BI203 exam 2

Nuclear sub-compartments

(1) Replication and transcription factories (2) Nucleolus

Oxidative phosphorylation

- 10 NADH and 2 FADH2 --> 34 ATP - Two general steps: (1) establish an electrochemical gradient across inner membrane; (2) couple energy stored in gradient to synthesis of ATP - Electrochemical gradient established is of protons (H+) --> ~10-fold higher concentration in intermembrane space - The phospholipid bilayer is impermeable to ions, so protons can cross the membrane only through protein channels - Electrochemical gradient across inner membrane establishes pH gradient and voltage difference - pH 7 of intermembrane space, pH 8 for matrix - Gradient = stored energy

Vesicle docking and fusion

- A Rab protein on the vesicle membrane binds to a tethering factor associated with the target membrane - This is followed by the formation of complexes between SNAREs on the vesicle and target membranes - The coiled-coil domains of the SNAREs zip together, bringing the vesicle and target membranes into close proximity --> this destabilizes the membranes, and the membranes fuse

Importins

- A family of receptor proteins that bind NLS and transport proteins into the nucleus - Importins work in conjunction with the GTP-binding protein Ran, which controls directionality of movement - Importins bind NLS, then interact with cytoplasmic filaments of nuclear pore complex, which directs transport of the importin/cargo complex into nucleus - Many importins and exportins are members of a family of nuclear transport receptors called karyopherins --> karyopherins have specific substrates that they recognize and transport

How do cells couple the flow of protons down the electrochemical gradient to the synthesis of ATP?

- ATP synthase couples the flow of protons down gradient to synthesis of ATP - ATP synthase is complex V - ATP synthase consists of two multisubunit components, F0 and F1, which are linked by a slender stalk - F0 spans the lipid bilayer, forming a spinning channel through which protons cross the membrane --> spinning helps drive protons to matrix - One subunit of F1 also spins and harvests the free energy derived from proton movement down the electrochemical gradient by catalyzing the synthesis of ATP from ADP + Pi - Some parts of ATP synthase come from mitochondrial genome, other parts from nuclear genome - ATP synthesis requires continuous transport of ATP out of matrix and ADP + Pi into matrix

Signal hypothesis

- Amino acid sequences at the N-terminus of a growing polypeptide chain target the ribosome to the ER --> "signal sequence" - Known: (1) translation of secreted proteins takes place on ER-bound ribosomes - Known: (2) the protein was transferred across the membrane during synthesis - Known (3): secreted protein had 20 amino acids missing at its N-terminus compared to protein translated in vitro on free ribosomes - The signal is removed when the growing polypeptide chain enters the ER

Smooth ER and synthesis of phospholipids

- Because they are hydrophobic, membrane lipids are synthesized in association with already existing membranes rather than the aqueous cytosol - Most lipids are synthesized in smooth ER - Glycerol phospholipids are synthesized in the ER membrane from cytosolic precursors - Two fatty acids linked to co-enzyme A (CoA) carriers joined to glycerol-3-phosphate, yielding phosphatidic acid (PA), which is inserted in to the membrane --> a phosphatase then converts PA to diacylglycerol - Different polar head groups can then be attached to diacylglycerol to generate a variety of phospholipids - Synthesis of phospholipids on the cytosol side allows the hydrophobic fatty acid chains to remain buried in the membrane - New phospholipids are added only to the cytosolic half of the ER membrane --> some must be transferred to the other half

Transport by coated vesicles

- COPII-coated vesicles carry cargo from the ER to the Golgi, and clathrin-coated vesicles carry cargo outward from the trans-Golgi network - Clathrin-coated vesicles also carry cargo back from the plasma membrane to endosomes and other organelles such as the trans-Golgi network and lysosomes - COPI-coated vesicles retrieve ER-resident proteins from the ERGIC and cis Golgi and carry Golgi-resident enzymes back from the trans Golgi to earlier Golgi cisternae

Internal organization within the nucleus: chromosomes and higher-order chromatin structure

- Chromatin becomes highly condensed during mitosis to form the compact metaphase chromosomes - During interphase, most of the chromatin decondenses and is distributed throughout the nucleus - Chromosomes and chromatin are not randomly distributed throughout the nucleus in interphase cells - Individual chromosomes occupy distinct territories --> organized such that transcriptional activity of a gene is correlated with its position - DNA replication and transcription takes place in clustered regions within the nucleus - This organization was first suggested in 1885 and confirmed in 1984 by studies of polytene chromosomes in Drosophila salivary glands --> each chromosome was found to occupy a discrete region of the nucleus, called a chromosome territory - In situ hybridization with fluorescent probes specific for repeated sequences on individual chromosomes has been used to visualize the location of chromosomes within a nucleus - Centromeres share a common location within nucleus of interphase cells (so do telomeres) --> this is probably because these centromeric and telomeric locations require a certain amount of processing from other proteins or interactions with other proteins, so it will help the cell to have those proteins (ex: telomerase) in a centralized location - In interphase cells, the euchromatin is decondensed and transcriptionally-active, and is distributed throughout the nucleus - Heterochromatin is highly condensed and not transcribed, and is located to the periphery of the nucleus, where it anchors chromatin to the nuclear lamina called lamina-associated domains (LADs) - The genes within LADs are generally transcriptionally repressed - LADs correspond to heterochromatin - The nucleolus is also surrounded by heterochromatin (called nucleolus-associated domains or NADS) - DNA sequences found in NADs substantially overlap with those in LADs and are very likely to be associated with repetitive sequences

How do cells maintain the electrochemical gradient for oxidative phosphorylation?

- Energy from NADH and FADH2 - Machinery is the electron transport chain --> ETC couples released energy to pump H+ against their gradient - NADH --> NAD+ + H+ + 2e- - 2 e- + 1/2 O2 + 2 H+ --> H2O

Topology of the secretory pathway

- Ex: TM receptor protein synthesized in ER - The lumens of the ER and Golgi apparatus are topologically equivalent to the exterior of the cell - Consequently, those portions of polypeptide chains that are translocated into the ER are exposed on the cell surface following transport to the plasma membrane - Protein incorporated into membrane of vesicle as it moves from ER to Golgi - After moving through Golgi, protein is packaged into a vesicle again - Vesicle moves from Golgi to plasma membrane

Gaucher disease

- Gaucher disease is caused by deficiency of glucocerebrosidase, which catalyzes hydrolysis of glucosylceramide to glucose and ceramide - In the most common form of the disease, macrophages are the only cells affected - Their function is to eliminate aged and damaged cells by phagocytosis

Overview of glucose catabolism

- Glycolysis (cytosol): glucose --> pyruvate - Glycolysis yields net gain of 2 ATP and 2 NADH - Citric acid cycle (mitochondria): pyruvate --> acetyl CoA - CAC yields net gain of 2 ATP, 8 NADH, and 2 FADH2 - Oxidative phosphorylation (mitochondria) turns 10 NADH and 2 FADH2 into 34 ATP - Most of the energy from glucose catabolism is generated in mitochondria

How does the Ran cycle influence nuclear transport?

- High Ran/GDP levels in the cytoplasm (low Ran/GTP) - High Ran/GTP levels in nucleus (low Ran/GDP) - Importins bind NLS and transport cargo protein through nuclear pore complex - Ran/GDP does not come in with the imported proteins --> Ran/GDP is imported into the nucleus through its own transport complex formed with NTF2 and exportin (complex dissociates once in nucleus) - In the nucleus, Ran GTP exchange factor (Ran GEF) exchanges GDP for GTP --> this is not a phosphorylation event - Ran GEF is only located in the nucleus, which is why there is a high concentration of Ran/GTP in nucleus - Ran/GTP binds to importin and dislodges the cargo protein --> Ran/GTP-importin complex is transported back to cytoplasm - In the cytoplasm, Ran GTPase activating protein (Ran GAP) on fibrils hydrolyzes the GTP on Ran to GDP, releasing the importin - Ran GAP is only in the cytosol --> this is why Ran/GDP is high in cytosol - The Ran/GDP formed in the cytoplasm is then transported back to the nucleus by its own import receptor, where Ran/GTP is regenerated

Unfolded protein response (UPR)

- If an excess of unfolded proteins accumulates, a signaling pathway called the unfolded protein response (UPR) is activated - It leads to expansion of the ER and production of more chaperones - If protein folding can't be adjusted to a normal level, the cell undergoes programmed cell death - Unfolded proteins activate 3 receptors in the ER membrane: IRE1, ATF6, and PERK - IRE1 cleaves pre-mRNA of a TF (XBP1) --> XBP1 translocates to nucleus and stimulates transcription of UPR genes - ATF6 is cleaved to release the active ATF6 transcription factor - TFs from IRE1 and ATF 6 lead to increase chaperone synthesis and increased lipid synthesis enzymes (for expansion of ER) and increased ERAD proteins - . PERK is a protein kinase that phosphorylates translation factor eIF2, which inhibits general translation and reduces the amount of protein entering the ER - All 3 activate transcription of UPR target genes

Regulation of nuclear transport: NF-𝜅B

- In one mechanism of regulation of nuclear transport, TFs or other proteins associate with cytoplasmic proteins that mask their NLS, and so they remain in cytoplasm - TF NF-𝜅B is regulated by protein-protein interactions with I𝜅B --> complexed with I𝜅B in cytoplasm - In absence of appropriate stimuli, protein-protein interactions between NF-𝜅B and I𝜅B mask the NLS of NF-𝜅B - Extracellular stimuli causes phosphorylation of I𝜅B, which (1) targets I𝜅B for degradation and (2) releases NF-𝜅B (exposing its NLS) - If I𝜅B is phosphorylated and degraded by ubiquitin-mediated proteolysis, NF-𝜅B can enter the nucleus and activate transcription of its target genes

Orientations of membrane proteins

- Integral membrane proteins span the membrane via α-helical regions of 20-25 hydrophobic amino acids, which can be inserted in a variety of orientations - Some proteins will curl around so N terminus ends up in cytosol - Other proteins just have N terminus go into ER lumen - Proteins that span the membrane multiple times are inserted by an alternating series of internal signal sequences and TM stop-transfer sequences

Insertion of membrane proteins via internal transmembrane sequences

- Internal transmembrane sequences can lead to the insertion of polypeptide chains in either orientation in the ER membrane - The transmembrane sequence directs insertion of the polypeptide such that its amino (N) terminus is exposed on the cytosolic side - The transmembrane sequence exits the translocon to anchor the protein in the lipid bilayer and the remainder of the polypeptide chain is translocated into the ER as translation proceeds - Other internal transmembrane sequences are oriented to direct the transfer of the amino-terminal portion of the polypeptide across the membrane - Continued translation results in a protein that spans the ER membrane with its amino terminus in the lumen and its carboxy (C) terminus in the cytosol

Lysosomes

- Lysosomes: membrane-enclosed organelles that contain enzymes to break down all types of biological polymers - They are the digestive system of the cell - They can vary in size and shape depending on the materials that have been taken up for digestion - Lysosomal function is dependent on an internal pH of 5 --> lysosomal proton pumps maintain acidic pH by pumping protons into lysosome - Most lysosomal enzymes are acid hydrolases—active at pH 5 in lysosomes, but not in the cytoplasm (pH 7.2) --> this prevents uncontrolled digestion of cell contents if the lysosome membrane breaks down - Lysosomes contain about 60 different degradative enzymes - Mutations in genes that encode these enzymes result in lysosomal storage diseases—undegraded material accumulates in the lysosomes of affected individuals - General degradation of cell constituents & particles taken up by the cell - Lysosomes digest material taken up from outside the cell by endocytosis - Lysosomes are formed when transport vesicles from the trans-Golgi network fuse with a late endosome - Endosomes represent an intersection between the secretory pathway and the endocytic pathway - Lysosomes degrade cellular materials (proteins, organelles) by autophagy - Autophagy: turnover of the cell's own components --> important in embryonic development and programmed cell death - During autophagy, a small area of cytoplasm or organelle is enclosed in a vesicle (autophagosome), which fuses with a lysosome, and its contents are digested - Lysosomes degrade extracellular materials taken up by phagocytosis

Formation and fusion of a transport vesicle

- Membrane proteins and lumenal secretory proteins with their receptors are collected into selected regions of a donor membrane where the formation of a cytosolic coat results in the budding of a transport vesicle - The vesicle is transported by motor proteins along cytoskeletal filaments to its target - The transport vesicle then docks at its target membrane, the coat is removed, and the vesicle fuses with its target

Mitochondrial genome

- Mitochondria contain their own genetic system - Mitochondrial genomes are usually circular DNA molecules, present in multiple copies - Most encode only a few proteins that are essential for oxidative phosphorylation --> 13 proteins (of oxidative phosphorylation), 2 rRNAs (16S and 12S), 22 tRNAs (all components necessary for mitochondrial translation) - Mitochondrial genomes vary in size between different species - The mitochondrial genetic code is different from the universal code --> U in the tRNA anticodon can pair with any of the bases in the third codon position of mRNA, thus four codons are recognized by a single tRNA - Some codons specify different amino acids in mitochondria than in the universal code - Mitochondrial proteome: 1000-1500 different proteins, 99% encoded by nuclear genome

Nucleolus

- No membrane, but can be defined by heterochromatin - Site of rRNA synthesis, rRNA processing, and assembly of ribosomal subunits - Actively growing mammal cells have 5 to 10 million ribosomes that must be synthesized each time the cell divides - Nucleolus contains ~200 copies of the rRNA gene encoding 18S, 5.8S, and 28S rRNA - ~2000 copies of the 5S rRNA gene are located outside nucleolus - The 5.8S, 18S, and 28S rRNAs are transcribed as a single unit in the nucleolus by RNA polymerase I, yielding a 45S ribosomal precursor RNA - Transcription of the 5S rRNA takes place outside the nucleolus and is catalyzed by RNA polymerase III - Following each cell division, nucleoli become associated with the nucleolar organizing regions that contain the 5.8S, 18S, and 28S rRNA genes - Transcription of 45S pre-rRNA leads to fusion of small prenucleolar bodies - In most cells, the initially separate nucleoli then fuse to form a single nucleolus - Formation of ribosomes requires assembly of pre-rRNA with ribosomal proteins and 5S rRNA - Ribosomal proteins are produced in cytoplasm and imported to nucleolus, where they assemble with pre-rRNA prior to cleavage - Pre-ribosomal particles are then exported to cytoplasm, yielding 40S and 60S ribosomal subunits

How are proteins destined for the nucleus identified?

