Mol. bio ALL quiz 3

What are organelles

membrane bound compartments located within the cell 

• Self-replicating 

• Highly specialized for protein synthesis, segregation/transport, energy production, & protein degradation 

• Allows hydrophobic things to hide from the aqueous environment 

• Cytosol & mitochondria are the MAJORITY of the cell volume 

Compare the rough and smooth ER

Rough ER	Smooth ER
Studded with ribosomes  Located in ALL eukaryotic cells  Abundant in liver (for protein synthesis)	Production and storage of glycogen, steroids, and other macromolecules  Sequestration of calcium  NO ribosomes  Located in cells that specialize in lipid metabolism (cells that synthesize steroid hormones from cholesterol)  More in liver for lipoprotein synthesis  Membrane contains cytochrome enzymes for detox functions

What is the ER

• Interconnected network of tubules, vesicles, and cisternae (network) 

• Transportation system of eukaryotes 

• Protein translation 

• Folding & transport of proteins to be used in some cell membrane or to be exocytosed from cell 

• Rebuilding nuclear envelope after cytokinesis 

• N-linked glycosylation of proteins 

• Lots of proteins to help with folding 

What is the Golgi apparatus (function, structure, purpose)

synthesis of carbohydrates, sorting and exporting ER products, synthesis of glycosaminoglycans (GAGs) (protein modifications) 

• Function: Primarily modifies proteins delivered from the rough ER through glycosylation & phosphorylation 

  • Important in processing proteins for secretion 

• Involved in creating lysosomes & endosomes 

• Structure: Composed of membrane-bound stacks known as cisternae 

  • Between 5-8 

  • Has 5 functional regions (each has specific enzymes on membrane surface  

1. Cis-golgi network 

2. Cis-cisterna 

3. Medial-cisterna 

4. Trans-cisterna 

5. Trans-Golgi network 

   • Proteins move sequentially through the 5 regions 

   Purpose: adding things to protein chain to help it communicate or reach destination (processing) 

1. Secondary oligosaccharide processing

2. Glycosylation = necessary for protein folding 

Describe secondary oligosaccharide processing and glycosylation (in Golgi)

1. Secondary oligosaccharide processing = oligosaccs added in ER 

• Further glycosylation and phosphorylation occurs in Golgi with different enzymes at each region of Golgi (mucins and proteoglycan formation) 

2. Glycosylation = necessary for protein folding 

• Resistance to proteolytic digestion, sugars can be recognized by lectins important during development and cell-cell adhesion 

• n-linked = can be done in golgi or ER 

• o-linked glycosylation = specific on Threonine oxygen 

What are lyosomes

intracellular digestion 

• Carry out digestion function with acid hydrolases (~40) 

• Cell protected from lysosmal function by the lysosomal membrane 

  • Highly glycosylated & pH environment in lysozyme, making hydrolytic enzymes non-functional  

    • Lysosomal pH = maintenance of lysosomal pH via vacuolar H+ ATPase that pumps hydrogen ions into the lysosome  

What are endosomes

Before lysosomes form, endocytosis forms early endosomes and then late endosomes and eventually lysosomes

• On the way to lysosomes, endocytosed material must first pass through a series of organelles

  called endosomes

What are transport vesicles

form specialized coated regions of membranes 

• Coats include Clathrin (P -> G), COPII (ER -> G), & COPI (G -> ER) 

What are COPII-coated vesicles

ER to Golgi (Sar 1 -> adaptor protein -> outer coat proteins -> vesicle formed) 

1. Active Sar 1-GTP (membrane bound to ER) recruits COPII adaptor proteins and causes the membrane to deform  

2. The adaptor proteins will then recruit the outer coat proteins to help form the bud 

3. Exit signal on cytosolic tails of cargo proteins interact with membrane cargo receptors and soluble ER proteins & complex into budding transport (interacting with Sar 1-GTP and COPII proteins) 

