Bio Midterm 1

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Eukaryotic cells

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* Has nucleus that contain most of the genetic material (chromosomes)
* Estimated 10 trillion cells in an adult human
* Appeared about 2 billion years ago
* Same genetic heritage but differ in function (muscle cells, skin cells, neurons)

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Prokaryotic cells

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* Do not contain nucleus
* 10 times eukaryotic cells (100 trillion)
* 2-3 percent of our body mass
* Provide essential functions throughout our physiological systems
* Most are not harmful

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Microbiome

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a population of microbiotic organisms, microorganisms, or

microbes within our body. Includes prokaryotic bacteria also small eukaryotic organisms

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Microorganism

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organisms that are not visible to the naked eye but only visible under a microscope

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Four Classes of Macromolecules

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1. Nucleic acids (DNA & RNA):

2. Proteins (structural elements of a cell and perform metabolic activities (enzymes))

3. Polysaccharides/ Carbs (plant cell wall and sources of stored energy)

4. Phospholipids (primary component of the cell membrane)

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Cell membrane

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* Made up of lipid macromolecules
* Has hydrophobic and hydrophilic properties that allow “stacked” lipid bilayers to form
* 5-10 nm in thickness

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Phospholipid

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* Main component of a cell membrane
* Consist of a glycerol molecules that is linked to a phosphate and 2 fatty-acids
* Said to be amphipathic (hydrophobic and hydrophilic)
* Tails consist of a pair of hydrocarbon chains (typically 16 or 18 carbons in a single chain)
* Bonds connecting the carbons may be single bonds (saturated) or double bonds (unsaturated)

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Steroids (cholesterol)

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* Characterized by a 4-hydrocarbon ring structure
* Like phospholipids, they contain hydrophilic head group and hydrophobic tail

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Organization of Cell Membranes

Phospholipids

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* Form lipid bilayers and lipid micelles (important for absorption of fat-soluble vitamins and complex lipids in the human body)
* Hydrophilic heads interact with water in the extracellular or intracellular environment
* Tails will interact with tails to form hydrophobic cores
* Energy not used

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Cell Membranes are “Fluid”

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* Can move laterally within the cell membrane lipid bilayer (left, right, forward, backward)
* Can not flip without a great deal of energy
* Region of lower fluidity is called a lipid rafts (found to gather proteins involved in the same metabolic pathway or a collection of receptors on the surface of the cell)

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Many Factors Affect the Fluidity of Cell Membranes

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1. Number of carbons in the fatty acid tails
2. Temperature
3. The presence or absence of cholesterol

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1. Unsaturated or saturated fatty acids*

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* Double bonds produce kinks or bends in the chain which has the effect of pushing neighboring phospholipids further apart and increasing fluidity
* Kinks affect overall permeability

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1. Number of carbons in the fatty acid tails

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* Longer chains packed together more tightly than the shorter chains, reducing the fluidity of the membrane

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1. Temperature

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* Higher temps promote fluidity
* Lower temps decrease fluidity
* * cold adapted organisms tend to have more unsaturated phospholipids in their membranes that help maintain fluidity

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1. The presence or absence of cholesterol

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* Found in every cell in our body
* Make up about 50% of the molecules found in the bilipid membranes
* Constrain fluidity of the membrane by packing closely to neighbouring phospholipids
* Helps maintain fluidity at lower temperature by keeping solidified phospholipids apart

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Cell Membranes are Selectively Permeable

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* Small, non-polar molecules and hydrophobic molecule can pass through the phospholipid bilayer relatively quickly
* Charged and larger polar molecules have greater difficulty in moving across the lipid bilayer of the cell membrane, if at all

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The Fluid Mosaic Model

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* Cell membranes are a mosaic of lipids, proteins, and carbohydrates
* Proteins can be attached on interior or exterior of the cell or actually embedded in the bilayer
* Transmembrane proteins embedded within the phospholipid bilayer of the cell membrane can facilitate transport across the membrane
* Peripheral membrane proteins are temporarily associated with a biological membrane

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Passive diffusion (simple diffusion)

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* From areas of high to low concentration (along a gradient)
* Small molecule crossing the phospholipid bilayer
* Into or out of the cell
* Does not require energy

Ex: lipid soluble molecules, gases, uncharged polar molecules, and water

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Passive transport (facilitated diffusion)

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* Movement of small molecules with a concentration gradient
* Use of proteins embedded in the cell membrane
* Osmosis (transport of water across a membrane)
* Aquaporin (protein channels) allow rapid transport (at over 10x the rate that water takes to move through the bilayer) of water and are exclusively permeable to water
* Does not require energy

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Isotonic environment

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external/internal environment has the same concentration of solute

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Hypotonic environment

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extracellular fluid has lower solute concentration than inside of the cell, this results in excessive movement of water into the cell Can lead to net gain of water causing cells to swell and sometimes burst,

creating ghost cells

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Active transport

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* Molecules move against the concentration gradient
* Use of proteins embedded in the cell membrane
* Does require energy from the hydrolysis of ATP
* Primary active transport (direct expenditure of ATP-dependent)
* Secondary active transport (indirect expenditure of ATP energy)