- Nuclear proteins contain nuclear localization signals - Common NLS characteristics: rich in basic AAs (lysine and arginine) - These signals are recognized by nuclear transport receptors - Mutation blocked localization of T antigen to nucleus --> transfer of T antigen NLS to cytosolic proteins resulted in their nuclear localization - NLSs can be bipartite --> composed of AA sequences separated by non-NLS amino acids - T antigen NLS is a single stretch of AAs

Protein disulfide isomerase (PDI) and peptidyl prolyl isomerase

- PDI and peptidyl prolyl isomerase act as chaperones by catalyzing protein folding - PDI catalyzes disulfide bond formation - PDI is abundant in the ER, where an oxidizing environment allows (S-S) linkages - Peptidyl prolyl isomerase catalyzes isomerization of peptide bonds that involve proline residues - Isomerization between the cis and trans configurations of prolyl-peptide bonds could otherwise be a rate-limiting step in protein folding - Proline is a ring AA, so it's less likely to fold properly - Trans conformation of proline is ideal

RNA polymerase

- Principle enzyme responsible for RNA synthesis - Like DNA polymerase, RNA pol reads DNA 3' to 5' and synthesizes RNA in the 5' to 3' direction - Unlike DNA pol, RNA pol can initiate de novo

Chromatin at promoters and enhancers

- Promoters and enhancers are devoid of nucleosomes, leaving their DNA available to transcription factors --> these regions can be digested with DNase (DNase hypersensitive sites) - Monomethylation and trimethylation of lysine happens in specific places --> can be used as a marker of a specific genomic location (i.e., promoter or enhancer) - The nucleosomes flanking promoters are marked by trimethylated H3 lysine 4 (H3K4me3) --> allows promoters to be free of histones, available for transcription complex - The nucleosomes flanking enhancers are marked by the monomethylated form of H3 lysine 4 (H3K4me1) --> enhancers free of histones, available for TFs - Could identify promoter or enhancer sequences using antibodies specific for these modifications

Glycoprotein folding by chaperones and ERAD

- Protein folding in the ER is slow and inefficient, and many are misfolded - They are rapidly degraded by the ER-associated degradation (ERAD) process: misfolded proteins are identified, returned to cytosol, and degraded by ubiquitin-proteasome system - As the glycoprotein exits the translocon, chaperones bind and assist in folding - A chaperone has a folding sensor that checks for proper folding by monitoring exposed hydrophobic regions - If the protein is correctly folded (no exposed hydrophobic regions), it proceeds to exit the ER - However, if too many hydrophobic regions are exposed, indicating improper folding, the protein is targeted back to the cytosol through a ubiquitin ligase complex in the ER membrane - The protein is ubiquitylated at the cytosolic side of this complex and degraded in the proteasome - If an excess of unfolded proteins accumulates, a signaling pathway called the unfolded protein response (UPR) is activated - UPR leads to expansion of ER and production of more chaperones - If protein folding can't be adjusted to a normal level, cell undergoes programmed cell death

Nuclear export

- Ran also controls nuclear export of proteins via regulation of exportin trafficking - RNAs are transported to the cytoplasm as ribonucleoprotein complexes (RNPs) - Karyopherin exportins transport tRNAs, rRNAs, and miRNAs - Proteins destined for nuclear export contain a nuclear export signal (NES) - NESs are recognized and bound by exportins - Binding of Ran/GTP stabilizes the complex and transports it out of the nucleus - In the cytoplasm, GTP hydrolysis and release of Ran/GDP leads to dissociation of cargo protein --> conversion of Ran/GTP to Ran/GDP by Ran GAP releases cargo protein - Ran/GDP is transported back to the nucleus by its own transport receptor termed NTF2

Regulation of nuclear transport

- Regulation of protein transport is a mechanism for controlling protein activity in the nucleus - Regulation of import and export of transcription factors is a way of controlling gene expression - Two examples of regulated gene expression through controlled access of TFs to nucleus: (1) NF-𝜅B --> protein-protein interactions mask the NLS from importins; (2) Pho4 --> phosphorylation blocks NLS-importin interactions

Mechanism of telomerase action

- The mechanism was determined in 1985 in studies of the protozoan Tetrahymena - Telomeric DNA naturally has 3' overhang - Telomere 3' overhang binds to telomerase RNA via complementary base pairing --> template is longer than overhang - Telomerase uses reverse transcriptase activity to make DNA to extend the 3' overhang based on the RNA template - Primase will make an RNA template complementary to the elongated 3' overhang and polymerase will fill in the DNA - RNA primer is removed, leading to another 3' overhang - DNA on new strand is now one repeat unit longer than it was before

Structure of the nuclear envelope

- The nuclear envelope separates the nuclear contents from the cytoplasm - It controls traffic of proteins and RNAs through nuclear pore complexes, and plays a critical role in regulating gene expression - The nuclear envelope consists of two nuclear membranes, an underlying nuclear lamina, and nuclear pore complexes

Colon cancer and DNA repair

- There are two described forms of inherited colon cancer: familial adenomatous polyposis (extremely rare) and hereditary nonpolyposis colorectal cancer/HNPCC (more common) - Mutations in the human homologs of MutS and MutL (MSH and MLH) cause inherited colon cancer (HNPCC), one of the most common inherited diseases - Defects in these genes result in a high frequency of mutations in other cell genes, and the likelihood that some will eventually lead to the development of cancer by affecting genes that regulate cell proliferation - A key characteristic of colon cancer is that it develops gradually over several years - The initial stage of colon cancer development is the outgrowth of small benign polyps, which eventually become malignant and invade the surrounding connective tissue

Principles of transport across the nuclear envelope

- These principles apply to localization of proteins to subcellular locations, including mitochondria, ER, and the nucleus - (1) Proteins destined for a particular subcellular location will contain a common AA sequence "tag" --> for nuclear proteins = nuclear localization signal (NLS) - (2) That tag will be recognized and bound by a transporter protein --> for nuclear proteins = importins - (3) That transporter protein will mediate transport of its cargo to destined location

Translocation of phospholipids across the ER membrane

- This requires passage of polar head groups through the membrane, facilitated by membrane proteins called flippases - This ensures even growth of both sides of the phospholipid bilayer

Endocytosis and lysosome formation

- Three types of endosomes: early endosomes, recycling endosomes, late endosomes - Early endosomes fuse with endocytic vesicles from the plasma membrane - They separate molecules for recycling from molecules destined for degradation in lysosomes - Molecules to be recycled are passed to recycling endosomes and back to the plasma membrane

Regulation of transcriptional elongation

- Transcription is initiated following phosphorylation of the RNA polymerase II C-terminal domain (CTD) at serine 5 by TFIIH - Factors involved in the initial stages of mRNA processing associate with the phosphorylated CTD - During transcription, the activity of RNA pol II can be paused by negative regulatory factors NELF and DSIF - Continuation of transcription (productive elongation) results from the phosphorylation of NELF, DSIF, and serine 2 of the polymerase CTD by P-TEFb - Phosphorylated NELF dissociates from the complex and is replaced by P-TEFb, and additional factors required for elongation and processing associate with the polymerase

Binding of translational repressors to 5' UTR sequence: regulation of Ferritin translation in response to iron levels

- Translational repressors inhibit translation by binding to 5' or 3' UTR sequences - An example of this is the regulation of Ferritin translation - Ferritin is synthesized for iron storage - When iron is absent, iron regulatory protein (IRP) binds to the iron response element (IRE) in the 5' UTR, blocking translation - When adequate iron is present, translation will proceed

All cells arise from pre-existing cells

- True for all cells, prokaryotes to complex eukaryotes - Occurs via cell division - Accurate transmission of genetic information (i.e., DNA sequence) is essential - Requires that the parental cell accurately replicates its genome - For mammals, a ~3 billion bp sequence must be replicated --> 50 trillion times to produce one human

Overview of structure of mitochondria

- Two membranes - Outer membrane is highly permeable - Inner membrane is highly impermeable - Two compartments - Inner and outer membranes are separated by an intermembrane space - The inner membrane has numerous folds (cristae), which extend into the interior (matrix) - Cristae increase surface area of inner membrane of mitochondria so that more reactions can take place - The membranes and compartments have specialized functions and compositions - The matrix is the site of CAC - The matrix contains the genetic system and enzymes for oxidative metabolism - Inner membrane is site of oxidative phosphorylation - Most mitochondrial proteins are translated in cytosol and imported post-translationally

Regulation of nuclear transport: Pho4

- Yeast TF Pho4 is regulated directly by phosphorylation - Pho4 is phosphorylated at a serine adjacent to its NLS, which blocks its binding to importin - Dephosphorylation of Pho4 allows importin to bind the Pho4 NLS - Pho4 is transported into the nucleus where it activates transcription of its target genes

Global regulation of translation via regulation of eIF2

- eIF2 is regulated by binding to GTP/GDP - Cells must regenerate eIF2-GTP - eIF2B exchanges GDP for GTP on eIF2 - This cycle continues when cells are healthy and in the presence of growth factors --> growth factors are extracellular molecules that promote the health of cells - eIF2 and eIF2B are inhibited by phosphorylation by regulatory protein kinases when cells are in stressful conditions (e.g., absence of growth factors) - This inhibits translation globally

Global regulation of translation via regulation of eIF4E

- eIF4E binds the 5' cap - eIF4E is regulated by protein-protein interactions with 4E-binding protein - Growth factors activate protein kinases that phosphorylate 4E-BP, so it is unable to bind eIF4E - In the absence of growth factors, the non-phosphorylated 4E-BPs bind to eIF4E and inhibit translation --> this blocks eIF4E from interacting with other eIFs and blocks global translation initiation

mRNA export from nucleus

- mRNA transport does not involve karyopherins and is independent of Ran - A distinct transporter complex moves the mRNA through the nuclear pore - Helicase on the cytoplasm side releases the mRNA from some (but not all) proteins in complex and ensures unidirectional transport

Fundamental principles of cells

1. Each cell has a "life of its own" 2. Cells are dynamic, living units that respond to their environment 3. Cells have specialized function(s) --> different cell types make up different tissues --> cells all have same DNA but different functions, so they must be expressing different proteins 4. Cells must cooperate and communicate with each other for the sake of the organism 5. All cells arise from pre-existing cells --> true for all cells (prokaryotes to complex eukaryotes)

How many genes are in the human genome?

20,000-25,000

What is the primary function of mitochondria?

Generate ATP from the breakdown of sugars (especially glucose)

Splicing therapy for Duchenne muscular dystrophy

MUSCULAR DYSTROPHY: - Muscular dystrophies are a group of over 30 diseases that lead to progressive weakening and degeneration of skeletal muscle - Duchenne muscular dystrophy (DMD) is the most common of these disorders, responsible for about 50% of cases - DMD is caused by mutations in an X-linked gene, so it primarily affects males - Symptoms are evident by 4 years of age, lose ability to walk by 12, death usually in 20s DYSTROPHIN: - Mutations responsible for DMD affect the largest gene in the human genome (DMD), which spans 2.4 Mb and includes 79 exons - DMD gene encodes a large protein called dystrophin, which contains 3685 AAs and plays key role in maintenance of skeletal muscle - Dystrophin links actin filaments responsible for muscle contraction to TM proteins in plasma membrane of muscle cells --> these TM proteins bind components of ECM, helping maintain cell stability during muscle contraction - Muscle degeneration in DMD results from absence of dystrophin due to mutations that lead to premature termination of translation of DMD mRNA - These mutations are usually deletions of multiple exons that cause frameshifts in the spliced DMD mRNA so translation is terminated prematurely and no protein is produced - A milder form of disease (Becker muscular dystrophy) results from in-frame deletions in DMD that lead to production of abnormal but partially functional proteins EXON SKIPPING THERAPY: - Premature termination of translation in DMD patients results from out-of-frame termination codons that are encountered in exons downstream of a deletion - Rationale of exon skipping therapy is to inhibit splicing to these exons, thereby preventing their inclusion in mRNA and restoring reading frame in an exon downstream - This approach is possible for about 50% of the deletions responsible for DMD - Most frequent deletions include exon 50, as part of deletions of exons 45-50, 47-50, 48-50, and 49-50 --> these deletions result in premature termination within exon 51 - If splicing to exon 51 is prevented, the normal reading frame is restored by splicing to exon 52, and a partially functional protein is produced - This can be accomplished with an antisense oligonucleotide that blocks a splicing enhancer in exon 51, shifting splicing to in-frame exon 52 and producing an internally deleted but still functional protein

How many cells are in the human body?