4. A subsequent membrane fusion event pinches off the coated vesicle 

5. Vesicles shed COPII coat and fuse with each other with SNARES, forming vesicular tubular clusters 

What are COPI-coated vesicles

Proteins without this signal can leak out of the ER 

• Retrieved by COPI proteins with retrieval signals (KDEL & KKXX) 

• KDEL receptors in tubular clusters and Golgi capture the escaped protein and carry them back to ER with COPI coated vesicles

How does Clathrin work

Golgi to the plasma membrane

• Receptors on trans-Golgi for M6P put clathrin-coated protein and transport it to the lysosome 

How does ER to Golgi work

• If proteins are properly folded & assembled, they’ll be packaged into COPII coated transport vesicles that bud from the ER membrane and go to the Golgi  

• Proteins get into the transporter by... 

  • Selective process = exit (transport) signal on proteins that COPII recognizes 

    • Proteins without this signal can leak out of the ER 

      • Retrieved by COPI proteins with retrieval signals (KDEL) 

      • KDEL receptors in tubular clusters and Golgi capture the escaped protein and carry them back to ER with COPI coated vesicles 

    • Loss of transporters can lead to various disorders (fat absorption, protein secretion) 

  • Some proteins only need a receptor on the ER membrane (because are soluble) 

How does the process to the golgi work? What is homotypic fusion?

• After the vesicle forms from the ER, the COPII proteins are released and the vesicles begin to fuse forming vesicular tubular clusters 

• Homotypic fusion = utilizes SNARE proteins (a t-SNARE and v-SNARE are required to force lipid bilayers closed and expel water) 

How does homotypic fusion work

• Homotypic fusion = utilizes SNARE proteins (a t-SNARE and v-SNARE are required to force lipid bilayers closed and expel water) 

  • SNARE = transmembrane proteins that catalyze membrane fusion reactions 

    • v & t- SNARES are wrapped together 

    • NSF protein (N-ethylmaleimide sensitive factor/fusion protein) prys v and t-SNARE apart 

    • v & t-SNARE bind with other v and t-snare on a different vesicle 

    • They fuse/wrap together 

    • Repeated  

What are vesicular tubular clusters

Vesicular tubular clusters = multiple vesicles fuse together so only 1 large one is transported to the Golgi 

• Pulled along microtubules to Golgi Cis face 

What is Co-translation translocation

Co-translational translocation = in rER, proteins are imported before they are completely translated 

• Ribosome is attached to ER (via special particles and sequences) 

• uses SRP

What is post-translational translocation

Post-translational translocation = Free ribosomes complete protein synthesis and release prior to the protein being translocated to a mitochondrial/nuclear membrane (NOT ER PROCESS) 

What is a signal sequence

a short sequence of amino acids at the N-terminal that direct secreted proteins to the ER & is then cleaved off by signal peptidase 

• Different for different polypeptides but contains several nonpolar AAs 

• Found in both transmembrane (destined for ER) and water-soluble (secreted or stay within organelle) proteins 

• Used in both co-translational and post-translational translocation 

• Not part of the final protein because it gets cleaved off by signal peptidase 

  • Signal peptidase cleaves of N-terminus 

What is a signal recognition particle (SRP)

guides signal sequence to ER translocator and SRP receptor (integral membrane protein on ER membrane – in blue) 

• Has a signal sequence binding site lined with methionine (flexible) allowing it to accommodate many signal sequences 

• One end binds to signal sequence & other blocks E site (stopping translation) 

  • ONLY USED IN CO-TRANSCRIPTIONAL TRANSLOCATION 

• Contains an RNA and 6 protein subunits 

• Rod-like shape that wraps around the large ribosomal subunit  

Where do different parts of membrane proteins remain

Membrane proteins: 

• Alpha helicies = Hydrophobic segments in the peptide chain are anchored in the protein membrane by a stop-transfer signal (stay inside away from the aqueous environment) 

• Water soluble proteins = stay in the cytosol 

• Single-pass transmembrane proteins = pass through the membrane only once (hydrophobic segment stays in the membrane) 