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Sodium potassium pump:

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* Every 3 sodium ions that are pumped out of the cell, 2 potassium ions are pumped in
* ATP gives up an energetic phosphate group to the transport protein
* Phosphate group binds and causes a change in shape to the transport protein releasing the sodium ions
* Once the pump binds 2 potassium ions, the phosphate group is released from the transport protein
* The protein returns to its original shape and releases the potassium ions into the cell

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Mitochondria

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* Cellular respiration (aerobic)
* 50 to over a million present in a cell
* Outer membrane surrounds the organelle
* Inner membrane connected to a series of structures called crysta
* Energy stored in the bonds of carbohydrates
* Where most ATP is synthesized

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Chloroplasts

*

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* Double membrane around exterior
* Filled with hundreds of flattened and stacked membrane called thylakoids (organized into piles called grana)
* Within the thylakoid membrane that pigments and enzymes participate in photosynthesis
* Energy stored in the bonds of carbohydrates

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Evolution

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* Both may have entered the cell from external origins
* Allowed ancestral prokaryotes to compartmentalize the genetic information into a nucleus
* Obtain greater control over regulation of genes and processing of proteins
* Became hosts to aerobic prokaryotes and photosynthetic prokaryotes (which we now know as mitochondria and chloroplasts, respectively)

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Endosymbiotic Theory of Organelle Evolution

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Analysis of the genes and proteins shows significant similarity of those of bacteria

Ex: Mitochondria and Chloroplasts contain their own circular genomes

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Glycolysis

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* Anaerobic
* Occurs in the cytosol of the cell
* 2 Pyruvate (3 carbon compound)
* 2 ATP
* 2 NADH (electron donors)

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Pyruvate Oxidation

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* 2 Acetyl CoA
* 2 NADH
* 2 CO2

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Kreb Cycle

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* 2ATP
* 2 FADH2
* 4 CO2
* 6 NADH

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Electron Transport Chain (ETC)

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* Resides in the extensive inner mitochondrial membranes
* Most ATP forms when the electron donors (NADH,FADH2) move through the protein complexes of the ETC
* As electrons move through the membranes, protons are pumped into the intermembrane spaces of the mitochondria
* Creates an electrochemical concentration gradient across the inner mitochondrial membrane that drives protons through the ATP synthase protein channels that are embedded in the membranes
* Resulting in the synthesis of an additional 32 ATP

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Carbohydrates

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* Polymerize via glycosidic linkages to form larger chains
* Enzymes catalyze the condensation reaction between hydroxyl (OH) groups
* Reaction usually occurs between the OH group of carbon 1 of one molecule and carbon 4 on the other
* Either alpha 1-4 glycosidic linkage or 1-4 beta glycosidic linkage
* Glucose, fructose, and galactose
* Lactose, sucrose, maltose, and more.

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Polysaccharides

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* Can include disaccharides
* More commonly starch and glycogen
* Two types of polysaccharides: unbranched (amylose) and branched (amylopectin) (1-6 glycosidic linkages, often used for energy in our own bodies)
* Storage polysaccharide that is found in all photosynthetic plants
* Alpha 1-4 glycosidic linkages between alpha-glucose monomers
* We store the digested carb as highly branched glycogen polysaccharide helices

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Triphosphate (ATP)

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* Adenine, attached to a ribose sugar, and 3 phosphate groups
* Phosphate groups have 4 negative charges in close proximity
* Charges repel each other, giving electrons of the phosphate groups a very high potential energy
* A hydrolysis reaction releases the energy
* ATP is used for mechanical, transport, and chemical work

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The Anatomy of a Camel

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* Hump consists of fat (tristearin C57H110O6) and can weigh up to 80 lbs
* Extreme tolerance to water loss (up to 30-40% of their body weight)
* Minimize water losses by condensing water in the nostrils

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Geese

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* Canadian geese lose up to half of their body weight in fat
* Canadian geese can fly up to 3280 ft
* Bar-headed geese can fly up to 29500 ft and 65 km/h

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Lipolysis

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* Fat molecules are ~3 times as much ATP than per molecule of glucose
* Breakdown of lipids (Adipocyte)
* Hydrolysis of triglycerides into glycerol and free fatty acids
* Vesicles contain fatty acids
* Membrane transporters transport fatty acids at a high rate into circulation and into myocyte
* Undergo circulatory or cytosol transport (T)
* Entry into the mitochondria is through specific transporters and FFA undergo -oxidation with each pass through the reaction spiral that yields acetyl-CoA
* Acetyl-CoA then enters the tricarboxylic acid cycle (TCA) producing reducing equivalents for production of ATP and the subsequent generation of CO2
* -oxidation produces more acetyl-CoA than glycogen
* Forks at the bottom are sarcomere (where muscle contraction occurs) contains actin and myosin which are motor proteins whose interactions produce movement, and in this particular case, muscle contraction.