~50 trillion (50,000,000,000,000)

Promotors

- How does RNA polymerase know where to synthesize RNA? --> RNA pol is directed to the beginning of genes via interactions with gene promotors - Promotor = region of DNA upstream of the transcription start site (TSS) where RNA pol binds to initiate transcription of a gene - How does RNA pol know to bind the promotor? --> promotors contain specific DNA sequences that recruit RNA pol

Initiation of translation using eukaryotic initiation factors (eIFs)

- eIFs are proteins that associate with the ribosome and help initiate translation - In eukaryotes, initiation is complex and requires at least 12 proteins - eIF2 binds the initiator methionyl-tRNA and has to be associated with GTP --> hydrolyzes GTP to GDP during translation - Some eIFs are associated with small subunit - Poly-A binding protein (PABP) binds the poly-A tail (if poly-A tail is long enough) and associates with a bunch of other proteins and eIF4E to form the initiation complex - PABP is like quality control --> mRNA has to have a long enough poly-A tail in order to form initiation complex and be translated - eIF4E specifically binds the 5' cap --> ensures mRNA is properly capped - eIF2, PABP, and eIF4E all get together with the small subunit to make a complex with methionyl-tRNA - Once this complex is made, PABP leaves --> 3' end of mRNA relaxes - Complex begins scanning the 5' UTR for AUG --> requires one ATP per codon scanned - When AUG is found, GTP on eIF2 is hydrolyzed and methionyl-tRNA gets involved - Initiation complex falls apart and large subunit is recruited - eIF2 is involved in dissociating the small subunit initiation complex - eIF5B provides energy (GTP) to recruit the large subunit/assemble ribosome complex - End initiation with methionyl-tRNA associated with AUG, small subunit, and large subunit together - eIF2 is regulated by binding to GTP/GDP (allosteric modulation) --> when associated with GTP, it's in a high energy form and can initiate translation; when bound to GDP, it can no longer start translation

What else does DNA replication at the replication fork require, other than the polymerization of nucleotides?

1. PARENTAL dsDNA NEEDS TO BE SEPARATED AND STABILIZED AS ssDNA - Separation: helicase catalyzes the unwinding of DNA (requires ATP to break H bonds) - Helicase is located at the replication fork - Stabilization: single-stranded DNA binding proteins (SSB) stabilize the unwound DNA, keeping it single-stranded so that it can be copied by DNA polymerase 2. DNA POL NEEDS TO BE LOADED AND STABILIZED ON THE ssDNA TEMPLATE - This is done by polymerase accessory proteins - Sliding-clamp proteins (proliferating cell nuclear antigen/PCNA in eukaryotes) function to load the polymerase onto the primer and maintain stable association with the DNA template - If PCNA were not present, DNA pol might still work, but it would be much slower - Clamp-loading proteins (replication factor C/RFC in eukaryotes) use energy from ATP hydrolysis to open the sliding clamps and load them onto the DNA template --> loads clamp (which then grabs polymerase) to the location where dsDNA turns into ssDNA - Clamp-loading protein interacts with helicase and can address sliding clamps on both strands 3. "SUPERCOILING" OF PARENTAL DNA AHEAD OF THE REPLICATION FORK MUST BE RELIEVED - As DNA unwinds, the DNA ahead of the replication fork is forced to rotate, which would cause DNA molecules to twist - Although eukaryotic chromosomes are composed of linear rather than circular DNA molecules, their replication also requires topoisomerases --> otherwise, the complete chromosomes would have to rotate continually during DNA synthesis - Topoisomerase I and II relieve knots ahead of replication fork - Topoisomerases have built-in endonuclease ad ligase activity --> can break DNA and then repair it - Transient break serves as a swivel to allow free rotation of DNA strands - Only difference between topoisomerase I and II is whether it makes a transient break on one strand of DNA or both (one strand for I, two strands for II) Histone modifications/chromatin remodeling also regulates transcription and DNA replication (much less known)

Numbers of origins and rates of synthesis

E. COLI: - 1 origin - 4 x 10^6 bp of DNA replicated in 30 minutes (~100,000 bp per minute) IF HUMANS HAD ONLY 1 ORIGIN: - 3 x 10^9 bp DNA would take ~30,000 minutes (3 weeks) to replicate at the same speed - However, eukaryotic replication is 10 times slower due to packaging of chromatin, so it would take ~6 months EUKARYOTIC CELLS: - Multiple origins - Yeast: approximately 1 origin per 40 kb - Human: approximately 1 origin per 50-300 kb

Specialized roles of DNA polymerases during DNA replication in prokaryotes vs. eukaryotes

PROKARYOTES: - In E. coli, most of the synthesis is done by DNA pol III on leading and lagging strand - Primase makes the RNA primer, DNA pol III fills in the rest - DNA pol I removes RNA and fills gap with DNA - DNA pol I is a 5'-3' exonuclease --> primer is removed starting from gap at 5' end - DNA pol I is a 5'-3' polymerase --> new nucleotides are added 5' to 3' - Fragments are ligated by DNA ligase EUKARYOTES: - In mammals, the leading strand is synthesized by DNA pol 𝜀 - Primase initiates RNA primer, which is extended by DNA pol α - DNA pol 𝛿 fills in the rest - RNase H (5'-3' exonuclease) removes the RNA primer - DNA pol δ (5'-3' polymerase) fills in the gap with DNA - Fragments are ligated by DNA ligase

Three ways to regulate protein function

(1) Binding of small molecules (allosteric regulation) (2) Phosphorylation and other modifications (3) Protein-protein interactions

Two levels of cell mechanisms in place to regulate translation

- (1) Translational regulation of specific mRNAs via translational repressor proteins, regulated polyadenylation, and RNA interference with microRNAs - (2) "Global" regulation of translation (i.e., of all mRNAs) via regulation of initiation factors eIF2 and eIF4E - Regulation of translation plays a key role in gene expression - Regulation includes translation al repressor proteins and noncoding microRNAs - Global translational activity is modulated in response to stress, nutrient availability, and growth factor stimulation

How are amino acids attached to tRNA?

- 20 different aminoacyl tRNA synthetases each recognize: (1) a particular AA; (2) the "cognate tRNAs" to which the AA should be attached - Attachment occurs in two steps and is energy-dependent (ATP) - Step 1: the AA is joined to AMP, forming aminoacyl AMP - Step 2: the AA is transferred to the 3' CCA end of the tRNA and AMP is released - tRNA with AA attached is often referred to as a charged tRNA

Chaperone proteins

- 3D protein conformation results from interactions between the side chains of amino acids - All information for the correct conformation is provided by the AA sequence - Proteins can fold spontaneously into their correct conformation, but this occurs far too slowly to operate alone within a cell - Protein folding is facilitated by a class of proteins called chaperones - Chaperones act as catalysts that assist the self-assembly process without becoming part of the folded protein - Chaperones have two modes of action: (1) stabilize unfolded polypeptide chains to prevent aggregation (Hsp70 family) --> critically important within cell because of high concentration of other proteins; (2) provide an isolated environment within which correct folding takes place (chaperonins)

Mismatch repair

- A DNA repair mechanism (aka, "pathway") by which cells can repair mismatched base pairs incorporated during DNA replication - The mismatch repair system scans newly replicated DNA, and enzymes excise and replace mismatched bases - Originally identified in E. coli, where it's carried out by three proteins: MutS, MutH, and MutL - Mismatch repair reduces the error frequency during DNA replication by about a thousandfold MECHANISM IN E. COLI: - MutS recognizes the mismatch and forms a complex with MutL and MutH - MutH endonuclease then cleaves the unmethylated new strand adjacent to the mismatch at a GATC sequence opposite a methyl group - DNA is unwound and repaired - MutS and MutL direct excision between nick and mismatch - How does the mismatch repair machinery identify the correct strand to fix? --> E. coli methylates As within GATC (about once every 4 kb) after replication EUKARYOTES: - Mismatch repair pathway is present in eukaryotic cells but with some differences - Strand specificity is not determined by methylation, but dictated by the presence of single strand breaks in newly replicated DNA -- the ends of growing strands - Eukaryotes have MutL Homolog (MLH) and MutS Homolog (MSH) --> dukaryotes lack MutH homolog because the mechanisms is no longer dependent upon identifying methylation - MSH and MLH bind to the mismatched base and direct excision of the DNA between a strand break and the mismatch

Enhancers

- A type of cis-acting element - Also bind transcription factors that regulate transcription, but function independent of proximity and orientation to TSS - Enhancers can be located almost anywhere in the genome, including on different chromosomes - Ex: the SV40 enhancer --> has promoter element and upstream regulatory sequences, as well as enhancer with TF binding sites - When proteins bind to enhancers, transcription will be enhanced - Enhancers are independent of orientation to TSS (can be forwards or backwards) - Forward: enhancer is going in the same direction as the promoter - Backwards: enhancer is going in the opposite direction as the promoter - Enhancer is independent of proximity to TSS --> transcription rate stays the same whether enhancer is nearby, far away, upstream, or downstream of gene - Enhancers can be more than 50 kb away from the promoters they regulate in mammalian cells --> this is physically possible due to DNA looping - DNA looping allows a TF bound to a distant enhancer to interact with proteins associated with the RNA pol/Mediator complex at the promoter - DNA is held in place structurally by cohesin proteins - Cohesin binds upstream of the promoter and somewhere near the enhancer to bring those DNA sequences relatively close enough so that whatever protein is involved can bind the enhancer and Mediator at the same time - Enhancers often contain binding sites for multiple transcription factors that act cooperatively --> ex: immunoglobulin enhancer (active in lymphocytes, but not other cell types) - Immunoglobulin enhancer is partly responsible for tissue-specific expression of the immunoglobulin genes - Enhancers usually have multiple sequence elements that bind different regulatory proteins that work together to regulate gene expression - Immunoglobulin heavy-chain enhancer has at least 9 enhancer binding sites for antibody genes because during an immune response, we want lots of antibodies to be generated quickly - Mutation in any one of the enhancer binding sites in immunoglobulin gene reduces transcription to its target gene, but does not block it altogether - Enhancers account for 10% or more of human genomic DNA, emphasizing the importance of these elements - Many mutations linked to human diseases affect enhancers rather than protein-coding sequences - In any given cell type, multiple enhancers work together to regulate individual genes

Upstream regulatory sequences

- A type of cis-acting element - Located close to TSS (~50-1000 bp upstream), typically 5-6 bp long - The role of upstream regulatory sequences is to bind additional proteins/trans-acting factors (transcription factors) that regulate transcription --> ~2500 of the 20-25,000 human genes encode TFs - Upstream regulatory sequences are basically just TF binding sites - Ex: herpes virus thymidine kinase gene (virus gene expressed in eukaryotes) --> TATAA promotor element at -25; upstream regulatory sequences -50 to -100 and include CAT box and GC box

Formation of a clathrin-coated vesicle

- After being delivered to the trans-Golgi membrane, Arf/GDP is activated to Arf/GTP by a guanine nucleotide exchange factor (Arf/GEF) - Arf/GTP recruits an adaptor protein, which binds to the cytosolic tail of a transmembrane receptor with its lumenal cargo and also serves as a binding site for assembly of a clathrin coat - Clathrin consists of three protein chains that associate with each other to form a basketlike lattice that distorts the membrane and drives vesicle formation - Dynamin constricts the neck of the vesicle and drives membrane fission

Protein misfolding diseases

- Alzheimer's disease, Parkinson's disease, and type 2 diabetes are associated with aggregation of misfolded proteins - The misfolded proteins form fibrous aggregates called amyloids, characterized by B-sheet structures - Defects in protein folding are responsible for protein misfolding diseases - Cystic fibrosis is caused by a mutation that results in one AA deletion that leads to improper folding of protein CFTR --> CFTR transports Cl- ions across epithelial cell membranes - Alzheimer's disease is characterized by two aggregate types in brain tissue: neurofibrillary tangles (misfolded tau proteins) and amyloid plaques (aggregates of misfolded amyloid-B protein [AB])

Histone acetylation

- Amino-terminal tail domains of core histones are rich in lysine (positive charge) and can be modified by acetylation - Acetyl groups (COCH3) are added post-translationally by histone acetyltransferase (HAT) - Addition of acetyl groups neutralize positive charge of lysines on the NH3+ at the end of the R group - Neutralization of charge relaxes chromatin condensation to produce active chromatin because the N-terminal tail is no longer able to wrap around the outside of the nucleosome and hold the DNA in place - Lysine acetylation is reversible --> acetyl group can be removed by histone deacetylase (HDAC) - HATs and HDACs interact with TFs to regulate gene expression - HATs are coactivators --> recruited to DNA by transcriptional activators (direct activator) --> this leads to chromatin decondensation --> this leads to transcriptional activation - HDACs are corepressors --> recruited by transcriptional repressors (direct repressor) --> this leads to chromatin condensation --> this leads to transcriptional repression - Histone modifying enzymes bind to the CTD of RNA pol II and modulate chromatin structure as RNA pol II transcribes

