yet methylated) and corrects it.

"Occurs predominantly in S phase of cell cycle."

"Defective in Lynch syndrome (hereditary nonpolyposis colorectal cancer [HNPCC])."

• Lynch syndrome (HNPCC) = a hereditary cancer syndrome where mismatch repair genes (MLH1, MSH2, etc.) are mutated.
• Without mismatch repair → errors accumulate → colorectal cancer, endometrial cancer, and others at young ages.

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PAGE 38 - MUTATIONS IN DNA

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"Degree of change: silent << missense < nonsense < frameshift."

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SINGLE NUCLEOTIDE SUBSTITUTIONS (Point Mutations):

TRANSITION vs TRANSVERSION:

"Transition - purine to purine (e.g., A to G) or pyrimidine to pyrimidine (e.g., C to T)."

• Transition = switching within the same class (purine stays purine, or pyrimidine stays pyrimidine)
• Examples: A→G, G→A (both purines), C→T, T→C (both pyrimidines)
• More common in nature

"Transversion - purine to pyrimidine (e.g., A to T) or pyrimidine to purine (e.g., C to G)."

• Transversion = switching between classes (purine → pyrimidine, or vice versa)
• Examples: A→T, G→C, C→A, T→G
• Less common but can be more mutagenic

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SILENT MUTATION:

"Codes for same (synonymous) amino acid; often involves 3rd position of codon (tRNA wobble)."

• Silent mutation = the DNA changes, but the PROTEIN doesn't change at all.
• How? Because of codon degeneracy! Remember, most amino acids have multiple codons. So a base change (especially at the 3rd position - due to wobble) often produces a codon that still codes for the SAME amino acid.
• Example: GAG → GAA = both code for Glutamic acid. No change in protein.

Non-medico analogy: You change one letter in a word, but the meaning stays the same: "colour" → "color" - still means the same thing.

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MISSENSE MUTATION:

"Results in changed amino acid (called conservative if new amino acid has similar chemical structure). Examples: sickle cell disease (substitution of glutamic acid with valine)."

• Missense mutation = one base change → WRONG amino acid incorporated into the protein.
• The protein may still work (if the new amino acid is chemically similar = conservative missense) or may be dysfunctional.

• In the β-globin chain of hemoglobin, position 6: Glutamic acid (charged, hydrophilic) is replaced by Valine (nonpolar, hydrophobic)
• This is because of a single A→T transversion in the codon: GAG → GTG (at DNA level)
• Result: The hemoglobin molecule clumps together under low-oxygen conditions → red blood cells become sickle-shaped → they block small blood vessels → pain crises, organ damage

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NONSENSE MUTATION:

"Results in early stop codon (UGA, UAA, UAG). Usually generates nonfunctional protein. Stop the nonsense!"

• Nonsense mutation = a base change converts a normal amino acid codon into a STOP codon.
• Stop codons = UGA, UAA, UAG (in mRNA). These signal "STOP making protein here."
• If a stop codon appears early (before the protein is complete), you get a truncated (shortened) protein that usually doesn't work.

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FRAMESHIFT MUTATION:

"Deletion or insertion of any number of nucleotides not divisible by 3 (or if divisible by 3, split across adjacent codons) → misreading of all nucleotides downstream. Protein may be shorter or longer, and its function may be disrupted or altered."

• The genetic code is read in groups of 3 (codons). The reading frame is like a train track - if you remove or add track segments that aren't a multiple of 3, the whole track gets shifted.
• Insertion or deletion of 1 or 2 nucleotides shifts the reading frame → every single codon downstream is now WRONG → catastrophic effect on protein

• Duchenne Muscular Dystrophy = frameshift mutations in the dystrophin gene → no functional dystrophin protein → progressive muscle wasting
• Tay-Sachs disease = frameshift in the HEXA gene
• Cystic fibrosis = deletion of 3 nucleotides (ΔF508, which is divisible by 3, so technically not a frameshift, but still very serious)

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SPLICE SITE MUTATION:

"Retained intron in mRNA → protein with impaired or altered function. Examples: rare causes of cancers, dementia, epilepsy, some types of β-thalassemia, Gaucher disease, Marfan syndrome."