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Ketogenesis:

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* Ketone bodies can be converted to acetyl CoA for further catabolism in the Krebs Cycle

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* Proteins:

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* provide structural support and act as catalysts that facilitate chemical reactions

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* nucleic acids

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* encode and transmit genetic info

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* Lipids:

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* make up cell membranes, store energy and act as signalling molecules, consist of fatty acids bonded to other organic molecules

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* polymers:

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* complex molecule made up of repeated simpler units by covalent bonds
* protein is a polymer of amino acids
* nucleic acids are polymers of nucleotides
* carbs are polymers of simple sugars

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* Amino Acid Structure:

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* central carbon: alpha carbon covalently to four groups (V)
* 1. carboxyl group 2. amino group 3. a hydrogen atom 4. Side chain: differs from each AA
* the identity of an amino acid is determined by the structure and composition of the side chain

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* peptide bond

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* The carbon atom in the carboxyl group of one aa is joined to the nitrogen atom in the amino group of the next by covalent linkage: peptide bond
* the formation of a peptide bond involves the loss of a water molecule
* this because in order to form a C-N bond, the carbon atom must release an 02- and the N atom must lose two H+
* 20 amino acids

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* Transcription

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* existing proteins create a copy of DNA in the form of RNA

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* Translation:

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* RNA is read to determine what building blocks to use to create pr

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* central dogma

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* This pathway from DNA >> RNA >> Protein is called the central dogma
* The central dogma describes the basic flow of information in a cell

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* gene:

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* The DNA sequence that corresponds to a specific protein product

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* Exceptions to dogma

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* transfer from RNA to DNA ( HIV >> AIDS)
* transfer from RNA to RNA ( replication of the genetic material of influenza

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* In prokaryotes, transcription and translation occur in the ?

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* cytoplasm,

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* in eukaryotes, Transcription and translation occuR IN?

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* transcription occurring in the nucleus and translation in the cytoplasm.

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* exocytosis

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* vesicles can bud off and fuse with components of the endomembrane system, creating a set of interconnected spaces. They can even fuse with the plasma membrane

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* endocytosis

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* A vesicle can bud off from the plasma membrane, bringing material from outside the cell into a vesicle, which can then fuse with other organelles

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* nuclear envelope

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* defines the boundary of the nucleus (Fig. It consists of two membranes
* These two membranes are continuous with each other at protein openings called nuclear pores. These pores act as gateways that allow molecules to move into and out of the nucleus, and thus are essential for the nucleus to communicate with the rest of the cell. In fact, the transfer of information encoded by DNA depends on the movement of RNA molecules out of the nucleus,
* The nuclear envelope with its associated protein pores regulates which molecules move into and out of the nucleus.

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* ER ENDOPLASMIC RETICULUM

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* produces and transports many of the lipids and proteins used inside and outside the cell. It is the site of production of most of the lipids

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* transmembrane proteins

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* transmembrane proteins and proteins destined for the Golgi apparatus, lysosomes, or export out of the cell are synthesized in the

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* Ribosomes

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* are the sites of protein synthesis, in which amino acids are assembled into polypeptides guided by the information stored in mRNA

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* vesicles

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* are an effective means of moving proteins that are either embedded in the ER membrane or free floating inside.

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SMOOTH EDONPLASMIC RETICULUM

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* is also the site of fatty acid and phospholipid biosynthesis. Thus, this type of ER predominates in cells specialized for the production of lipids.

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* Glycine

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* Glycine is different from the other amino acids because its R group is hydrogen, exactly like the hydrogen on the other side, and therefore it is not asymmetric.
* glycine is nonpolar and small enough to tuck into spaces where other R groups would not fit
* glycine increases the flexibility of the polypeptide backbone, which can be important in the folding of the protein

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* Proline’s

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* Proline’s linkage creates a kink or bend in the polypeptide chain and restricts rotation of the C–N bond, thereby imposing constraints on protein folding in its vicinity, an effect the very opposite of glycine’s.

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* Cysteine

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* has a –SH group. When two cysteine side chains can react to form an S–S disulfide bond, which covalently joins the side chains, forming cross-bridges that can connect different parts of the same protein or even different proteins.

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* primary structure AA

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* The sequence of amino acids in a protein is its

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* local secondary structures.AA

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* The sequence of amino acids ultimately determines how a protein folds. Interactions between stretches of amino acids in a protein form

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* tertiary structure.

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* Longer-range interactions between these secondary structures in turn support the overall three-dimensional shape of the protein, which is its

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* quaternary structure.

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* some proteins are made up of several individual polypeptides that interact with each other, and the resulting ensemble is the

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* β sheet

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* In a β sheet, the polypeptide folds back and forth on itself, forming a pleated sheet that is stabilized by hydrogen bonds
* The side chains project alternately above and below the plane of the β sheet. β sheets typically consist of 4 to 10 polypeptide chains aligned side by side
* the antiparallel configuration is more stable because the carbonyl and amide groups are more favorably aligned for hydrogen bonding.