Transcription in E. coli

- At the beginning, RNA pol is bound nonspecifically to DNA - Specific binding of σ subunit to -35 and -10 promoter sequences leads to closed promoter complex --> DNA not unwound - RNA pol then unwinds 12-14 bases of DNA to form an open promoter complex, allowing transcription - RNA pol is interacting with ~40 bp of DNA (TSS to -35 site) - After addition of about 10 nucleotides, σ subunit is released from RNA pol - Core polymerase continues to elongate primary RNA chain (not yet mRNA) - Transcription stops when RNA pol reaches a termination signal (not a stop codon) - Termination signal is pre-defined in the DNA --> adjacent complimentary sequences located at the end of the genes - Transcription of the GC-rich inverted repeat results in a segment of RNA that forms a stable stem-loop structure (GC has more H bonds) --> this disrupts RNA pol's association with the DNA template and terminates transcription - Transcription can also be terminated using a protein called rho, which can emulate the stem loop structure and kick off RNA pol

Nonstandard codon-anticodon base pairing

- Base pairing at the third codon position is relaxed, allowing G to pair with U, and inosine (I) in the anticodon to pair with U, C, or A - Phenylalanyl tRNA pairing: allows phenylalanyl (Phe) tRNA to recognize either UUC or UUU codons --> guanosine (codon or anticodon) pairing with cytosine (codon or anticodon) (3 H bonds) or uridine (codon or anticodon) (2 H bonds) - Alanyl tRNA pairing: allows alanyl (Ala) tRNA to recognize GCU, GCC, or GCA --> inosine (anticodon) pairing with uridine (codon) (2 H bonds), cytosine (codon) (2 H bonds), or adenine (codon) (2 H bonds) - Ability of inosine to bind to U, C, or A leads to redundancy in table/wobble position - Nonstandard pairing happens on the 5' end of the anticodon (1st position in anticodon, last position in mRNA)

Base-excision repair

- Base-excision repairs DNA when a base of a nucleotide is damaged or mutated by excising the single base - Ex: uracil can arise in DNA when dUTP is incorporated in place of thymine, or uracil can be formed in DNA by the deamination of cytosine - Excision of uracil in DNA is catalyzed by DNA glycosylase, which cleaves the bond between the uracil and the deoxyribose of the DNA --> DNA glycosylases also recognize and remove other abnormal bases - DNA glycosylase action results in the formation of an apyrimidinic or apurinic site (AP site) --> a sugar with no base attached - Purine bases can also be lost spontaneously - These sites are repaired by AP endonuclease --> the deoxyribose is removed, and the resulting gap is filled by DNA polymerase and ligase

Modification of chromatin structure via histone modification

- Because eukaryotic DNA is packaged in chromatin, chromatin structure is a critical aspect of gene expression - Histones can be modified several ways -- key mechanisms for regulating gene expression - Chromatin limits ability of DNA for transcription, affecting both TF binding and action of RNA pol - Many histone modifications are stably inherited when cells divide - If you modify the positive charges in histones, you modify how it interacts with DNA --> if histone isn't positively charged, it will loosen and let go of DNA - Core histones have two domains: (1) histone-fold domain (folded ball part of nucleosome); (2) amino-terminal tail domain (unfolded tail from amino end, ~25 AA long) - Amino-terminal tail domains can be subject to several types of covalent modifications that influence transcription (acetylation, methylation, phosphorylation)

Endonucleases vs. exonucleases

- Both cleave phosphodiester bonds from double-stranded sequences - Endonucleases cleave within a sequence of nucleotides - Exonucleases cleave at the end of a sequence of nucleotides - Exonucleases cleave with directionality --> 5' to 3' exonucleases cleave from the 5' end; 3' to 5' exonucleases cleave from the 3' end

Prokaryotic vs. eukaryotic mRNA

- Both prokaryotic and eukaryotic mRNAs contain UTRs at their 5' and 3' ends - Eukaryotic mRNAs also contain 5' 7-methylguanosine caps and 3' poly-A tails - Prokaryotic mRNAs are frequently polycistronic --> they encode multiple proteins, each of which is translated from an independent start site - Eukaryotic mRNAs are usually monocistronic, encoding only a single protein

Regulation of protein function through phosphorylation

- Catalyzed by enzymes called protein kinases - Protein kinases transfer phosphate groups from ATP to the hydroxyl groups of side chains of serine, threonine, or tyrosine --> protein-serine/threonine kinase and protein-tyrosine kinase - Phosphorylation is reversible --> it can activate or inhibit proteins in response to environmental signals - Phosphorylation is reversed by enzymes called protein phosphatases, which catalyze hydrolysis of phosphorylated AAs - Phosphorylation can regulate protein activity --> ex: regulation of eIF2 and eIF2B in response to cell stress - Phosphorylation can modulate proten-protein interactions --> ex: binding of transcription elongation and processing factors to the phosphorylated CTD of RNA pol II - Kinases and phosphatases often function in sequence within signal transduction pathways --> a mechanism that converts a mechanical or chemical stimulus to a cell into a specific cellular response - Sequential action of a series of protein kinases can transmit a signal from the cell surface to target proteins in the cell, resulting in changes in cell behavior in response to environmental stimuli - Ex: regulation of glycogen breakdown by protein phosphorylation --> in muscle cells, epinephrine signals the breakdown of glycogen to glucose-1-phosphate, providing energy for increased muscular activity --> this is catalyzed by glycogen phosphorylase, which is regulated by a protein kinase - The signaling pathway is initiated by allosteric regulation --> epi binds to a cel surface receptor, and cAMP binds to cAMP-dependent kinase --> signal is then transmitted to its target by the sequential action of protein kinases (phosphorylase kinase and glycogen phosphorylase) - Key features of signal transduction pathways: amplification and reversibility

How do chaperones stabilize unfolded polypeptide chains during translation and during transport into organelles?

- Chaperones bind to polypeptide chains that are still being translated on ribosomes - The chain must be protected from aberrant folding or aggregation with other proteins until synthesis of an entire domain is complete - Many chaperones were initially identified as heat-shock proteins (Hsp), expressed in cells subjected to high temperatures --> Hsp70 family (found in prokaryotes and eukaryotes) - Hsp stabilize and facilitate refolding of proteins that have been partially denatured - Chaperones bind to exposed short hydrophobic regions - Ex. during transport: partially unfolded proteins stabilized by chaperones are transported across the mitochondrial membrane --> chaperones in the mitochondrion then facilitate folding

Chromosomal domains and CTCF

- Chromatin within the nucleus is organized into looped domains formed by the interaction of CTCF and cohesin - Activity of any given enhancer is specific for the promoter of its target gene - Specificity is maintained partly by architectural proteins such as CTCF, which divide chromosomes into independent domains and prevent enhancers from acting on promoters located in an adjacent domain

What mechanisms are in place to ensure accurate DNA replication?

- Complementary base pairing: C-G and A-T --> hydrogen bonding only favors correct base pairing by ~100-fold - Selection by complementary base pairing has error rate of 1/100 - Mutation rates indicate that error frequency during replication is less than one incorrect base per 10^9 nucleotides --> this is much lower than would be predicted simply on the basis of complementary base pairing - DNA polymerase helps select the correct bases for insertion --> binding of correctly matched dNTPs induces conformational changes in DNA polymerase that lead to incorporation of the nucleotide - Base selection by DNA polymerase reduces error rate to 1/10^5 --> increases fidelity of replication about a thousandfold - Proofreading by DNA polymerase reduces error rate to 1/10^7 - 1/10^8 - If all else fails, mismatch repair corrects errors to less than 1/10^9

Chemiosmotic coupling and the ETC

- Components of the electron transport chain are organized into four complexes in the inner mitochondrial membrane NADH: - NADH is produced in the Krebs cycle in the matrix --> electrons from NADH enter the ETC at complex I - Complex I breaks down NADH to NAD+ + H+ --> these electrons change conformation of proteins in complex I - Complex I proteins then transport protons from matrix against gradient into intermembrane space (4 H+ transported) - Electrons are then transferred to coenzyme Q (ubiquinone), which carries electrons to complex III - Complex III receives electrons and changes conformation and pumps protons from matrix to intermembrane space (4 H+ pumped) - Complex III passes electrons to cytogrome c, which passes them on to complex IV - Complex IV (cytochrome oxidase) pumps H+ into intermembrane space (2 H+ pumped), and combines electrons to make H2O - ~10 protons pumped for each NADH molecule - Transfer of electrons from complexes I, III, and IV results in stepwise release of energy --> energy coupled pump protons out of matrix FADH2: - Complex II receives electrons from the citric acid cycle intermediate succinate and can pass on electrons the same way as with NADH (e.g., cytochrome c and ubiquinone) - Complex II does not pump H+ across membrane --> FADH2 is therefore less efficient as far as releasing energy and pumping H+ across - Electrons go to coenzyme Q and then to complex III - Complex III pumps 4 H+ - Electrons go from complex III to cytochrome c to complex IV - Complex IV pumps 2 H+ and creates H2O - ~6 H+ pumped per molecule of FADH2

Wobble hypothesis

- Crick proposed the wobble hypothesis in 1966 - The base in the first position on tRNA (the 5' end) is usually an abnormal base like inosine, pseudoeuridine, tyrosine, etc. - The 3rd position in codon is wobble position, 1st position in anticodon is wobble position - These abnormal bases can pair with more than one type of nitrogenous base in the third position of the codon of the mRNA - Wobble bases in tRNA: (C --> G), (A --> U), (G --> C/U), (U --> A/G), (I --> C/A/U) - Wobble bases in mRNA: (C --> G/I), (A --> U/I), (G --> C/U), (U --> A/G/I)

Proofreading by DNA polymerase

- DNA polymerases III (E. coli), ε and 𝛿 (eukaryotes) exhibit 3' to 5' exonuclease activity --> proofreading - DNA polymerase III incorporates wrong base --> polymerase excises mismatched base via 3'-5' exonuclease --> synthesis proceeds with incorporation of correct base - The need for proofreading likely provided the evolutionary pressure for: (1) DNA pol's dependency on an annealed primer; (2) 5' to 3' directionality of synthesis

How/where is replication of the genomic DNA initiated in the first place?

- DNA replication does not start at a replication fork --> it starts at origins of replication (ori) - Origins of replication are binding sites for proteins that initiate the replication process E. COLI: - Origin of replication in E. coli is a single 245 bp DNA sequence - An initiator protein binds to specific DNA sequences within the origin --> binding leads to synthesis of RNA primers and unwinding of DNA by helicase and SSB - Because bacteria has a circular genome, it ends up with two origins of replication, two replication forks, synthesis happening in both directions EUKARYOTIC: - Multiple origins are needed to replicate the much larger genomes of eukaryotic cells in a reasonable amount of time - Replication is initiated by a complex of proteins (ORC) that recognizes origin - Additional proteins and helicase are loaded - Replication forks move in opposite directions towards other replication bubbles

Examples of DNA damage induced by radiation and chemicals

- Damage can block replication or transcription, and lead to a high frequency of mutations - Exposure to UV light --> adjacent thymines in DNA become thymine dimer with cyclobutane ring --> cannot be broken by helicase or read by DNA polymerase; ring is so bulky it prevents double-stranded binding near it - Alkylation: addition of methyl group --> guanine turns into O6-methylguanine - Reaction with carcinogen --> bulky groups present in cigarette smoke stick to guanine residues

How can we confirm that a transcription factor actually binds DNA?

- Electrophoretic-mobility shift assay (EMSA) - A sample containing radiolabeled fragments of DNA is divided in two, and half of the sample is incubated with a protein that binds to a specific DNA sequence - Samples are then analyzed by electrophoresis in a nondenaturing gel so that the protein remains bound to DNA - Protein binding is detected by the slower migration of DNA-protein complexes compared to that of free DNA - Only a fraction of the DNA in the sample is actually bound to protein, so both DNA-protein complexes and free DNA are detected following incubation of the DNA with protein - Can show specificity of binding by mutating DNA or protein

Termination of translation using release factors

- Elongation continues until a stop codon (UAA, UAG, UGA) is translocated into the A site - There is no anticodon/tRNA that matches a stop sequence --> stop sequences are recognized by proteins that look like tRNAs (release factors) - In prokaryotic cells, RF1 recognizes UAA or UAG and RF2 recognizes UAA or UGA - In eukaryotic cells, eRF1 recognizes all three stop codons - When a stop codon is reached, release factor enters A site and causes ribosome to dissociate - mRNA can then be translated again and all the molecules involved can be recycled

Why does DNA polymerase synthesize DNA 5' to 3'?

- Energy to add nucleotide comes from 5' triphosphate on the nucleotide itself - If the wrong base is added and removed, the end looks like it did before the wrong base was added, so the correct base can then be added - If synthesis was 3' to 5', then excision of the incorrect terminal 5' nucleotide would prevent further incorporation of free nucleotides because the end would look different (1 phosphate instead of 3)

Eukaryotic RNA polymerases

- Eukaryotes have RNA polymerases I, II, and III --> gene family members with specialized - RNA pol II transcribes all protein-coding genes (mRNA) --> it requires initiation factors that (in contrast to bacterial σ factors) are not associated with the polymerase - RNA pol II also synthesizes miRNA and lncRNA - RNA pol III synthesizes tRNA - rRNA is synthesized by RNA pol I (5.8S, 18S, 28S) and RNA pol III (5S) - snRNA and scRNA are synthesized by RNA pol II and III - Mitochondrial genes are synthesized by mitochondrial RNA pol - Chloroplast genes are synthesized by chloroplast RNA pol

What defines the origins of replication in eukaryotes?