How does the SRP work

1. SRP (with ribosome/signal sequence) binds to SRP receptor on ER membrane 

2. The ribosome (with the polypeptide chain) moves and binds to the translocator on the ER membrane 

3. Peptide is completed within ER cistern

*ONLY WITH CO-TT

What happens after the ER

• Many proteins stay in the ER 

  • Have a KDEL (4 amino acid sequence = retention signal) sequence at C-terminus 

    • Also serves as a retrieval signal if accidently gets sent out of ER 

  • Must be folded properly to stay in the ER 

Ex: BiP = hold proteins for degradation in the ER 

Calnexin & calreticulin (heat shock proteins) 

• Others leave the ER (to the Golgi for further processing) 

  • Processed by adding oligosaccharides (glycosylation) 

What is glycosylation

adding oligosaccharides 

• Occurs at the amine group of an Asparagine 

• N-linked 

• Results in the removal of a mannose and glucoses (“trimming”) 

• Important for transmembrane proteins on extracellular side for cell-cell interactions 

• Oligosaccharide tags onto 90% of glycoproteins & are markers for protein folding 

 

How does movement through the golgi work

Moving through the Golgi:  

• Unclear process but it is known that vesicular transport forms into what is needed to get the job done 

• Directional 

What are secretory vesicles

Secretory vesicles bud from trans-Golgi network packaged into secretory vesicles 

• Secretory proteins first aggregate in vesicles 

• Vesicle contents released in response to certain signals  

What is proteoglycan assembly (and proteoglycan structure)

adding sugar groups to proteins (for stability/rigidity) 

• Golgi proteoglycans assembled in the Golgi 

  • For protection, communication, and adhesion 

• Glycosyl transferase enzymes link oligos to hydroxyl groups of certain amino acids in a protein backbone (addition of GAG chains) 

• Proteoglycans are important in extracellular matrix 

Proteoglycan structure:  

• Hyaluronic acid backbone 

• Link proteins connect core proteins 

• O & N-linked oligosaccharides are branching off 

What is Charcot-Marie-Tooth disease

motor nerve disorder appearing first in the legs and then arms. Eventually have sensory symptoms (can’t feel in limbs) 

• Loss of peripheral sensory and motor nerves (myelin disorder) 

  • Damaged myelin sheath 

• Common cause = duplication region in chr. 17p (has gene that makes myelin) 

  • 40 different possible genes involved  

What is Multiple sclerosis (MS)

CNS demyelinating disease (autoimmune) 

• No nerve impulses because myelin affected 

• Cause is unknown, but ALL possible causes stimulate the ERSR in myelin producing cells 

  • Virus? Gene defect? Both? Cells trying to remyelinate axons? 

What is Achondrogenesis

congenital disorder that results in defects in the structure of the golgi apparatus 

• Mutation in TRIP11 gene 

• Affects cartilage and skeletal development 

• Affected infants have extremely short limbs, narrow chest, short ribs that fracture easily, & lack of normal bond formation in skull/spine/pelvis 

What is DEAF 1555

hearing loss dur to an A to G mutation in mitochondrial DNA 

What is MELAS 3242

deafness, stroke, or diabetes 

What is LHON

optic neuropathy (blindness) 

What is MERRF

myotonic epilepsy and ragged-red fiber disease 

What is NARP 8993

Leigh’s encephalopathy 

What is ADPD 4336

Alzheimer/Parkinsons 

What do problems in the ER lead too

too many mis/unfolded proteins accumulating in/near ER causing it to become stressed 

• Can start an apoptotic response within cells to eliminate affected cells 

• ERSR or UPR = inhibits translation of many proteins, but upregulates chaperones (issue with BiP) 

• Occurs mostly in nervous system where myelinating cells produce much plasma membrane in the myelinating process 