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* Protein sorting

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* is the process by which proteins end up where they need to be to perform their function. Protein sorting directs proteins to the cytosol, the lumen of organelles, the membranes of the endomembrane system, or even out of the cell entirely.

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* signal sequences

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* Proteins produced on free ribosomes start off in the cytosol and are sorted to their final destination after translation. These proteins are often directed to their proper cellular compartments by means of particular amino acid sequences called signal sequences

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* nuclear localization signal,

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* there are several types of signal sequences that direct proteins to different cellular compartments. Proteins with no signal sequence remain in the cytosol. Most proteins with a signal sequence at their amino ends are targeted to mitochondria or chloroplasts. Proteins targeted to the nucleus usually have signal sequences located internally. The signal sequence for the nucleus, also called a nuclear localization signal, enables proteins to move through pores in the nuclear envelope.

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* proteins destined for the lumen or for secretion are fed into the ER lumen as they are synthesized, whereas the proteins destined for membranes are inserted in the ER membrane as they are synthesized

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note

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* signal-recognition particle (SRP)

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* signal sequence is recognized almost immediately after synthesis by an RNA–protein complex known as a

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* chaperone proteins

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assist with protein folding

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* motor proteins,

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Motor proteins associate with microtubules and microfilaments to cause movement.

* Motor proteins cause muscle contraction by moving the actin microfilaments inside muscle cells. the cytoplasm of muscle cells shown in is packed with actin microfilaments that are anchored to the ends of the cell. Myosin, a motor protein found in muscle cells, binds to actin and undergoes a conformational change. As a result, the actin microfilaments slide relative to myosin, causing the cell to shorten, or contract

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* kinesin and dynein

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* Kinesin is similar in structure to myosin and transports cargo toward the plus end of microtubules at the periphery of the cell By contrast, dynein carries its load away from the plasma membrane toward the minus end

MICROTUBLES

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Aquaporins

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* aquaporins allow water to flow through the plasma membrane more readily by facilitated diffusion
* The diffusion of water is known as osmosis. As in any form of diffusion, water moves from regions of higher water concentration to regions of lower water concentration
* water concentration drops as solute concentration rises. Therefore, it is sometimes easier to think about water moving from regions of lower solute concentration to regions of higher solute concentration.

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* topoisomerase II,

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* Underwinding is caused by an enzyme, topoisomerase II, that breaks the double helix, rotates the ends, and then seals the break. Underwinding creates strain on the DNA molecule, which is relieved by the formation of supercoils,

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* positive supercoils

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*

*

* **DNA is negatively supercoiled

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* nucleosome

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* Eukaryotic DNA is first wrapped around a group of histone proteins called a nucleosome
* Each nucleosome consists of two molecules, and each molecule consists of four different histone proteins. The histone proteins are rich in the amino acids lysine and arginine, whose positive charges neutralize the negative charges of the phosphates along the backbone of each DNA strand.

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* 30-nm chromatin fiber

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* The nucleosomes and associated DNA are then coiled to form a structure called the

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* chromosome condensation,

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* When the chromosomes in the nucleus condense in preparation for cell division, each chromosome becomes progressively shorter and thicker as the 30-nm fiber coils in a manner that is still not fully understood called chromosome condensation, an active, energy-consuming process

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* scaffold

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* Without histones to coil around, the DNA spreads out in loops around a supporting protein structure called the chromosome

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* Including the sex chromosomes, humans have 23 pairs of chromoso

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**

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* pyrimidine

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* The pyrimidine bases have a single ring and include thymine (T), cytosine (C), and uracil (U).

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* purine

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* The purine bases have a double-ring structure and include adenine (A) and guanine (G).

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* Nucleotides are composed of three components:

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a 5-carbon sugar, a nitrogen-containing compound (base) and one or more phosphate groups

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* phosphodiester bond,

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* nucleotides is connected by a phosphodiester bond, which forms when a phosphate group in one nucleotide is covalently joined to the sugar unit in another nucleotide, involves the loss of a water molecule
* the phosphodiester linkages in a DNA strand give it polarity,

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* nucleoside

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* The combination of sugar and base

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* major groove and the minor groove

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* The outside contours of the twisted strands form an uneven pair of grooves, called the major groove and the minor groove. These grooves are important because proteins that interact with DNA often recognize a particular sequence of bases by making contact with the bases via the major or minor groove or both.

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* %A = %T and %G = %C.