• Exons = the coding parts of a gene (get included in final mRNA)
• Introns = the non-coding parts of a gene (get removed/spliced out during RNA processing)
• Splice site = the specific DNA sequence (GT...AG at the boundaries) that tells the splicing machinery where to cut and join.
• Splice site mutation = mutates the GT or AG boundary sequence → the splicing machinery can't find the correct cut site → the intron is NOT removed (retained) and stays in the mRNA.
• The intron contains random stop codons or frameshifts → abnormal protein

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SLIPPED STRAND MISPAIRING:

"Occurs when DNA polymerase slips and either inserts or removes one or more additional nucleotides by mistake in an area of repetitive nucleotides. Anticipation occurs secondary to insertion of increased repeats across generations. Example: CAG repeat expansion in Huntington disease."

• In areas of DNA where the same sequence repeats many times (e.g., CAGCAGCAGCAG...), DNA polymerase can "slip" and loop out some repeats, either:
  • Deleting some repeats (making the region shorter), or
  • Inserting extra repeats (making the region longer)
• Trinucleotide repeat expansion = if repeats keep getting LONGER with each generation, this can cause disease.

• Normal people have ~10-35 CAG repeats in the Huntingtin gene
• Huntington patients have >36 CAG repeats
• More repeats = earlier onset, more severe disease
• The CAG repeat encodes glutamine → too many glutamines = toxic protein that kills neurons in the brain → movement disorder, dementia, death

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PAGE 39 - LAC OPERON & FUNCTIONAL ORGANIZATION OF EUKARYOTIC GENE

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THE LAC OPERON

"Classic example of a genetic response to an environmental change. Glucose is the preferred metabolic substrate in E. coli, but when glucose is absent and lactose is available, the lac operon is activated to switch to lactose metabolism."

• E. coli = the common gut bacterium (Escherichia coli). It is the "model organism" for studying gene regulation.
• E. coli's priority: Use glucose first (the easiest energy source). Only switch to lactose when glucose runs out AND lactose is available. This is economical - don't waste energy making enzymes unless needed.
• The Lac operon = a set of three genes (LacZ, LacY, LacA) that work together to metabolize lactose. They are all regulated together as a unit (this is what an "operon" is - a group of genes under shared control).

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Mechanism of switching on:

"Low glucose → ↑ adenylate cyclase activity → ↑ generation of cAMP from ATP → activation of catabolite activator protein (CAP) → ↑ transcription."

1. Low glucose = the cell is starving for its preferred fuel
2. ↑ Adenylate cyclase = this enzyme is activated when glucose is low
3. Adenylate cyclase converts ATP → cAMP (cyclic AMP - a signaling molecule)
4. cAMP activates CAP (Catabolite Activator Protein, also called CRP - cAMP Receptor Protein)
5. CAP-cAMP complex binds to the CAP site on the lac operon DNA (upstream of the promoter)
6. CAP-cAMP enhances RNA polymerase binding → ↑ transcription of lacZ, lacY, lacA genes

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"High lactose → unbinds repressor protein from repressor/operator site → ↑ transcription."

• When lactose is present, it is converted to allolactose (an isomer)
• Allolactose acts as an inducer - it binds to the Lac repressor protein
• When the repressor has allolactose bound, it CHANGES SHAPE and can no longer grip the operator region of the DNA
• The repressor falls off the operator
• RNA polymerase can now transcribe the lac genes → lactose-metabolizing enzymes are made

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FUNCTIONAL ORGANIZATION OF A EUKARYOTIC GENE

1. Enhancer/Silencer (far upstream or even within introns)
2. Promoter (CAAT box + TATA box region)
3. 5' UTR (Untranslated Region)
4. Exon 1 - GT - Intron 1 - AG - Exon 2 - GT - Intron 2 - AG - Exon N
5. AATAAA (polyadenylation signal)
6. 3' UTR
7. Silencer (can be far downstream)

• RNA polymerase reads the DNA and makes a long RNA copy called heterogeneous nuclear RNA (hnRNA) or pre-mRNA
• Contains BOTH exons AND introns

• The introns are cut out
• Note the GT...AG rule: Every intron starts with GU (GT in DNA) and ends with AG. This is how the splicing machinery recognizes where introns begin and end.

• After splicing, the mature mRNA has:
  • 5' Cap = 7-methylguanosine cap added to the 5' end (cotranscriptionally, while being made)
  • AUG start codon = where translation begins
  • Protein coding region = sequence of codons
  • Stop codon = where translation ends
  • AAUAAA = polyadenylation signal
  • Poly-A tail = ~200 adenosines added to the 3' end (posttranscriptionally, after making)

• The mature mRNA leaves the nucleus and goes to the cytosol
• Ribosomes bind and translate it into protein

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PAGE 40 - REGULATION OF GENE EXPRESSION, RNA PROCESSING, RNA POLYMERASES

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REGULATION OF GENE EXPRESSION

PROMOTER:

"Site where RNA polymerase II and multiple other transcription factors bind to DNA upstream of gene locus (AT-rich upstream sequence with TATA and CAAT boxes, which differ between eukaryotes and prokaryotes)."