  • Involved in disorders like CMTD & MS 

What do disorders in the Golgi lead to

• Inclusion-cell disease = Group of congenital glycosylation disorders caused by mutation in genes encoding glycosylating enzymes or transport proteins (lysosomal storage disease because no hydrolases in lysosomes) 

  • Lethal by age 2 

  • Proteins are excreted instead of going to lysosomes  

• Alzheimer disease = neuronal golgi fragments and atrophies (amyloid build) 

What do mitochondrial diseases lead to

blindness, heart failure, GI problems, tremors, poor balance 

• Not enough energy produced 

• Cells and organs don’t function 

• Enough mitochondria must be affected for symptoms to show up (more than just 1 or a few) 

• Tissues highly dependent on oxidative respiration are the most affected 

  • Brain, heart, muscle 

What are the lysosome degradation pathways

1. Endocytosis = cells take in components of the plasma membrane and extracellular space and deliver them to internal compartments called endosomes 

2. Phagocytosis = the plasma membrane is directed to wrap around the particle to be engulfed until it fuses with itself, resulting in an enclosed phagosome inside the cell (bacteria) 

3. Autophagy = This pathway engulfs parts of the cytosol or whole organelles into a newly assembled compartment, which then fuses with lysosomes to deliver its contents for degradation 

• Deletion of obsolete cell parts via lysosomal digestion 

4. Macropinocytosis = a process whereby the plasma membrane protrudes from the cell and engulfs a portion of the surrounding extracellular fluid into a macropinosome. 

What is lysosomal hydrolase sorting

Enzymes have a mannose-6-phosphate group attached in N-linked oligosaccharides 

• Receptors on trans-Golgi for M6P put clathrin-coated protein and transport it to the lysosome 

What is the mitochondria

 organelles that generate most of the cell’s supply of ATP 

• Free radical generation, apoptosis control, cell growth, & cell cycle regulation 

• ~2,000 per cell 

• Composed of compartments that carry out specialized functions (multi-layered) 

  • Outer membrane, intermembrane space, inner membrane, cristae, and matrix 

• Involved in formation of hydrogen gradient using fat and sugar and producing water and CO2 

• Electron transport process done by the citric acid cycle 

  • CAC makes 2 CO2, 3 NADH, 1 GTP, and 1 FADH2 

• Maternally inherited 

  • That's why there is a different range of disease severity when mitochondria affected 

What is the outer membrane of the mitochondria

contains complexes of integral membrane proteins that form channels through which a variety of molecules and ions move in and out (simple receptors)

What is the inner membrane of the mitochondria

contains 5 complexes of integral membrane proteins  

1. NADH dehydrogenase (complex I) 

2. Succinate dehydrogenase (Complex II) 

3. Cytochrome c reductase (complex III) 

4. Cytochrome c oxidase (complex IV) 

5. ATP synthase  

GOAL OF COMPLEX IS TO CREATE A PROTON GRADIENT and ultimately ATP 

What is the mitochondrial matrix

contains mixture of soluble enzymes that catalyze the respiration of pyruvic acid and other small organic molecules (mtRNA (majority protein coding) & tRNA) 

• Pyruvic acid is oxidized and decarboxylated to produce CO2 and a fragment of acetate which is donated to oxaloacetate to form citric acid 

What is mitochondrial DNA

made of 37 genes that encode for... (majority protein-coding genes) 

• 2 ribosomal RNAs, 22 tRNAs, 13 polypeptides embedded in the inner membrane, 7 subunits that make up the NADH dehydrogenase, 3 subunits of cytochrome c oxidase, 2 subunits fo rATP synthase, & cytochrome b 

What is exocytosis and the 2 types

proteins destined for cell export are in trans golgi transport vesicles that fuse with the plasma membrane of the cell 

1. Constitutive secretory pathway

2. Regulated secretory pathway

What is the constitutive secretory pathway

Constitutive secretory pathway = operates continuously in unpolarized cells (fibroblasts, WBCs) 

• No signals required for proteins to leave 

What is the regulated secretory pathway

Regulated secretory pathway = cells that secrete proteins rapidly to different domains of the cell surface (more complex) (secretion triggered) 

Describe the image

word

What makes up a polypeptide

• Backbone = the regular structure that forms when amino acids are linked together via a peptide bond. Consists of repeating units of NHs, CHs, C=O, NH, CH etc. 