- Eukaryotic origins of replication were first studied in the yeast S. cerevisiae --> they were identified as sequences that can support replication of plasmids in transformed cells - Origins are recognized by a complex of proteins (origin of replication complex/ORC) --> ORC proteins are initiators of replication in all eukaryotes - In S. cerevisiae, the ORI = "ARS element" (autonomously replicating sequences) - ARS is ~100 bp long that contains ACS (ARS consensus sequence) - ACS: (A/T)TTTA(T/C)(A/G)TTT(A/T) - ACS is the binding site of ORC --> ORC recruits other proteins, including MCM DNA helicase - S. pombe (type of yeast) has an origin ~1 kb long with AT-rich ORC binding sites - Drosophila and mammals have origins that span up to 10-50 kb with no identified ORC binding sites - Replication origins in other eukaryotes are much less well defined than ARS elements of S. cerevisiae --> origins in higher eukaryotes may be defined by 3D chromatin structure rather than DNA sequence

Excision repair

- Excision repair can be used for a wide variety of DNA damage - Three types: (1) mismatch repair, (2) base-excision, (3) nucleotide-excision

5' capping of pre-mRNA

- First post-translational modification of pre-mRNA - The 5' end of the transcript is modified by addition of a 7-methylguanosine cap by guanylyl transferase - Guanylyl transferase is associated with the CTD of RNA pol II and modifies the transcribed RNA as transcription takes place - Cap is linked through a 5'-to-5' cap - 5' cap has two roles: (1) mediates interactions with ribosomes to initiate translation; (2) stabilizes the mRNA for translation (mRNA would otherwise be degraded) - If a transcript is not modified with a 5' cap, it will not be translated

Transcription of RNA pol III genes

- Genes for tRNAs, 5S rRNA, and some of the small RNAs (U6 snRNA for spliceosome) are transcribed by RNA pol III - They are expressed from three types of promotors: TFIIIA, TFIIIB, and TFIIIC

Lipid addition to the outer face of plasma membrane proteins

- Glycolipids (GPI anchors) - Proteins can be anchored to the outer face of the plasma membrane by glycolipids attached to their C-terminus - GPI anchors are joined to the C-terminal AA by an ethanolamine, which is linked to an oligosaccharide that consists of three mannose and one glucosamine residues - The oligosaccharide is in turn joined to the inositol head group of phosphatidylinositol - The two fatty acid chains of the lipid are embedded in the plasma membrane

Glycosylation

- Glycosylation adds carbohydrate chains to proteins to form glycoproteins - The carbohydrate moieties play important roles in protein folding in the ER, in targeting proteins for transport, and as recognition sites in cell-cell interactions - Glycosylation starts in the ER before translation is complete - O-linked oligosaccharides are added to serine or threonine within the Golgi apparatus - The sugar joined to the AAs is usually N-acetylgalactosamine for O-linked - O-linked oligosaccharides usually consist of only a few carbohydrate residues, which are added one sugar at a time - The carbohydrate chains of N-linked glycoproteins are attached to asparagine - The sugar joined to the AAs is N-acetylglucosamine for N-linked - The first step in formation of N-linked glycoproteins is the addition of a 14-sugar oligosaccharide in the ER

Golgi apparatus

- Golgi apparatus (Golgi complex): proteins from the ER are processed and sorted for transport to endosomes, lysosomes, the plasma membrane, or secretion - Most glycolipids and sphingomyelin are synthesized in the Golgi - The Golgi is composed of flattened membrane-enclosed sacs (cisternae) and associated vesicles - It has polarity: proteins from the ER enter at the cis face, usually oriented toward the nucleus. They exit from the trans face - The Golgi has 4 regions: (1) cis compartment—receives molecules from the ERGIC; (2) medial and (3) trans compartments—most modifications are done here; (4) trans-Golgi network—the sorting and distribution center - The mechanism of protein movement through the Golgi is an area of controversy - The stable cisternae model: proteins are carried between cisternae in transport vesicles - The cisternal maturation model: proteins are carried within the cisternae, which gradually mature and progressively move through the Golgi in the cis to trans direction - Vesicles return Golgi resident proteins back to earlier Golgi compartments - Sorting of cargo into budding vesicles at the Golgi is dependent on post-translational modifications that occur within the Golgi - Golgi is composed of several cisternae and associated vesicles - Specific protein processing events take place in specific sub-compartments - Two key modifications within the Golgi: O-linked glycosylation and modification of N-linked sugars added in the ER

RNA polymerase in bacteria

- In E. coli, RNA pol has 5 subunits: α (2 copies), 𝛽, 𝛽', ɷ, σ - α, 𝛽, 𝛽', and ɷ subunits make up the "core polymerase" in E. coli and are responsible for polymerase activity - In E. coli, the σ subunit is necessary for initiation - Most bacteria have several different σ subunits that direct RNA pol to different start sites under different conditions - During elongation, RNA pol maintains an unwound region of about 15 bp - High-resolution structural analysis shows the 𝛽 and 𝛽' subunits form a crab-claw-like structure that grips the DNA template --> a channel between these subunits contains the polymerase active site

Cis-acting elements

- In addition to promoters, most genes are regulated by additional cis-acting elements - Two types: upstream regulatory sequences and enhancers

What accounts for the differences in structure and function between different cell types?

- In each cell type, only a subset of the 20-25,000 genes are actually expressed - Some genes are expressed in many (or all) cell types - Some genes are expressed only in particular cell types - Regulation of gene expression is critical - Ex: Telomerase is higher in germ cells and embryonic cells, lower in adult somatic cells - Ex: many of the genes encoding the DNA replication machinery (e.g., sliding-clamp proteins) are expressed during DNA replication, but not during G1, G2, and M (mitosis) phase

mRNA degradation

- In eukaryotes, mRNA half-lives vary (less than 30 minutes to 20 hours) - Short-lived mRNAs code for regulatory proteins, levels of which can vary rapidly in response to environmental stimuli - mRNAs encoding structural proteins or central metabolic enzymes have long half-lives - Degradation of eukaryote mRNAs is initiated by shortening the poly-A tails - After deadenylation, mRNAs that keep 5' cap are degraded from 3' to 5' direction - mRNAs that undergo decapping after deadenylation are degraded from 5' to 3' direction - Rapidly degraded mRNAs often contain specific AU-rich sequences near the 3' ends -- binding sites for proteins that can either stabilize them or target them for degradation - These RNA-binding proteins are regulated by extracellular signals, such as growth factors and hormones - Degradation of many mRNAs is regulated by miRNAs, which stimulate degradation as well as inhibit translation

RNA processing and turnover

- In eukaryotes, pre-mRNAs are extensively modified before export from the nucleus - Throughout processing, transport, translation, and degradation, mRNA molecules are associated with proteins to form messenger ribonucleoprotein particles (mRNPs) - Transcription and processing are coupled - Transcription yields a pre-mRNA (aka, primary RNA transcript) - The CTD of RNA pol II plays a key role by serving as a binding site for the enzymes involved in mRNA processing - mRNA processing entails three steps: (1) 5' capping; (2) 3' polyadenylation; (3) splicing

Prokaryotic vs. eukaryotic signals for translation initiation

- Initiation sites in prokaryotic mRNAs are characterized by a Shine-Dalgarno sequence that precedes the AUG initiation codon - Base pairing between the Shine-Dalgarno sequence and a complementary sequence near the 3' terminus of the 16S rRNA (small subunit) aligns the mRNA on the ribosome - Eukaryotic mRNAs are bound to the 40S ribosomal subunit (small subunit) by their 5' 7-methylguanosine cap - The ribosome then scans along the mRNA until it encounters an AUG initiation codon

First experimental approach to test the signal hypothesis

- Isolated rough microsomes (ER with ribosomes) and smooth microsomes through centrifugation - Rough microsomes have greater density - Compared in vitro translation of a secreted protein (igG light chain) on rough microsomes and free ribosomes - Translation on free ribosome --> protein gets translated and ends up with protein containing signal sequence (slightly larger) - Translation with microsomes present --> protein is smaller and ends up inside microsome

DNA polymerase

- Key enzyme for DNA replication - All DNA polymerases add a deoxyribonucleoside 5'-triphosphate to the 3' hydroxyl group of a growing DNA chain (the primer strand) --> catalyzes the formation of phosphodiester bond - Energy for DNA polymerase to add nucleoside comes from two phosphates off the nucleoside itself - All DNA polymerases synthesize 5' to 3' --> evolutionarily conserved because fundamentally advantageous - All DNA polymerases can only extend a pre-existing polynucleotide (aka, primer), which must be bound to a template strand by complementary base pairing - DNA pol gene families: prokaryotic (5 pol genes) and eukaryotic (~15 pol genes) - Prokaryotic DNA pol: I, II, III, IV, V - Eukaryotic DNA pol: alpha (α), epsilon (ε), delta (δ)

How does modulation of the length of the poly-A tail affect translation rates?

- Length of poly-A tail can be regulated by proteins that bind 3' UTR --> can either shorten or extend the tail - Poly-A tail regulates translation via PABP, which binds eIF4G - Translational regulation is very important during early development - Many mRNAs with short poly-A tails are stored in oocytes --> translation is activated at fertilization or later stages - Lengthening the poly-A tails allows binding of PABP, which stimulates translation

The ubiquitin-proteasome pathway

- Major pathway of protein degradation in eukaryotes - Rapid, regulated degradation of proteins involved in cell proliferation, cell cycle, programmed cell death - Targeted degradation of proteins by the proteasome (large, multi-subunit protease complex) - Proteins are targeted by the addition of ubiquitin ("ubiquitination") to lysine residues - Ubiquitin is highly conserved in all eukaryotes - Three enzymes involved in ubiquitination of proteins: (1) E1 (Ub-activating enzyme); (2) E2 (Ub-conjugating enzyme); (3) E3 (Ub-ligase enzyme) - E1 attaches ubiquitin on E2 --> E2 sticks ubiquitin on protein --> E3 recognizes the target protein and helps E2 - E3 is a critical determinant of targeted degradation --> lots of E3 in the cell, some E2, not a lot of E1 - Ubiquitin is attached to the amino group of the side chain of a lysine residue, then more are added to form a chain --> the longer the chain, the more likely it is to be recognized by the proteasome - Polyubiquitinated proteins are recognized and degraded by a large protease complex, the proteasome - Proteasome cuts proteins (using ATP) into ~5-15 AA, which can then be broken down further and recycled - Ex. of targeted cell cycle proteins: cyclins that regulate progression through the division cycle of eukaryotic cells - Entry into mitosis requires activation of Cdk1 --> Cdk1 is activated by cyclin B --> transition from metaphase to anaphase requires inactivation of cyclin B - Entry of cells into mitosis is in part by cyclin B, a regulatory subunit of Cdk1 protein kinase --> the active B-Cdk1 complex induces entry into mitosis - Degradation of cyclin B by the proteasome then leads to inactivation of the Cdk1 kinase, allowing the cell to exit mitosis and return to interphase

Identification of eukaryotic regulatory sequences using gene-transfer assays

- Many cis-acting DNA sequences regulate expression of eukaryotic genes - These regulatory sequences have been identified by gene transfer assays - Regulatory sequences are ligated to reporter genes that encode easily detectible enzymes, such as firefly luciferase --> this reflects how strongly it's transcribed - The regulatory sequence directs expression of the reporter gene in cultured cells - Can use this to evaluate strength of promoter and how easily RNA pol can access it by cloning the regulatory sequence into plasmids and compare promoters - Can also combine promoters and include other proteins through another plasmid (e.g., expressing repressor protein, CAP protein, etc.)