• Promoter = the "start button" of a gene. Located UPSTREAM (5' side) of the gene.
• TATA box = an AT-rich sequence (~25-30 bp before the transcription start site). Basal transcription factors bind here to recruit RNA polymerase II.
• CAAT box = another AT-rich sequence (~80 bp upstream). Increases efficiency of transcription.
• Mutations in the promoter → dramatically reduce transcription. The "start button" is broken.

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ENHANCER:

"DNA locus where regulatory proteins ('activators') bind, increasing expression of a gene on the same chromosome."

• Enhancer = a DNA region that, when bound by activator proteins, increases transcription of its target gene.
• Key point: Enhancers can be located VERY FAR from the gene they regulate - thousands of base pairs away, upstream OR downstream, or even within an intron of another gene!
• The DNA loops around so the enhancer region is brought close to the promoter.

Non-medico analogy: The enhancer is like a remote booster for a radio station. It doesn't have to be next to the transmitter to strengthen the signal.

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SILENCER:

"DNA locus where regulatory proteins ('repressors') bind, decreasing expression of a gene on the same chromosome."

• Silencer = opposite of enhancer. Repressor proteins bind here and decrease transcription.
• Can also be located far from the gene.

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EPIGENETICS:

"Changes made to gene expression (heritable mitotically/meiotically) without a change in underlying DNA sequence."

• Epigenetics = "above genetics" - changes to gene expression that:
  1. Do NOT involve changing the DNA sequence (no mutations)
  2. Can be passed on to daughter cells (heritable)
• Primary mechanisms:
  • DNA methylation (discussed earlier - silences genes)
  • Histone modification (acetylation, methylation - discussed earlier)
  • Noncoding RNA (microRNA, long noncoding RNA - regulate gene expression post-transcriptionally)

Analogy: Epigenetics is like adding sticky notes to a book without changing the actual text. The notes say "skip this chapter" or "read this chapter twice," but the book itself is unchanged. Yet those notes can be copied when the book is photocopied.

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RNA PROCESSING (Eukaryotes)

"Initial transcript is called heterogeneous nuclear RNA (hnRNA). hnRNA is then modified and becomes mRNA."

• hnRNA (pre-mRNA) = the raw, unprocessed RNA transcript directly from DNA. Long, containing introns.

The following modifications occur in the nucleus:

"Capping of 5' end (addition of 7-methylguanosine cap; cotranscriptional)"

• A 7-methylguanosine (7-MeG) cap is added to the 5' end of the pre-mRNA while it's still being transcribed.
• Functions of 5' cap:
  1. Protects the mRNA from degradation by exonucleases
  2. Required for recognition by ribosomes (needed to start translation)
  3. Helps in nuclear export of mRNA

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"Polyadenylation of 3' end (~200 As → poly-A tail; posttranscriptional)"

• After transcription, an enzyme adds ~200 adenosine (A) nucleotides to the 3' end = Poly-A tail
• Functions of Poly-A tail:
  1. Protects mRNA from degradation
  2. Facilitates nuclear export
  3. Helps in translation efficiency
  4. The longer the poly-A tail, the more stable the mRNA
• Signal for polyadenylation: The sequence AAUAAA (in the mRNA) = polyadenylation signal. The poly-A polymerase finds this signal and adds the A's after it.

"Poly-A polymerase does not require a template. Mutations in polyadenylation signal → early degradation prior to translation."

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"Splicing out of introns (posttranscriptional)"

• Introns are removed after transcription.
• Done by a complex molecular machine called the SPLICEOSOME (made of small nuclear RNAs = snRNAs).
• GT...AG rule: Introns start with GU and end with AG (in mRNA terms).

"Capped, tailed, and spliced transcript is called mRNA."

"mRNA is transported out of nucleus to be translated in cytosol."

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"mRNA quality control occurs at cytoplasmic processing bodies (P-bodies), which contain exonucleases, decapping enzymes, and microRNAs; mRNAs may be degraded or stored in P-bodies for future translation."