• Amino acid side = attached to repetitive backbone

  • Some are polar, nonpolar, or hydrophobic 

• PROTEIN FOLDING BASED ON THE DISTRIBUTION OF SIDE CHAINS AND BONDS FORMED BETWEEN DIFFERENT PARTS OF POLYPEPTIDE 

• There's interactions between side chains: Hydrogen bonds, Van der Waals, & electrostatic attractions 

What are the hydro. interactions within a polypeptide chain/protein

Hydrophobic interactions inside 

• Non-polar side chains inside in the core 

Hydrophilic interactions outside

• Polar side chains gather on the outside 

What is a primary structure

sequence of a chain of amino acids (linear) 

• Involves amino acid sequence 

What is a secondary structure

amino acids interact with each other to fold into a repeating pattern (protein folding) 

• Two regular folding patterns caused by hydrogen bonding are found in parts of proteins (secondary structures) 

• made up of alpha helix and beta sheets

Protein domains = small units of protein that fold independently of each other 

• • Different protein domains of a single protein perform different functions 

  • Ex: SRC = protein kinase that adds phosphate groups to other proteins using ATP as the substrate in cell signaling (has 3 domains: 1 catalyzes & 2 are regulatory) 

• Polar side chains gather on the outside 

• Non-polar side chains gather at the core 

What is an alpha helix

specific folding patterns because of hydrogen bond formation 

• A single helix resulting when a single polypeptide chain twists around itself to form a cylinder 

  Ex: Keratin (fingernail protein) 

What is a beta sheet

antiparallel chains  

• Different amino acids join to form this type of protein structure, and it is the specific amino acids that make this structure  

• Ex: fibroin (silk component) 

What is a tertiary structure

3D folding pattern; attractions between different secondary structures 

• Alpha & beta sheets connecting together  

• The combination of different primary and secondary structures 

What is a quaternary structure

Other peptide chains interact with main protein chain 

• NOT all proteins reach this point 

• Different unconnected proteins join together to form one protein  

Ex: hemoglobin made of different globins 

What are fibrous proteins

filamentous proteins (considered to be intermediate filaments) 

• Hold things together (structural things) 

  • Long fiber 

Ex: Fibrinogen, collagen, troponin 

What are globular proteins

tend to be rounded and bulky  

• Large, bulky & compact 

Ex: hemoglobin, enzymes, plasma proteins

Why is proteins binding to ligands important

Structure = function 

• Biological properties are determined by what a protein binds to (physical interactions) 

• All proteins bind with high specificity 

Ligand = substance bound by a protein (binds to receptor)

• Weak, non-covalent bonds hold a ligand to a protein 

Ligand-binding site = cavity in the protein surface formed by amino acids 

• Allows binding to be specific  

What is an enzyme

proteins that bind one or more ligands (substrates) and convert them into products 

• Catalysts permit cells to make and break covalent bonds (speed up reactions 

  Ex: alpha amylase binding starches and sugars to make monosaccharides 

• Usually end in –ase 

Types: Hydrolase, nuclease, proteases, synthases, ligases, kinases, phosphatases

What do hydrolases do

Hydrolases = catalyze a hydrolytic cleavage reaction  

Types

Nucleases = break down nucleic acids by hydrolyzing bonds between nucleotides 

Proteases = break down proteins by hydrolyzing bonds between amino acids  

What does synthases do

synthesize molecules in anabolic reactions by condensing two smaller molecules together 

What does ligase do

join together 2 molecules in an energy dependent process 

What does kinase do

catalyze the addition of a phosphate group to a molecule 

What does phosphatase do

catalyze the hydrolytic removal of a phosphate group from a molecule  

What does protein phosphorylation do

proteins regulated by adding or removing phosphate (activate/repress protein) 