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**

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* histone

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* Histone proteins are found in all eukaryotes, and they interact with double-stranded DNA without regard to sequence. In addition, histone proteins from any organism can form nucleosomes with the DNA of any other organism.
* histones H3 and H4 are the most conserved
* Histo

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* RNA world hypothesis:

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* RNA molecules can actually act as enzymes that facilitate chemical reactions. Because RNA has properties of both DNA (information storage) and proteins (enzymes), many scientists think that RNA, not DNA, was the original information-storage molecule

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* All nucleic acids are synthesized by addition of nucleotides to the 3′ end. That is, they grow in a 5′-to-3′ direction, also described simply as the 3′ direction. Because of the antiparallel nature of the DNA–RNA duplex, the DNA template that is being transcribed runs in the opposite direction, from 3′ to 5′

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**

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* polymerization

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* The polymerization reaction releases a phosphate–phosphate group (pyrophosphate), shown at the lower right which also has a high-energy phosphate bond that is cleaved by another enzyme. Cleavage of the pyrophosphate molecule makes the polymerization reaction irreversible,

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Transcription in Prokaryotes

Initiation

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* RNA polymerase attaches to specific promoter regions of DNA
* Promoter regions (start of the specific gene) indicate the transcriptional starting point where RNA synthesis actually begins
* -10 region consensus sequence TATAAT is positioned -10 nucleotides upstream
* -35 region additional sequence TTGCCA -35 nucleotides up stream of the transcription start site, can enhance the rate of transcription and are proteins called sigma factors which directly recognize and bind the promoter region
* RNA polymerase core enzyme binds to a sigma subunit to create a holoenzyme that is capable of binding to and unwinding the double-stranded DNA helix to allow transcription to occur
* Different sigma factors are able to identify specific promoters to the RNA polymerase, and therefore assist with turning genes on and off when needed

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Transcription in Prokaryotes Elongation

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* RNA polymerase separate the two strands of the DNA double helix, forming the transcription bubble
* Ribonucleotides are then able to enter the RNA polymerase and assemble in a complementary fashion to the DNA template strand
* Template strand is threaded through a separate channel to keep strands apart
* RNA polymerase restores the double helix once the transcript is produced
* Release and cleavage of the pyrophosphate group during the phosphodiester bond formation, renders this polymerization reaction of RNA transcript elongation irreversible by hydrolysis
* Appropriate hydrogen bonding is ensured before the high-energy phosphate bond is cleaved when adding a nucleotide

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Termination

Transcription in Prokaryotes

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* 2 types of terminator sequences
* consist of inverted nucleotide repeat sequences which fold back on themselves to form a G–C rich hairpin loop along the same mRNA strand
* Pauses the RNA polymerase and leads to the release of the mRNA transcript
* use a specific prokaryotic protein (Rho factor) which can bind to and subsequently utilize ATP energy to move along the formed RNA transcript while unwinding it from the DNA template
* Which is able to destabilize the interaction between RNA and DNA template, leading to the release of the transcript and the transcription complex

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Transcription in Eukaryotes

Initiation

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* Specific proteins called general transcription factors are required to mediate the binding of RNA polymerase to a promoter and to initiate transcription
* TFIID (largest general transcription factors) binds to -25 region to position itself near the initiation site of transcription
* -25 region
* -35 region

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Elongation

Transcription in Eukaryotes

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* RNA polymerase I and III transcribe structural, non-coding RNAs
* RNA polymerase I transcribes the genes for the ribosomal RNAs (rRNA)
* RNA polymerase II transcribes the messenger RNAs (mRNA)
* RNA polymerase III transcribes the genes for the transfer RNAs (tRNA) and other small regulatory RNA molecules

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Termination

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* Occurs by many different processes, depending upon the exact polymerase utilized
* RNA polymerase I: eukaryotic termination factor in a similar manner as prokaryotic rho-dependent termination
* RNA polymerase II: depends on a poly(A)-dependent mechanism of termination (coupled with mRNA maturation and vice versa)
* RNA polymerase III: mechanism that resembles the rho-independent termination in prokaryotes

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Post-transcriptional Modification

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* mRNA must be modified in the nucleus to produce mature mRNA
* RNA cannot leave the nucleus before it has been modified
* Also true for tRNA and rRNA
* Ensure export of the mRNA from the nucleus
* Help protect against ribonuclease enzyme that target Phosphodiester bonds
* Help with attachment of the ribosome and initiation of translation

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Addition of the 5’ CapTranscription in Eukaryotes

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* Involves the attachment of a modified guanosine (7-methylguanosine) to the mRNA through an unusual 5’ to 5’ triphosphate linkage
* In order to attach the 7-methylguanosine, the terminal 5’ phosphate is removed from the mRNA molecule by a phosphatase enzyme, and another enzyme, guanosyl transferase enzyme, catalyzes the attachment of the 7-methylguanosine 5’ cap

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Addition of the Poly (A) Tail

Transcription in Eukaryotes

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* Adenine nucleotides added to the 3’ end of the mRNA transcript following recognition of a polyadenylation signal sequence (AATAAA) that is transcribed from the DNA template strand near the end of the gene sequence
* Once this signal is transcribed, the mRNA is cleaved and a poly (A) polymerase enzyme is able to add between 150-200 adenine nucleotide bases to the 3’ end of the RNA transcript (process is called polyadenylation)
* Polyadenylation is coordinated with termination of transcription

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Processing RNAs in Eukaryotes