Lipid addition to inner face plasma membrane proteins

- Many proteins associated with the inner face of the plasma membrane are modified by addition of lipids - N-myristoylation: initiating methionine is removed, leaving glycine at the N-terminus of the polypeptide chain --> myristic acid (14C fatty acid) is then added - Prenylation affects Ras proteins and proteins of the nuclear envelope (nuclear lamins), which have cysteine residues followed by three other acids at the C terminus - The first step in prenylation is addition of 15C farnesyl group to side chain of cysteine (farnesylation) --> this step is followed by proteolytic removal of the 3 C-terminal AAs and methylation of cysteine, which is now at C terminus - Palmitoylation: palmitic acid (16C fatty acid) is added to the side chain of an internal cysteine residue

Regulation of protein function through protein-protein interactions

- Many proteins consist of multiple subunits --> interactions between them can regulate protein activity - Ex: regulation of cAMP-dependent protein kinase --> cAMP-dependent protein kinase has two regulatory and two catalytic subunits in the inactive form - cAMP binds to the regulatory subunits, which induces conformational change and dissociation of the complex --> the free catalytic subunits are then enzymatically active protein kinases - cAMP acts as an allosteric regulator by altering protein-protein interactions

Repair of O6-methylguanine

- Methylation of the O6 position of guanine forms O6-methylguanine, which pairs with thymine instead of cytosine - This can be repaired by an enzyme (O6-methylguanine methyltransferase) that transfers the methyl group from O6-methylguanine to a cysteine residue in its active site

E. coli lac operon

- Most transcriptional regulation in bacteria operates at initiation - Studies of gene regulation in the 1950s used enzymes involved in lactose metabolism - The lac operon is the prototypical example of negative and positive control of transcription - Lac operon is a set of 3 genes expressed as a unit (i.e., an operon) that encodes enzymes that carry out lactose metabolism: (1) B-galactosidase (z; cleaves lactose into glucose and galactose); (2) lactose permease (y; transports lactose into the cell); (3) transacetylase (a; inactivates toxic thiogalactosides that are transported into the cell along with lactose) - The enzymes are only expressed when lactose is present - Two loci control transcription --> (1) o/operator (adjacent to transcription initiation site); (2) i (outside the operon, encodes a protein that binds to the operator) NEGATIVE CONTROL: - Mutants that don't produce i gene product express the operon even when lactose is not available --> this implies that the normal i gene product is a repressor, which blocks transcription when bound to o - The lac operon is subject to negative control by repressor protein --> repressor binds to the operator only when lactose is not present - When lactose is present in normal cells, it binds to the repressor, preventing it from binding to the operator, and the genes are expressed POSITIVE CONTROL: - Regulatory proteins activate transcription - Glucose is the preferential source of energy --> E. coli will express B-galactosidase only if glucose is not available - E. coli links expression of the lac operon to glucose levels through CAP (catabolite activator protein), which recruits RNA pol to the lac operon promoter - CAP is regulated by cAMP and must be bound to cAMP to activate transcription - cAMP is derived from ATP through adenylyl cyclase, which is inhibited by a product of glucose (a-ketoglutarate) - If glucose decreases, levels of cAMP increase - cAMP binds to the regulatory protein CAP (catabolite activator protein) - This stimulates CAP to bind to its target DNA sequence upstream of the lac operon --> CAP facilitates binding of RNA pol to the promotor

Nuclear lamina

- Nuclear lamina is a fibrous mesh that provides structural support from the inside (cytoskeletal structure) - Located beneath inner and outer membranes (especially inner) - Consists of fibrous proteins (lamins) and other proteins - Lamins are a class of intermediate filament proteins that associate to form higher order structures - Two lamins interact to form a dimer --> the a-helical regions wind around each other to form a coiled coil - The lamin dimers associate with each other head-to-tail to form the lamina - Side-by-side association of polymers gives higher order structure - Nuclear lamina is anchored to the inner nuclear membrane via protein-protein interactions and addition of lipids (prenylation) - Lamins bind to inner membrane proteins such as emerin and lamin B receptor (LBR) --> they are connected to the cytoskeleton by LINC protein complexes - Nuclear lamins bind chromatin indirectly via interactions between lamin-associated proteins and histones H2A and H2B --> this is a way of anchoring chromatin to particular locations

Nucleotide-excision repair in mammalian cells

- Nucleotide-excision repair removes damaged bases as part of an oligonucleotide - Used almost exclusively for thymine dimers - Excinuclease, an enzyme complex, can directly excise an oligonucleotide (12 nucleotides) --> excinuclease cuts single strand - Helicase is required to unwind the DNA for excision - The resulting gap is filled by DNA polymerase and sealed by ligase - People born with defects in this repair system will develop skin cancer if they are exposed to sunlight - The cell also utilizes nucleotide excision repair to correct defects caused by mutagenic substances, among other things

What happens once proteins are in the ER?

- Once in the ER, proteins are further trafficked to their final destination via the Golgi apparatus - Bip is a chaperone protein that helps proteins fold in the ER - Most proteins will spend less than 10 minutes in the ER - Many proteins undergo post-translational modifications in the ER before being transported to the Golgi - Proteins are glycosylated (N-linked glycosylation) in the ER while translation is still in progress - The oligosaccharide is synthesized on a lipid (dolichol) carrier - Glycosylation helps prevent protein aggregation in the ER and provides signals for subsequent sorting - Some proteins are attached to the plasma membrane by glycolipids called glycosylphosphatidylinositol (GPI) anchors - GPI anchors are assembled in the ER membrane and added to the carboxy terminus of some polypeptides - GPI-anchored proteins are transported as membrane components via the secretory pathway --> their orientation within the ER dictates they will be exposed on the outside of the cell

Synthesis is always 5' to 3', but only one strand runs 5' to 3' in the direction of the replication fork migration. How do cells replicate the opposite strand?

- One strand is synthesized continuously (leading strand) and one is synthesized discontinuously (lagging strand) - The lagging strand is formed from short (1-3 kb), discontinuous pieces of DNA that are synthesized backward (Okazaki fragments) - Okazaki fragments are joined together by DNA ligase - Lagging strand is called the lagging strand because it requires more enzymes and is therefore less efficient than leading strand

Ribosomes

- Peptide bond formation is catalyzed by ribosomes - Ribosomes are named according to their sedimentation rates in ultra-centrifugation --> 70S for bacterial and 80S for eukaryotic - Cells have many ribosomes, illustrating the importance of protein synthesis --> E. coli has about 20,000 ribosomes; growing mammalian cells can have 10 million - All ribosomes consist of a large subunit and small subunit --> each subunit is composed of multiple polypeptides and ribosomal RNAs (rRNAs) - The subunits of eukaryotic ribosomes are larger and have more proteins than prokaryotic ribosomes - Eukaryotic ribosome has a 60S large subunit and 40S small subunit, with four different rRNAs (28S, 18S, and 5.8S encoded by the same gene; 5S) - 60S subunit is composed of 28S, 5.8S, and 5S rRNAs and ~46 proteins - 40S subunit is composed of 18S rRNA and 33 proteins - RNA pol I transcribes 28S, 18S, and 5.8S; RNA pol III transcribes 5S - RNA pols I and III produce a pre-rRNA transcript, which is then processed via cleavage into mature rRNAs - Mature rRNAs are then ready to complex with ribosomal proteins in the nucleolus - rRNAs within the large subunit catalyze peptide bond formation (can do this even after 90% of ribosomal proteins have been removed) - It was first thought that rRNAs played only a structural role in ribosomes, but it was later shown that rRNAhas catalytic activity - Ribosomes build polypeptides one amino acid at a time - Once the ribosome reaches a stop codon, it leaves the mRNA --> mRNA is free to fold into its functional conformation - Ribosomes require help from proteins to carry out translation via three distinct stages

Direct repair of thymine dimers

- Photoreactivation is one mechanism of repairing UV-induced pyrimidine dimers - Energy from visible light activates a photoreactivating enzyme that breaks the cyclobutane ring structure, reversing the dimerization reaction - Many types of cells use photoreactivation, but it is not universal --> placental mammals lack this mechanism

Patterns of histone modification

- Post-translational histone modifications (methylation, phosphorylation, acetylation) affect gene expression by altering chromatin properties, and by providing binding sites for proteins that activate or repress transcription - Distinct patterns of histone modifications are characteristic of transcriptionally active and inactive chromatin - Active chromatin: acetylation of a number of lysine residues, methylation of some lysine and arginine (also positive) residues, phosphorylation of other residues - Inactive chromatin: no acetylation, methylation on lysine residues that would otherwise be acetylated (competing), no phosphorylation - Methylated H3 lysine-9 and -27 residues are binding sites for proteins that induce chromatin condensation and formation of heterochromatin

How does DNA synthesis finish?

- Problem: DNA polymerase can't copy the 3' end of the lagging strand template after the RNA primer is removed (because there is no primer ahead to extend from) - If it were dependent only on DNA pol, chromosomes would get shorter with every round of replication - Solution: extension of the old strand --> provides "space" for primase to create an RNA primer for DNA pol - Telomeres are simple-sequence repeats (TTAGGG) --> they are maintained by telomerase, which catalyzes synthesis of telomeres in the absence of a DNA template - Telomerase is a reverse transcriptase (a class of DNA polymerases) - Telomerase carries its own template RNA, which is complementary to the telomere repeat sequences --> provides an RNA template to make DNA that will match the simple-sequence repeats in telomeres - Multiple copies of the telomeric repeat sequences can be generated to maintain telomeres

Promotors in bacteria

- Promotors in bacteria are six nucleotides long and are located at 10 and 35 base pairs upstream of the transcription start site - -10 element: 5'-TATAAT-3' - -35 element: 5'-TTGACA-3' - Consensus sequences are the bases most frequently found in different promotors - Experiments show the functional importance of -10 and -35 regions: (1) genes with promotors that differ from the consensus sequences are transcribed less efficiently; (2) mutations in these sequences affect promoter function; (3) the σ subunit binds to both regions - σ subunit binding assembles the rest of RNA polymerase

Protein degradation

- Protein levels in cells are determined by rates of synthesis and rates of degradation - Half-lives of proteins vary greatly --> differential rates of degradation are important in cell regulation - Many regulatory protein have short half-lives --> this allows levels to change quickly in response to external stimuli - Faulty or damaged proteins are recognized and degraded - Two pathways for protein degradation: (1) the ubiquitin-proteasome pathway --> rapid, regulated degradation of proteins; (2) lysosomal proteolysis --> slow, gradual turnover of proteins and other cell constituents

Once in the ER, proteins are further trafficked to their final destination via the Golgi apparatus. How are these proteins trafficked through the cell?

- Protein transit from ER to final destination is mediated by vesicular transport - Four steps - (1) Sorting of cargo into vesicle bud - (2) Proteins and phospholipids are exported from the ER in vesicles that bud from a specialized region of the ER, the ER exit site (ERES) - (3) Vesicle docking to target membrane - (4) The vesicles fuse to form the ER-Golgi intermediate compartment (ERGIC), then move to the Golgi apparatus - Proteins targeted for export have peptide and carbohydrate signals that direct their packaging into transport vesicles - Lumenal ER proteins targeted for the Golgi are bound by transmembrane proteins that are selectively packaged into vesicles - Unmarked proteins in the ER can also be packaged and transported to the Golgi by a default pathway - Proteins that function within the ER are recognized in the ERGIC or Golgi and transported back to the ER --> these proteins, such as BiP, have a targeting sequence (KDEL or KKXX) at the carboxy terminus that directs retrieval back to the ER

Transport from the Golgi apparatus

- Proteins are sorted in the trans-Golgi network and transported in vesicles to their final destinations - Proteins can be transported to the plasma membrane either directly or via recycling endosomes - In addition, proteins can be sorted into distinct secretory granules for regulated secretion - Alternatively, proteins can be targeted to late endosomes, which develop into lysosomes

Transcription of the ribosomal RNA gene

- RNA pol I is the enzyme that carries out transcription for ribosomal genes - 18S, 5.8S, and 28S ribosomal subunits are organized on one gene close together (separated by spacer DNA) and are often located in tandem within the genome - Two transcription factors, UBF (upstream binding factor) and SL1 (selectivity factor 1), bind cooperatively to the rDNA promoter and recruit RNA pol I to form an initiation complex - Generally have a chain of polymerases reading the sequence to generate multiple copies - Transcription yields a large RNA molecule (45S pre-rRNA), which is then cleaved into 28S, 18S, and 5.8S rRNAs - 5S subunit is transcribed by RNA pol III

Overview of eukaryotic transcription of mRNA genes

- RNA pol II binds promoter of gene - Assemble general TFs, TBP, Mediator - Transcription factors (activators and repressors) get involved, bind to specific sequences, and interact with Mediator to affect ability of RNA pol II - Also have regulation of gene expression via regulation of chromatin structure (HATs, HDACs) - Serines in CTD of RNA pol II needs to be phosphorylated --> provides scaffold for proteins to process during transcription - RNA pol II reads DNA 3' to 5', writes 5' to 3' - Primary RNA transcript is processed (5' capping, splicing, 3' polyadenylation) - Ultimately, the mature RNA transcript will be transported out of the nucleus and translated into a protein by ribosomes

RNA interference (RNAi)

- RNAi, mediated by short double-stranded RNAs, is used as an experimental tool to block gene expression at the level of translation - In cells, it is an important mechanism of translational regulation - Eukaryotic genomes contain genes encoding dsRNA (miRNAs), which regulate gene expression by RNAi - RNAi is mediated by small interfering RNAs (siRNAs) and microRNAs (miRNAs) - siRNAs are produced from dsRNAs by the nuclease Dicer - miRNAs are transcribed by RNA pol II, then cleaved by nucleases Drosha and Dicer - One strand of miRNA or siRNA is incorporated into an RNA-induced silencing complex (RISC) - siRNAs generally pair with their targets and induce cleavage of the mRNA - Most miRNAs form mismatches in the 3' UTRs that repress translation - The miRNA/RISC complex represses translation and targets the mRNA for degradation by stimulating deadenylation - There are about 1000 human miRNAs --> each targets ~100 different mRNAs - About 1/3 of human genes are regulated by miRNAs - siRNA has perfect pairing, which leads to cleavage - Mismatched pairing leads to repressed translation

Regulation of protein function through the binding of small molecules (allosteric regulation)