• Adding a phosphate group causes conformational change because the charges on the protein are changed 

  • Forms binding sites for other molecules 

• Adding or removing the phosphate group can either activate or repress the protein 

What does protein kinase do

adds phosphate group 

What does phosphatase do

removes phosphate group 

What are motor proteins

to move molecules in or around cells 

• All work done by hydrolysis of ATP 

EX: 

• Muscle contraction 

• Cell movement 

• Chromosomal movement during cell division 

• Moving neurotransmitter vesicles around neurons 

• Moving intracellular particles 

• Moves polymerases along a strand of nucleic acid 

What is membrane transport

specific proteins pass through membranes (both cellular and organelle membranes) to act as channels or binders 

• Cell receptors = bound to cell membrane and catch extracellular signals  

  • Have affinity for different things 

  • Trigger a reaction inside the cell/organelle 

  • Ex: glycoprotein cell receptors 

What are some protein functions

Protein functions:  

1. Tissue nutrition 

2. Water distribution (balance) 

3. Plasma buffer 

4. Substance transport 

5. Structural support 

6. Antibodies, coagulation, hormones 

7. Enzymes 

8. Movement 

9. Membrane receptors and transport 

*PROTEINS ESSENTIAL FOR FUNCTION (too little or too much can result in disease) 

What does abnormally folded proteins result in

esults in many disease because protein aggregates form due to a loss of cellular quality control 

• Normal proteins eventually begin to aggregate, forming amyloid fibrils 

What is amyloid

when normal proteins begin to aggregate together because there are other proteins that are mutated (lost cellular quality control)  

• Cross-beta filament proteins (multiple sheets stacking together) 

• Particularly sensitive in the brain 

• Not ALL amyloid is bad 

• Are protease resistant  

• Ex: Alzheimer, type 2 diabetes, Parkinson, Huntington, rheumatoid arthritis, & cardiac arrhythmias 

What is prion disease (PrP*)

An infectious agent that is comprised entirely of a misfolded protein (PRoteinaceous and Infectious viriON) 

• Hypothesized to infect and propagate by refolding abnormally into a structure that is able to convert normal molecules of the protein into the abnormally structured form (ALTER PROTEINS) 

• Induce amyloid 

• Able to convert normal PrP proteins into the infectious isoform (PrP*) by changing their conformation 

  • Pulls other proteins/PrP to misshape with it 

  • Ex: Mad cow disease, Creutzfeld-Jacob disease, & scrapie 

What is Prion P (PrP)

a normal protein found on the membrane of cells (especially neurons) 

• Has 209 amino acids and is MAINLY an alpha-helical structure 

the bottom box like protein is an amyloid

What are some prion related diseases

Mad cow disease, Creutzfeld-Jacob disease, & Scrapie 

• Results when normal PrP are converted into PrP* 

What are CpG islands

surround housekeeping genes (need to be kept on all the time – important) that code for proteins that keep cells viable 

• Helps maintain cellular specialization 

• CpG methylation is important for efficient gene repression to be passed onto daughter cells 

• Surround the promoter 

  • 20,000 of CpGs mark the 5’ end of transcription units (typically around the promoter) 

• This ratio probably reflects a balance between methylated CG loss by DNA repair and CG gain by random mutation. The CG sequences that remain are very unevenly distributed in the genome; they are present at 10 times their average density in selected regions

Epigenetic inheritance

heritable changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence 

• Epi = in addition to => environmental 

• Can be affected by the environment 

  Ex: Lactose tolerance 

  • LPH metabolizes lactose (encoded by the LCT gene on chr. 2) 

    • LCT expression regulated by polymorphism in upstream gene MCM6 (if polymorphism is missing, then lactose intolerance happens) 

  • LPH is high in European populations and lower in African populations => likely because they need more vitamin D than Africans  