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* Exons are the sequences of the mRNA that are necessary for coding the sequences of amino acids in the protein
* Introns do not code for anything and need to be removed or excised from the mRNA
* Exons need to be joined or spliced together prior to translation through a process called RNA splicing

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RNA Splicing and the Spliceosome

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* Occurs at specific short nucleotide sequences that are situated at each end of an intron
* Splicesomes are composed of five small nuclear ribonucleoproteins (or snRNPs) that are made up of small nuclear RNAs and proteins, and are able to recognize the splice sites within the intron
* RNA catalyze splicing
* Donor site attacks acceptor site, releasing a loop and tail (lariat)

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mRNA Export: Nuclear Pore

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* Nuclear pore complexes in the nuclear membrane that act as gateways for molecules to move into and out of the nucleus
* Protein-lined channels that pass through both membranes of the nuclear envelope (both the inner and outer membrane)
* In addition to transporting RNA from the nucleus to the cytoplasm, nuclear pores can also transport proteins, carbohydrates, and important signaling molecules into the nucleus

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The Genetic Code

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* George Gamow determined that a 3-base code (triplet) is the smallest group of nucleotides that will accommodate the need to code for 20 amino acids

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Deciphering the Code

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* Marshall Nirenberg and Johann Matthaei used a cell-free system to decipher the first letter of the code in 1961
* RNA template, nucleotides, ribosomes, amino acids and an energy source
* Three nucleotides make a codon

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The Standard Code

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* Non-template strand referred to as the coding strand
* Template strand referred to as the non-coding strand
* Only AUG codes for methionine and is the start codon
* Only UCG codes for tryptophan
* UAA, UAG, and UGA are translation stop codons

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Reading Frames

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* Addition or removal of one single nucleotide will cause a frame shift mutation
* Addition or removal of a codon conserves the codon sequence before or after the removal

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Nucleus

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* Double membrane-bound domain that contains chromosomes which pack and control DNA molecules
* Most ribosomal RNAs are manufactured in the nucleolus where they bind to proteins to form the ribosomal subunits (which are exported into the cytoplasm)
* Double membrane of the nuclear envelope has embedded nuclear pore complexes that allows for material to flow into (DNA + RNA building blocks) and out (RNA and ribosomes) of the nucleus

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* Bound ribosomes

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* Free ribosomes translate proteins in the cytosol and remain soluble
* Bound ribosomes can attach to the endoplasmic reticulum

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Side Chains and Their Properties

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* Amino acids polymerize to form the primary structure when a bond forms between the carboxyl group of one amino acid and the amino group of another
* C-N bond that results from this condensation reaction is called a peptide bond
* When linked the amino acids are referred to as residues and the formed polymer is referred to as a polypeptide
* Properties are determined by their side chain (R group)
* Side chain differ in size, shape, and chemical properties
* Hydrophobic (may aggregate in an aqueous environment as a result of thermodynamically-stable hydrophobic interactions)
* Hydrophilic (may be charged-polarized and capable of forming ionic bonds, while other side chains favour the formation of hydrogen bonds)
* It is these interactions between the side chains that will allow the formation of a stable three-dimensional shape called a protein

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Alpha Helix

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* Amino and carboxyl groups are attached by covalent bonds called peptide bonds to form the polypeptide chain
* Coiled by the formation of hydrogen bonds between the carbonyl of the carboxyl group on one amino acid residue and the amide of the amino group of another amino acid residue four positions away
* Resulting in the R-groups sticking out of the helix

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Bound Ribosomes

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* mRNAs that encode proteins that are destined for the endomembrane system include a special signal sequence that once translated causes the ribosomes to become bound to the endoplasmic reticulum
* Special signal sequence is synthesized by the ribosome at the very initial part of the protein
* Once this signal sequence appears, it binds a signal recognition particle (SRP) which then binds to a signal recognition particle receptor (SRPR) in the ER membrane
* Polypeptide continues to be translated and is able to enter into the lumen (or the central canal) of the ER
* Once inside the ER the signal sequence is removed and translated polypeptide is now able to undergo important changes that allow for the final stages of protein processing and maturation to occur

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Protein Misfolds

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* Cystic fibrosis is a genetic disease that causes the accumulation of mucus in many organs
* Linked to mutations that lead to malfunction of an important cystic fibrosis transmembrane conductance regulator (CFTR) ion channel protein

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Cystic Fibrosis

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* Autosomal recessive matter (has to get the gene from both mother and father)
* Symptoms: persistent cough, thick mucous, wheezing, shortness of breath, chest infection and/or pneumonia, bowl disturbances (i.e. obstruction), salty “tasting” sweat
* Narrowing of the lumen of the trachea

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Airway Walls

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* Ciliated cells (must be in aqueous environment)
* Goblet/mucous cells (keep mucus flowing and from not getting into the epithelial cells)

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CFTR (Cystic fibrosis transmembrane conductance regulator)