• P-bodies = membrane-less organelles in the cytoplasm where mRNA quality is checked.
• If mRNA is defective or no longer needed, it gets degraded here.
• If mRNA needs to be "stored" temporarily (e.g., during stress), it can be parked in P-bodies.

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"Kozak sequence - initiation site in most eukaryotic mRNA. Facilitates binding of small subunit of ribosome to mRNA."

• Kozak sequence = a short consensus sequence surrounding the AUG start codon in eukaryotic mRNA.
• Example: (GCC)ACCAUGG (the AUG is the start codon)
• The ribosome scans the mRNA from the 5' cap and recognizes the AUG within this Kozak context to begin translation.
• Mutations in the Kozak sequence → ribosome can't recognize the start site → ↓ translation → ↓ protein production.

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RNA POLYMERASES

"No proofreading function, but can initiate chains. RNA polymerase II opens DNA at promoter site."

• Unlike DNA polymerases, RNA polymerases have NO proofreading ability. They make more errors.
• However, unlike DNA polymerases, RNA polymerases can START a new chain without needing a primer!

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EUKARYOTIC RNA POLYMERASES (3 types):

"RNA polymerase I makes rRNA, the most common (rampant) type; present only in nucleolus."

• RNA Pol I = makes rRNA (ribosomal RNA)
• rRNA = the most abundantly made RNA (makes up ~80% of total cellular RNA)
• This makes sense - ribosomes are needed in huge quantities for protein synthesis
• Location: Only in the nucleolus (a specific area inside the nucleus)
• Mnemonic: "rampant rRNA" - RNA Pol I makes the most abundant RNA

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"RNA polymerase II makes mRNA (massive), microRNA (miRNA), and small nuclear RNA (snRNA)."

• RNA Pol II = the most important one for gene expression! Makes:
  • mRNA (messenger RNA) = encodes protein sequences (the "instruction manual" for making proteins)
  • miRNA (microRNA) = small RNAs that regulate gene expression post-transcriptionally
  • snRNA (small nuclear RNA) = components of the spliceosome (used for splicing introns)
• Mnemonic: RNA Pol II makes Massive mRNA, miRNA, snRNA

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"RNA polymerase III makes 5S rRNA, tRNA (tiny)."

• RNA Pol III = makes small, functional RNAs:
  • 5S rRNA = a component of the large ribosomal subunit
  • tRNA (transfer RNA) = the adaptor molecules that carry amino acids to the ribosome during translation
• Mnemonic: RNA Pol III makes tiny tRNA

• I = rRNA (component of ribosome structure)
• II = mRNA (the message being translated)
• III = tRNA (brings amino acids to the ribosome)

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"α-amanitin, found in Amanita phalloides (death cap mushrooms), inhibits RNA polymerase II. Causes dysentery and severe hepatotoxicity if ingested."

• Amanita phalloides = the "death cap mushroom" - one of the most poisonous mushrooms in the world.
• Its toxin α-amanitin specifically poisons RNA Pol II → mRNA production stops → protein synthesis fails → severe liver damage (hepatotoxicity) and death.
• No antidote. Liver transplant is sometimes the only option.

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"Dactinomycin inhibits RNA polymerase in both prokaryotes and eukaryotes."

• Dactinomycin (Actinomycin D) = an antibiotic and chemotherapy agent
• Intercalates (inserts itself) between DNA base pairs → blocks the movement of RNA polymerase → stops transcription in ALL organisms (both bacteria and human cells)
• Used as a chemotherapy drug for certain cancers (Wilms tumor, Ewing sarcoma)

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PROKARYOTIC RNA POLYMERASES:

"1 RNA polymerase (multisubunit complex) makes all 3 kinds of RNA."

• Bacteria are simpler - one RNA polymerase makes rRNA, mRNA, AND tRNA
• It is a multisubunit complex (core enzyme: 2α + β + β') + sigma factor (σ)
• The sigma factor is what allows the bacterial RNA polymerase to recognize promoter sequences.

"Rifamycins (rifampin, rifabutin) inhibit DNA-dependent RNA polymerase in prokaryotes."

• Rifampin/Rifabutin = antibiotics that specifically block bacterial RNA polymerase
• They don't inhibit human RNA polymerase (selectivity!)
• Clinical use: Treatment of tuberculosis (TB) - rifampin is the backbone of TB treatment
• Also used for meningitis prophylaxis (Neisseria meningitidis and Haemophilus influenzae contacts)

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SUMMARY TABLE (Your Quick Reference)

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