• cis and trans epigenetic mechanisms

What are cis-epigenetic mechanisms

within the same DNA strand/molecule 

1. DNA methylation 

2. Histone modification 

What are trans epigenetic mechanisms

something outside the sequence that’s affecting the gene 

1. Positive feedback loop activated 

2. Conformation change to aggregated state 

What is imprinting

= silencing 

• A small minority of genes are expressed if inherited from mother, some expressed if inherited from father 

• Imprinting genes = one gene is silenced by methylation and cannot be transcribed 

  • Normal inheritance = 2 working copies of a gene (one from each parent) 

• Imprinting DOESN’T cause a disease, the other remaining gene does 

• Disease: Prader-Willi & Angelman syndrome (imprinting of gene in chr. 15 & a deletion) 

What is RNA interference (RNAi)

Short single-stranded non-coding RNAs of 20-30 nucleotides that guide or interact with other RNAs in the cell 

• Can inhibit translation of mRNA by catalyzing its destruction 

• Can mess with RNA and template during transcription and form repressive chromatin on the DNA 

• Proteins involved in RNAi are also involved in breaking apart dsRNA to form 23 nucleotide long siRNA 

• 3 types (miRNA, siRNA, piwiRNA) 

What are non-coding RNAs

lncRNAs = act as scaffold molecules (hold proteins together – after splicing) 

• Longer than 200 nucleotides 

• Ex: Telomerase RNA, Xist RNA, silencing RNA, gene regulators

What is CRISPR

Viral DNA fragments are incorporated into bacterial genome, acting as a vaccine to make small noncoding RNAs (crRNA) 

• crRNAs destroy the virus if it reinfects the bacteria 

  • Now using miRNA/siRNA (these are the Cas proteins that will interact with crRNA) 

    • Cas proteins need a PAM sequence to recognize bacteria’s genome and cleave in the right place 

What does DNA methylation do

Methylation = methylating Cytosine 

• marks genes that are “imprinted” 

• Requires a methyl transferase to pass along the methylation to correct daughter cells 

• Regulate gene expression (without changing DNA sequence) 

• Methylation of CpG sequences is important in efficient gene repression that can be passed on to daughter cells 

  • Helps maintain cellular specialization 

• Occurs right after DNA replication (inherited) 

  • Methyl transferase recognizes methylated C at CpG and adds it to new daughter strand 

What is deamination

Deficient in mammals because the methylated cytosine mutate into a T over time (via accidental deamination) 

• The remainder, C, are present in CpG islands in selected regions of the genome 

What are the 3 classes of RNAi

miRNA, siRNA, piRNA

What is micro RNA (miRNA)

Regulate some of human protein-coding genes by forming base pairs (complementary bp usually 7bp long in 3’ UTR of mRNA) with certain mRNAs & fine tune translation 

• Shut down method 

  • Small but mighty 

• Regulate gene expression by repressing translation via mRNA degradation 

  • miRNA calls other gene regulators to further reduce translation (mRNA of interest MUST have short common sequence in their 3’ UTR) 

• Made by RNA pol. II with a cap and poly-A tail. Proteins (Argonaute) are then added to form RISC (RNA-induced silencing complex) 

  • RISC = proteins + miRNA complex 

• Involved in heterochromatin formation using methylated histones

What are small interfering RNA (siRNA)

Look for viral or transposable RNA element (transcriptional repressor) (defense mechanism?) 