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* Gene was discovered in 1989 by Francis Collins, Lap-Chee Tsui and Jack Riordan
* Found on the long arm of chromosome 7
* Folded in the ER
* Target is the cell membrane to pump Cl- out of the cell to keep water at the optimal volume for the airway surface liquid (ASL)
* 1480 amino acids in a normal CFTR gene

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Patients with Cystic Fibrosis

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* CFTR protein doesn’t work causing the inside of the cell to be hypertonic and depleting the ASL that the ciliated cells need
* 1479 amino acids in a CFTR gene for someone with CF
* The most common mutation is amino acid #508 (PHE) is missing
* The result of this missing amino acid causes the protein to fold incorrectly
* Protein cannot leave the ER causing degradation
* Or Protein cannot function properly at the cell membrane not allowing Cl- to leave the cell

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Pharmacological Intervention of cf

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* Chaperons (Correctors) help fold and allow the protein to surface to the cell membrane
* Chaperons (Potentiators) improve CFTR function to transport Cl-

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Splicing Recognition Consensus Sequence

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* In the intron
* GU at the 5’ end
* AG at the 3’ end
* A Branch point

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Spliceosome

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* Has a cavity that the mRNA goes in
* Splicing is actually done by RNA
* Small nuclear ribonucleoproteins (snRNPs)
* Survival of Motor Neuron (SMN) protein involved in the assembly of snRNPs

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Spinal Muscular Atrophy (SMA)

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* Number one genetic killer of children under the age of 2
* Inherited neuromuscular condition
* A Motor Neuron Diseases (MND) damaged nerve cells that control muscles
* 1 in 40 people carry the SMN1/ Delta 7-SMN1
* Occurs when an individual inherits two mutated genes

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Translation Factors

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* mRNA
* Initiation factors
* Elongation factors
* Release factors
* Aminoacyl tRNA synthetases
* tRNA
* Ribosome (ribosomal RNA + ribosomal proteins)

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tRNA Structure

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* Made up of a single RNA strand ranging between 70-90 nucleotides in length
* Hydrogen bonding between complementary nucleotide bases that form four double-helical segments and three characteristic loops
* Anticodons are written in the 3’-5’ direction
* The “A” of the CCA nucleotide sequence at the 3’ is the point of attachment for an amino acid during tRNA molecule activation

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Aminoacyl tRNA Synthetases

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* Specific to the type of tRNA and the corresponding amino acid that it will bind
* Once bound to the active site, the enzyme catalyzes the covalent attachment of the tRNA molecule to its amino acid using the energy from ATP hydrolysis
* Charged tRNA (tRNA attached to an amino acid)

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Note:

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* Approximately 45 tRNA molecules
* Some tRNA can bind to more than one codon
* Flexibility of the third nucleotide pairing referred to as a wobble

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The Process of TranslationInitiation

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* Translation initiation complex forms towards the 5’ cap of the mRNA and then scans the mRNA until an AUG start (eukaryote)
* Initiation factors bind to the 5’ cap of the mRNA
* Recruitment of the small ribosomal subunit
* Other initiation factors will bind to the tRNA that is charged with methionine
* Partially assembled complex will move along until an AUG is encountered
* Large subunit of ribosome is then able to bind to the rest of the complex using the energy from GTP hydrolysis
* Translation initiation complex will assemble at one or more ribosome binding sites called Shine-Dalgarno sequences, located a few base pairs upstream of the start codon (prokaryote)

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The Process of Translation

Elongation

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* Polypeptides are synthesized from the carboxyl end to the amino end
* Charged tRNA are delivered to the aminoacyl (A) site with a GTP-bound elongation factor
* When correct codon-anticodon pairing has been made, the GTP is then hydrolyzed and the aminoacyl end for the tRNA is release from the elongation factor
* tRNA move down a site to the peptidyl (P) site where a peptidyl-transferase reaction occurs
* Peptide bond formation is catalyzed by the enzymatic activity of an RNA molecule

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The Process of Translation

Termination

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* GTP-bound elongation factors cause the deacylated-tRNA to move from the P-site to the E-site
* Next aminoacyl-tRNA to add to the A-site will then allow for the release of the deacylated-tRNA from the E site
* GTP-bound release factors will bind to the A-site and hydrolyze the dissociation of the translation complex

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Proteome

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* Genome 20 – 25000 genes
* Proteome 1,000,000 proteins
* This suggest that single genes can encode multiple proteins
* Complexity of our proteome relative to our genome is largely attributed to RNA processing and post-translational modifications

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Detecting a signal

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* Stimulus (high blood glucose)
* Sensor (pancreas)
* Effector (insulin, which causes body cells to take up glucose, along with muscle and liver to take up glucose and store it as glycogen)
* Response (decrease in blood glucose)

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Glucose is Absorbed into the Bloodstream

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* Majority of glucose is absorbed in the microvilli cells of the small intestine
* Small amount of glucose is absorbed in the mouth across thin epithelial surfaces