- Regulation of enzymes by small molecules that bind to a site distinct from the active site, changing the conformation and catalytic activity of the enzyme - Most enzymes are controlled by changes in conformation, often as a result of binding small molecules - This type of regulation is common in controlling metabolic pathways by feedback inhibition - Binding of a small molecule can be inhibitory or activating - Ex: eIF2 is subject to allosteric regulation by GTP/GDP binding --> GTP is activating because it promotes substrate binding and GDP is inhibitory because it inhibits substrate binding - Allosteric regulation by GTP/GDP is a key regulatory mechanism for many proteins --> collectively called GTP-binding proteins - Ex: GTP-binding Ras oncogene proteins - The Ras proteins alternate between active GTP-bound and inactive GDP-bound forms - The major effect of GTP binding vs. GDP binding is alteration of the conformation of two regions of the molecule, designated the switch I and switch II regions - Mutations in Ras genes contribute to 25% of human cancers --> Ras proteins are altered to be locked in the active GTP-bound conformation and continually signal cell division - Many transcription factors are regulated by allosteric regulation --> ex: the lac operon - Steroid hormones (e.g., estrogen) regulate transcription factors in mammalian cells

Elongation of translation using eukaryotic elongation factors (eEFs)

- Ribosomes have three binding sites: P (peptidyl), A (aminoacyl), and E (exit) sites - The initiator methionyl tRNA binds to the P site - The next aminoacyl tRNA binds to the A site by pairing with the second codon of the mRNA - An elongation factor (EF-Tu in prokaryotes, eEF1𝛼 in eukaryotes) complexed to GTP brings the aminoacyl tRNA to the ribosome (powered by GTP) - Selection of the correct aminoacyl tRNA determines the accuracy of protein synthesis - Base pairing alone can't account for the accuracy of protein synthesis - A "decoding center" in the small ribosomal subunit recognizes correct codon-anticodon base pairs and discriminates against mismatches - Insertion of the correct aminoacyl tRNA at A triggers a conformational change that induces the hydrolysis of GTP/eEF1𝛼 and release of the elongation factor - The peptide is then formed, catalyzed by the large ribosomal subunit - Translocation --> he initiator tRNA (uncharged) is now at the P site, aminoacyl tRNA in A site - Translocation is energy-dependent process --> uses eEF2 bound to GTP - Initator tRNA is released and recycled - Elongation is mediated by eEF2, coupled to GTP hydrolysis - Need one eEF1𝛼 and one eEF2 for each AA added - As elongation continues, translocation moves the peptidyl tRNA from A to P, and uncharged tRNA from P to E - A new aminoacyl tRNA binds A site and induces release of the uncharged tRNA from the E site - Translocation requires another elongation factor (EF-G in prokaryotes, eEF2 in eukaryotes) and is coupled to GTP hydrolysis - This continues until a stop codon is translocated into the A site

Stages of translation

- Ribosomes require help from proteins to carry out translation via three distinct stages (initiation, elongation, termination) - Initiation: small ribosomal subunit binds at 5' UTR and initiates polypeptide synthesis at the start codon with methionyl tRNA --> large subunit then joins, forming a functional ribosome - Translation initiation requires several eukaryotic Initiation Factors (eIFs) - Elongation: polypeptide chain elongates by successively adding amino acids - Translation elongation requires several eukaryotic Elongation Factors (eEFs) - Termination: when a stop codon is encountered, polypeptide is released and ribosome dissociates

3' polyadenylation of pre-mRNA

- Second post-translational modification of pre-mRNA - At the 3' end, a poly-A tail is added by poly-A polymerase through polyadenylation - Poly-A polymerase is associated with the CTD of RNA pol II and modifies transcribed RNA as transcription takes place - Poly-A tail is not actually at the very end of the transcript --> it's somewhat 5' of the 3' end - Every gene contains a poly-A site, which has three sequence elements: (1) hexanucleotide AAUAAA...CA; (2) GU-rich downstream element; (3) upstream element - Cleavage and polyadenylation of mRNA 3' ends is directed by these three sequence elements - Downstream region is recognized and cut off --> endonuclease cleaves right after AAUAAA...CA - Poly-A polymerase then recognizes the new 3' end and adds about 200 adenines to form the poly-A tail - Addition of poly-A tail has two functions: (1) regulates translational rates; (2) regulates mRNA stability - If poly-A tail is too short, the mRNA cannot be translated

How do signal sequences target proteins to the ER?

- Signal sequences are required for the sorting of proteins to several compartments - Signal sequences (about 20 AAs) include a stretch of hydrophobic residues and are usually located at the amino terminus of the polypeptide chain - Cotranslational targeting: as they emerge from the ribosome, signal sequences are recognized by SRP (signal recognition particle), which directs nascent proteins to the ER via interactions with the SRP receptor - Nascent = proteins in process of being translated by a ribosome - SRPs consist of 6 polypeptides and a small cytoplasmic RNA (SRP RNA) - SRP binds ribosome and signal sequence, inhibiting further translation - The entire complex binds to an SRP receptor on the rough ER membrane - The SRP is then released, and the ribosome binds to a membrane channel or translocon - Insertion of signal sequence opens translocon by moving a plug away from the channel - The signal sequence is inserted into the translocon, and translation resumes - Elongation of polypeptide drives its transfer through the translocon - The signal sequence is cleaved by signal peptidase and released into the ER lumen - Early studies showed that N-terminal signal sequences target proteins for co-translational import into ER --> more recent research has shown that some proteins are targeted to the ER post-translationally - In post-translational translocation (more common in yeast), polypeptides are targeted to the ER when translation is complete - The signal sequences are recognized by different receptors on the translocon - Hsp70 and Hsp40 chaperones keep the polypeptide chains unfolded so they can enter the translocon

Nuclear pore complex

- Sole channels for all transport into and out of nucleus - Nuclear membranes are phospholipid bilayers permeable only to small nonpolar molecules - Nuclear pore complexes are the only channels for small polar molecules, ions, and macromolecules - Nuclear pore complexes are large --> about 30 proteins (nucleoporins) or NUPs - RNAs synthesized in the nucleus must be exported to cytoplasm for protein synthesis - Proteins needed for nuclear functions must be imported form synthesis sites in cytoplasm - Electron microscopy shows pore complexes have 8 subunits organized around a large central channel made up of FG-NUPs, which are rich in phenylalanine and glycine - FG-NUPs work as a sort of filter so larger proteins can't get through unless there's some sort of energy involved - 8 spokes are connected to rings at the nuclear and cytoplasmic surfaces (nuclear ring and cytoplasmic ring) --> spoke-ring assembly surrounds a central channel - 8 protein filaments extend from the rings, forming a basket-like structure on the nuclear side - Nuclear basket attached to nuclear ring - Two modes of passage, dependent on size - Small molecules and small proteins (<20-40 kD) can pass freely in either direction via passive diffusion - RNAs and most proteins (>20-40 kD) are selectively transported, which requires energy

Transcriptional repressors

- Some transcriptional repressors also act via protein-protein interactions with the general transcription machinery --> these are sometimes referred to as "active repressors" - Two domains: DNA-binding domain and repression domain - Another way transcriptional repressors work is blocking activation (or elongation) of transcription by RNA pol II (ex: repressor protein in lac operon) --> can do this by competing with activator for same binding site

Binding of translational repressors to 3' UTR sequences

- Some translational repressors bind to specific sequences in the 3' UTR - Some bind to initiation factor eIF4E, interfering with its interaction with eIF4G and inhibiting initiation of translation - These repressors bind to specific sequences in the 3' UTR of some mRNAs

Initiation of translation at internal ribosome entry sites

- Some viral and eukaryotic mRNAs have IRES where translation can initiate independent of the 5' methyl cap - Internal ribosome entry sites (IRES) in some mRNAs can be recognized by eIF4G in complex with eIF4A, followed by recruitment of the 40S ribosomal subunit complexed to the initiator methionyl-tRNA bound by eIF2 - Alternatively, IRESs in other mRNAs are recognized directly by the 40S ribosomal subunit

Overview of protein sorting and transport

- Sorting and targeting of proteins to appropriate destinations are important tasks - The endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes are involved in protein processing and are connected by vesicular transport - The endoplasmic reticulum (ER) is a network of membrane-enclosed tubules and sacs (cisternae), extending from the nuclear membrane throughout the cytoplasm - The membrane is continuous and is the largest organelle of most eukaryotic cells - The ER has two domains that perform different functions: (1) rough ER covered by ribosomes on outer surface; (2) smooth ER has no ribosomes and is involved in lipid metabolism - Nuclear proteins are imported into nucleus by importins following translation on free ribosomes - Proteins synthesized on free ribosomes stay in the cytosol or are transported to the nucleus and other organelles - Proteins synthesized on membrane-bound ribosomes are translocated directly into the ER - Proteins destined for several other compartments are trafficked differently --> ex: plasma membrane proteins, secreted proteins, ER proteins, Golgi proteins, lysosomal proteins - These proteins are trafficked to their respective compartments through a common pathway: the secretory pathway - In eukaryotic cells, initial sorting takes place while translation is in progress

Chaperonins

- Sub-class of chaperones - Provide an isolated environment within which correct folding takes place - Chaperonins are found in both prokaryotic and eukaryotic cells - Chaperonins act in concert with Hsp70 chaperones to facilitate folding of newly translated proteins - After a polypeptide is translated, the partially folded intermediate is transferred to a chaperonin, where folding takes place --> requires energy from ATP - Chaperonins consist of subunits arranged in two stacked rings to form a double-chambered structure --> this isolates the protein from the cytosol and other unfolded proteins

If DNA polymerase requires a pre-existing primer, then how does the cell initiate synthesis of Okazaki fragments?

- Synthesis of Okazaki fragments is initiated by an RNA polymerase (primase) - Key: RNA polymerases can generate polynucleotides de novo (i.e., without a pre-existing primer) - Primers made are typically 10-20 bp long - Primase incorporates U instead of T because it's an RNA polymerase - Once the RNA primer is long enough, DNA polymerase can extend it - RNA primers are removed by exonuclease activity and the gap is filled in with DNA by DNA polymerase - Okazaki fragments are joined together by DNA ligase

How do transcription factors regulate transcription of genes?

- TFs act as either transcriptional activators or transcriptional repressors - One general mechanism: regulate the binding and/or activity of the general transcription machinery at a gene promoter (via direct protein-protein interactions or masking binding sites) - Another common mechanism: regulate the physical accessibility of a gene to the transcription machinery and/or process of transcription (via modification of chromatin structure --> modifications of histones or rearrangement of nucleosomes)

Initiation of transcription in eukaryotes

- Takes place on chromatin in the nucleus --> regulation of chromatin structure is important in regulating gene expression - RNA pol II is recruited to promotors by general transcription factors - In vitro systems identified 5 general TFs ("TFIIx") - TFIID is composed of multiple subunits, including TATA-binding protein (TBP) and other subunits (TBP-associated factors/TAFs) that bind to the lnr, DCE, MTE, and DPE sequences - After TFIID binds, TFIIB and TFIIF bind and are involved in recruitment of RNA pol II - TFIIE and TFIIH bind to form "pre-initiation complex" - TFIIH has two roles: (1) functions as a helicase; (2) has CTD kinase activity (to phosphorylate middle Ser of CTD) - Carboxy-terminal domain (CTD) of RNA pol II is 52 repeats (in humans) of Tyr-Ser-Pro-Thr-Ser-Pro-Ser --> phosphorylation of CTD (especially middle Ser) is necessary for RNA pol II to initiate transcription - CTD also serves as a binding site for a number of proteins carried along by RNA pol during transcription --> these proteins function to process primary RNA transcript to make it into mRNA - Additional factors contribute to transcription initiation within cells --> ex: Mediator (protein complex of 20+ subunits) facilitates interactions between RNA pol II and regulatory factors - Once CTD is phosphorylated, elongation and processing factors associate with it, and RNA pol is able to start transcribing - When RNA pol starts moving down the DNA, all TFs stay behind except TFIIF

Human telomeres and telomerase

- Telomerase activity maintains telomeres at their normal length - Most somatic cells don't have enough telomerase to maintain telomere length for an indefinite number of cell divisions - Telomeres gradually shorten as cells age, which eventually leads to cell death or senescence - Several premature aging syndromes are characterized by abnormally high rates of telomere loss; some are caused by mutations in telomerase - Conversely, cancer cells express abnormally high levels of telomerase, allowing them to continue dividing indefinitely

Proteolytic processing of insulin

- The mature insulin molecule consists of two polypeptide chains (A and B) joined by disulfide bonds - Insulin is synthesized as a precursor polypeptide (preproinsulin) containing an amino-terminal signal sequence that is cleaved by proteolytic enzymes (not chaperones) during transfer of the growing polypeptide chain to the ER - This cleavage yields a second precursor (proinsulin), which is converted to insulin by further proteolysis, removing the internal connecting polypeptide

Roles and events of the nucleus

- The nucleus is the main feature that distinguishes eukaryotic from prokaryotic cells - DNA replication - Transcription - Storage and maintenance of genetic information - RNA processing and RNP assembly - Separation of the genome from the site of mRNA translation plays a central role in eukaryotic gene expression

Transport to the plasma membrane of polarized cells

- The plasma membranes of polarized epithelial cells are divided into apical and basolateral domains - In this example (intestinal epithelium), the apical surface of the cell faces the lumen of the intestine, the lateral surfaces are in contact with neighboring cells, and the basal surface rests on a sheet of extracellular matrix (the basal lamina) - The apical membrane is characterized by the presence of microvilli, which facilitate the absorption of nutrients by increasing surface area - Specific proteins are targeted to the apical or basolateral membranes either in the trans-Golgi network or in a recycling endosome - Tight junctions between neighboring cells maintain the identity of the apical and basolateral membranes by preventing the diffusion of proteins between these domains

How are ATP, ADP, Pi (and other metabolites) transported across the inner mitochondrial membrane?