• Self-replicating: RNA pols use siRNA as primer to make more precursor siRNAs 

  • Broken apart and destroyed by RITS 

• Considered to be an ancient form of RNA interference  

What are Piwi-interacting RNA (piRNA)

bind to piwi proteins and protect the germ line from transposable elements 

Explain viral protection in relation to CRISPR

bacteria use non-coding RNA to destroy the DNA of invading viruses (via CRISPR) 

• Viral DNA fragments are incorporated into bacterial genome, acting as a vaccine to make small noncoding RNAs (crRNA) 

  • crRNAs destroy the virus if it reinfects the bacteria 

    • Now using miRNA/siRNA (these are the Cas proteins that will interact with crRNA) 

      • Cas proteins need a PAM sequence to recognize bacteria’s genome and cleave in the right place 

  • CRISPR is now used in plants and animals to manipulate genomes

What is Prader-Willi syndrome

aused by deletions of genetic region that includes small nuclear ribonucleoprotein polypeptide N gene 

• Maternal copy of gene is silenced (imprinted) 

  • Paternal gene is deleted 

• Increased appetite (over-weight) 

What is Angelman syndrome

Caused by a loss of expression of a single maternally expressed gene in the same region: UBE3A 

• UBE3A encodes for E3 ubiquitin ligase protein (involved in targeting proteins for degradation & is only imprinted in the brain) 

• Loss of UBE3A = abnormalities in normal protein degradation during brain development 

• The paternal gene imprinted 

  • Maternal is loss 

• “smiling” all the time 

What are transcription regulatory proteins?

recognize specific DNA sequences because of the surface features of the double helix (recognizes surface features) 

• Complex arrangements that respong to a variety of signals that are interpreted and integrated 

• Double helix features = major & minor groove 

  • Binds to the major grooves because it has the cis-sequences & other contact points 

• Contain structural motifs  

• Don't need to unwind DNA to read sequences 

• In bacteria: transcription is controlled by 1 regulatory protein (and have operons) 

• In eukaryotes: many proteins control transcription & can act over very long distances 

• Single transcription regulatory proteins 

  • Single proteins can make the final decision as to transcribe or not 

  • Can form an entire organ by beginning a cascade of other proteins & cell-cell interactions 

    Ex: eyeless drosophila (eyes instead of legs form) 

What are structural motifs

bind to the major groove based on the protein’s amino acid side chains (a recognizable, recurring 3D arrangement of secondary structure elements, such as helices and sheets, connected by loops)

• Found in transcription regulators 

Common motifs:  

1. Helix-turn-helix = 2 alpha helices with a recognition sequence 

• Homeodomains are a type of helix-turn-helix 

2. Zinc fingers = DNA binding proteins with a zinc (unique) moiety 

• zinc can hold alpha helix and beta sheet together 

• Zinc fills a two-alpha helix protein 

3. Protein protruding loops =  

4. Leucine zippers =  

What are cis-regulatory sequence

DNA sequences specify the time and place that each gene is to be transcribed when read by their specific bound transcription regulators 

• DNA sequences recognized within the same molecule 

• Control transcription and are upstream of the transcription initiation start point  

• Positive regulators = activators 

• Negative regulators = repressors 

• Many protein regulators control a single gene (many regulator sequences within that gene 

• DNA looping critical for proteins, sequences, and polymerase to interact 

  • “Complex switches” 

What is the significance of DNA looping

DNA looping critical for complex switches to interact (it ISN’T LINEAR) 

• Brings activators/repressors and sequences closer to the mediator and other TFs 

Gene control region (GCR)

consists of activator (and co-activator)/regulatory sequence/associated proteins, promoter region/factors/RNA pol, gene, more reg. proteins (chromatin remodelers) 

• Formed when a mediator is present and binds all the components together 

• RNA pol. II then transcribes all coding genes & non-coding RNA genes

How do cells control their own proteins? (list and describe)

• Transcriptional control = DNA to RNA transcript 

  • Controlled by DNA sequences (cis-regulatory sequences) near a gene upstream of the transcription initiation start point which are recognized by transcription regulator proteins 

• RNA processing 

  • RNA transcript to mRNA 

• RNA transport and localization 

  • mRNA being exported from the nucleus to the cytosol 

• Translation control 

  • mRNA to protein 

• Degradation control (both mRNA & protein) 

  • mRNA to inactive mRNA (degradation) or protein degraded into inactive protein 

• Protein activity control (phosphorylation) 

  • Making a protein into active or inactive