- The proton gradient drives metabolite transport through the transporters - ATP/ADP exchange is driven by the voltage - ATP carries a greater negative charge (-4) than ADP (-3), so ATP is exported from the mitochondrial matrix (negative) to the cytosol (positive), while ADP is imported into mitochondria (negative) - Pi (H2PO4-) and pyruvate import is coupled to OH- export, which is driven by pH difference - OH- in matrix is one reason why matrix pH is so high --> OH- wants to leave matrix and go to lower pH of intermembrane space - Pyruvate has -1 charge (acidic) --> energetically favorable to flow from matrix (ph 7) to intermembrane space (pH 8) - Pyruvate and OH- have same charge so flow isn't creating a voltage difference

Key study on secretory pathway

- The role of the ER in protein processing and sorting was first demonstrated by Palade and colleagues in the 1960s - Examined the path through which newly synthesized proteins are secreted from pancreatic acinar cells that secrete digestive enzymes into the small intestine - Method: (1) pulse-labeled newly synthesized proteins with radioactive "hot" amino acids --> location of the radiolabeled proteins was then determined by autoradiography - Method: (2) "chased" with non-radioactive "cold" amino acids - Method: (3) tracked location of radioactive protein through time - 3-minute label --> radioactivity found only in ER - 7-minute chase --> all radioactive proteins from ER were now in Golgi - 120-minute chase --> radioactivity in secretory vesicles or being released from cell - These experiments defined the secretory pathway: rough ER --> Golgi --> secretory vesicles --> cell exterior - Further studies showed a similar pathway for non-secreted proteins

Insertion of a membrane protein with a cleavable signal sequence

- The signal sequence is cleaved as the polypeptide chain crosses the membrane, so the amino (N) terminus of the polypeptide chain is exposed in the ER lumen - However, translocation of the polypeptide chain across the membrane is halted when the translocon recognizes a transmembrane sequence --> this allows the protein to exit the translocon laterally and become anchored in the ER membrane - Continued translation results in a membrane-spanning protein with its carboxy (C) terminus on the cytosolic side - Examples: LDL receptor and related family members

Structure of translocon

- The translocon consists of three transmembrane subunits - Lots of a-helices - Channel/pore with plug in the middle

Pre-mRNA splicing

- Third post-transcriptional modification of pre-mRNA --> generates mRNA - Splicing factors and spliceosome associate with CTD of polymerase II, so splicing happens during transcription --> association of splicing factors with CTD assures exons are joined in order 5' to 3' - mRNA splicing takes place in RNA/protein complexes called spliceosomes, which have five types of small nuclear RNAs (snRNAs) of 50-200bp --> U1, U2, U4, U5, and U6 - The snRNAs are complexed with 6-10 protein molecules to form small nuclear ribonucleoprotein particles (snRNPs) - snRNP snRNAs play two roles: (1) recognize and align the snRNPs at the branch point and splice sites; (2) directly catalyze the splicing reaction (this is known because the reactions can be catalyzed by U2 and U6 snRNAs in absence of protein) - Three sequence elements of pre-mRNAs are important: (1) 5' splice site; (2) 3' splice site; (3) branch point within intron - Pre-mRNAs contain similar consensus sequences at each of these positions - Sequences between introns that define splice sites are only a few bp long - Intron starts with GU and ends with AG --> these are very common in longer introns, so the information required for splicing is not necessarily lying in the intron itself (it also involves interactions with exons) - Introns frequently contain sequences that are similar to splice sites, but are not splice sites --> splicing factors help identify correct splice sites - Splicing factors: proteins that facilitate splicing by snRNPs by guiding them to correct splice sites (they do this by recognizing the exon sequences) - Splicing factors are from family SR - Some RNAs can self-splice --> they catalyze removal of their own introns in the absence of other protein or RNA factors - Splicing proceeds in two steps STEP 1: - SR splicing factors bind to specific sequences in exons and recruit U1 snRNP to 5' SS - First step in spliceosome assembly is binding of U1 snRNP to the 5' SS - Recognition of 5' SS involves base pairing between 5' SS consensus sequence and a complementary sequence at the 5' end of U1 snRNA - U1 cleaves at the 5' splice site GU - U2AF binds to pyrimidine-rich sequences at the 3' SS and U2 snRNP is recruited to branch point - U4, U5, and U6 are recruited and all snRNPs join 5' end of the intron to an adenine within the intron (branch point) to form a lariat-like loop intermediate - Once lariat is formed, U1 and U4 leave STEP 2: - Remaining snRNPs U2, U5, and U6 cleave at the 3' SS and ligate the exons - This excises the intron loop

Modification of chromatin structure via rearrangement of nucleosomes

- This is done via chromatin remodeling factors - Chromatin remodeling factors are protein complexes that move or eject histones to allow for binding of the transcriptional machinery - They alter contacts between DNA and histones and can reposition nucleosomes, change conformation of nucleosomes, or eject nucleosomes from DNA - Chromatin remodeling factors are coactivators --> recruited to DNA by activator proteins --> allows for displacement of nucleosome and binding of general TFs and RNA pol - Chromatin remodeling factors bind to the CTD of RNA pol II and modulate chromatin structure as RNA pol II transcribes

Transcriptional activators

- Transcription factors that activate transcription - Two domain structure --> domain 1 binds DNA at specific sequence (i.e., TF binding site); domain 2 activates transcription by interacting with general transcription machinery via protein-protein interactions - Transcriptional activators like Sp1 bind to regulatory DNA sequences and stimulate transcriptions --> domain 1 binds DNA and domain 2 interacts with Mediator - Mediator mediates regulation of transcription by TFs - These might facilitate better recruitment of RNA pol II to a promoter or facilitate better activation of RNA pol II at a promoter

Fundamentals of translation

- Translation is the synthesis of proteins as directed by mRNA templates, the first step in the formation of functional proteins - Polypeptide chains must fold into appropriate conformations and often undergo various processing steps, sorting, and transport - Gene expression is regulated at the level of translation in both prokaryotic and eukaryotic cells - There are also multiple controls on amount and activities of proteins, which ultimately regulate all aspects of cell behavior - During translation, the nucleotide sequence is read in triplets (codons) - Each codon codes for a specific amino acid - Proteins are synthesized from mRNA templates by a process that has been highly conserved throughout evolution - Codons are read 5' to 3' (different from transcription!) to direct polypeptide synthesis amino (N) to carboxy (C) terminus - If you're trying to write the protein that results from an mRNA transcript, start by looking for the start codon (AUG) - Translation ends at a stop codon - The 5' and 3' ends of mRNA are not translated into protein (called 5' and 3' untranslated regions, or 5' UTR and 3' UTR) --> larger than protein-coding sequence - The region that codes for amino acids is called the open reading frame (ORF) - Translation is carried out on ribosomes, with tRNAs serving as adaptors between codons and AAs - Protein synthesis involves interactions between the three types of RNA (mRNA, tRNA, rRNA), plus other proteins - Important prerequisite prior to translation is attaching the correct amino acids to the correct tRNAs - Translation requires an enzyme to catalyze peptide bond formation --> peptide bond formation is catalyzed by ribosomes

Deamination and depurination

- Two major forms of spontaneous damage to DNA - Deamination is the removal of an amine group (NH2) --> deamination of cytosine produces uracil; deamination of adenine produces hypoxanthine - Depurination: b-N-glycosidic bond is hydrolytically cleaved, releasing adenine or guanine --> dGMP to AP site

Inner and outer membranes of the nuclear envelope

- Two phospholipid bilayers and lumen (perinuclear space) - The outer membrane is continuous with the ER - The space between the inner and outer membranes (perinuclear space) is directly connected with the lumen of the ER - The inner membrane has integral proteins, including ones that bind the nuclear lamina

Transcriptional regulators

- Two types: cis-acting elements and trans-acting factors - Cis-acting elements are DNA sequences in the vicinity of a gene that regulate its expression (e.g., operator) - Trans-acting factors are factors (i.e., proteins) that regulate a gene's expression and are encoded by a different gene elsewhere in the genome (e.g., repressor)

tRNA

- tRNAs align amino acids with corresponding codons on the mRNA template - They are 70-80 nucleotides long and have characteristic cloverleaf structures resulting from base pairing (H bonding) between different regions - tRNAs have other nucleotides in addition to A, C, G, and U --> a lot of nucleotides have modified because tRNAs undergo a lot of processing - Each tRNA has an anticodon and a 3' overhang (AA attachment site) - tRNA sequences and modifications are not identical - All tRNAs are derived from individual tRNA genes with their own promoters, transcribed by RNA pol III - RNA pol III creates a pre-tRNA transcript, which undergoes processing steps to generate mature tRNA (with no AA attached yet) - All tRNAs fold into compact 3D L shapes to fit onto ribosomes during translation - They have the sequence CCA at the 3' end, and amino acids are covalently attached to the ribose of the terminal adenosine - The anticodon loop binds to the appropriate codon by complementary base pairing - Attachment of amino acids to specific tRNAs is mediated by enzymes called aminoacyl tRNA synthetases --> each of these 20 enzymes recognizes a single amino acid, as well as the correct tRNA to which it should attach - 20 enzymes for 20 amino acids, but ~45 tRNAs

Nuclear lamina diseases

MUSCULAR DYSTROPHY AND THE NUCLEAR ENVELOPE: - X-linked Emery-Dreifuss muscular dystrophy - Early in disease, elbows, neck, and heels of affected individuals become stiff, and there is often a conduction block in the heart - Symptoms seen by age 10 and include "toewalking" because stiff Achilles tendons and difficulty bending elbows - Heart problems develop by 20 - Gradual wasting and weakness of shoulder, upper arm muscles, and calf muscles - Mutations in TM protein emerin are responsible - Emerin is localized to inner nuclear membrane and absent in patients with this muscular dystrophy --> unexpected because mutations in nuclear envelope protein expressed in all cells is causing tissue-specific disease - Same dystrophy could also be inherited in a non-sex-linked manner - Families with non-sex-linked version had mutations in gene (LMNA) encoding A-type nuclear lamins - Mutations in one of two genes (one coding for inner membrane protein and one coding for major nuclear lamin) caused clinically identical muscular dystrophy MULTIPLE DISTINCT LAMINOPATHIES: - Laminopathies result from defects in nuclear lamina - There are now nearly 15 diseases known to arise from mutations in LMNA, including muscular dystrophies, cardiomyopathies, and premature aging syndromes - This is larger than the list of diseases arising from any mutations in any other human gene Mutations in other nuclear envelope proteins, including lamin B, lamin B receptor, and components of LINC complex are responsible for additional disorders - Two hypotheses for how mutations in these proteins cause so many distinct diseases MECHANICAL STRESS OR ABERRANT GENE EXPRESSION: - Mechanical stress hypothesis: mutations affecting nuclear lamina are thought to weaken the structural integrity of nucleus and connection to cytoskeleton - Mechanical stress hypothesis works best for muscular dystrophies because of stress placed on muscle cells --> suggests that lamin mutations make nucleus more vulnerable to stress transmitted via cytoskeletal interactions - Gene expression hypothesis: correct interaction of lamins with nuclear envelope is essential for normal tissue-specific expression of certain genes - Transcriptionally inactive genes are located preferentially at nuclear periphery, whereas expressed genes are concentrated in center of nucleus with cell-type specificity - Basis of these diseases could be a change in gene expression caused by defective chromatin localization - The lamins also interact with proteins in DNA replication and repair --> could provide further molecular basis for laminopathies

Replication and transcription factories

REPLICATION FACTORIES: - Most nuclear processes occur in distinct regions - DNA replication takes place in large complexes called replication factories, where replication of multiple DNA molecules takes place - These can be seen by labeling cells with bromodeoxyuridine (analog of thymidine), then staining with fluorescent antibodies - This staining overlaps with replication-specific protein PCNA TRANSCRIPTION FACTORIES: - Transcription occurs at clustered sites (transcription factories) that contain newly synthesized RNA - Coregulated genes, such as immunoglobulin genes from different chromosomes, may be transcribed in same factory

Golgi: modification of N-linked sugars added in the ER

The carbohydrate portions of glycoproteins are extensively modified in the Golgi - Specific processing of N-linked oligosaccharides in the Golgi targets glycoproteins to their final destination - N-linked oligosaccharides that were added in the ER are modified by a sequence of reactions catalyzed by enzymes in different compartments - Targeting of proteins to the plasma membrane entails sequential reactions --> ex: LDL receptor - the LDL receptor, critical for cholesterol metabolism and involved in hypercholesterolemia, is both N- and O-glycosylated - These modifications ultimately give the protein stability and also help direct the LDL receptor to its final destination, the plasma membrane - Specific processing of N-linked oligosaccharides in the Golgi targets glycoproteins to their final destination --> ex: lysosomal luminal proteins - N-linked oligosaccharide of lysosomal proteins are modified by mannose phosphorylation - Lysosomal luminal proteins are targeted by attachment of mannose-6-phosphate - They are recognized by a mannose-6-phosphate receptor in the trans Golgi network, which transports them to endosomes and on to lysosomes