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Insulin

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* Translated polypeptide that is coded in the insulin gene is 110 amino acids in length
* a-chain is 21 amino acids
* -chain is 30 amino acids
* These two amino acids chains form a dimer that makes up the functional insulin protein
* Processing of a single polypeptide of 110 amino acids to 2 polypeptides of 21 and 30 amino acids is achieved by post-translational modifications

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Post-translational Modifications

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* 110 amino acid precursor is know as preproinsulin and has a N-terminal signal sequence which interacts with signal recognition particles (SRP) to facilitate translocation into the lumen of the rough ER
* Cleavage of the signal sequence yields a proinsulin molecule
* Folding of the protein will occur with the help of chaperones in the ER
* Protein will move to the golgi apparatus where a small c-chain will be cleaved and form the mature insulin dimer of a and b chains
* N-terminal and C-terminal are able to bind to receptors on the target cells

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Other Post-Translational Modifications

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* Cleavage
* Folding
* Disulphide bridge formation
* Covalent attachment of other molecules
* Degradation of entire protein
* Phosphorylation is a reversible modification that involves the covalent attachment of a phosphate group to serine, threonine, or tyrosine amino acid residues in a protein by enzymes call kinases
* Methylation involves the covalent addition of a methyl group
* Acetylation involves addition of an acyl group

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Binding to Receptors on Target Tissues

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* Ability to bind to specific signals or ligands and produce a response
* Specific insulin receptors that fall into a family of receptors called receptor kinases
* Receptor kinases exist in monomeric form
* Dimerization (signaling molecules cause the receptors to pair up) which leads the activation of cytoplasmic domains of the receptor which have the ability to act like kinase proteins
* Kinase proteins engage in phosphorylation of specific amino acids which can lead to binding and activation of other important cytoplasmic proteins
* Intracellular signal ultimately leads to the activation of glucose transporter proteins at the cell surface, and as a result, the absorption of glucose into the cell

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Signals are Amplified

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* Initiation and maintenance of a signal is regulated by positive-feedback loops to keep the signal and amplification on
* Many elements in a signaling pathway can also activate negative-feedback loops which can lead to intracellular signal termination
* Double-negative feedback loop (an inhibitor of the signal can also be inhibited) provides fine control in a cell in response to an extracellular signal

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Alternative Splicing

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* Some exons are spliced along with the introns leading to the production of many isoforms (different types of mature mRNA) from the same pre-mRNA transcript

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Alternate Receptors

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* Skeletal muscle cells produce an insulin receptor with high affinity to insulin due to exon 11 being excised along with other introns
* Liver cells produce an insulin receptor with low affinity to insulin because exon 11 is included in its mature mRNA

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* promoters:

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* regions of typically a few hundred base pairs where RNA polymerase and associated proteins bind to the DNA duplex

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* TATA box

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* eukaryotic promoters contain a sequence similar to 5′-TATAAA-3′, which is known as a

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* general transcription factors

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* attract, or recruit, the RNA polymerase and its associated proteins to the site in eukaryotes

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* Pol II.

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* In eukaryotes, the RNA polymerase complex responsible for transcription of protein-coding genes is called

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* transcriptional activator protein

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* each of which binds to a specific DNA sequence known as an enhancer
* The transcriptional activator proteins recruit a mediator complex of proteins, which in turn interacts with the Pol II complex, and transcription begins.
* The initiation of transcription of any gene therefore depends on the availability of the transcriptional activator proteins that bind with the enhancers controlling the expression of the gene

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* polymerization reaction

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* The incoming ribonucleoside triphosphate is accepted by the RNA polymerase only if it undergoes proper base pairing with the DNA strand
* RNA polymerase orients the oxygen in the hydroxyl group at the 3′ end of the growing strand into a position from which it can attack the innermost phosphate of the triphosphate, competing for the covalent bond.
* The bond connecting the innermost phosphate to the next is a high-energy phosphate bond, which when cleaved provides the energy to drive the reaction that creates the phosphodiester bond attaching the incoming nucleotide to the 3′ end of growing chain.
* The polymerization reaction releases a phosphate–phosphate group (pyrophosphate). Cleavage of the pyrophosphate molecule makes the polymerization reaction irreversible.

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primary transcript:

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RNA transcript that comes off the template DNA strand

* In prokaryotes,The primary transcript is the mRNA

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* polycistronic mRNA:

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* Molecules of mRNA that code for multiple proteins

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*** the 5′ cap and poly(A) tail help to stabilize the RNA transcript. Single-stranded nucleic acids can be unstable and are even susceptible to enzymes that break them down. In eukaryotes, the 5′ cap and poly(A) tail protect the two ends of the transcript and increase the stability of the RNA transcript until it is translated in the cytoplasm

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888

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the large subunit of the ribosome includes three binding sites for molecules of transfer RNA (tRNA), which are called the A (aminoacyl) site, the P (peptidyl) site, and the E (exit) site.

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**

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aminoacyl tRNA synthetases

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Enzymes called aminoacyl tRNA synthetases connect specific amino acids to specific tRNA molecules. they are directly responsible for actually translating the codon sequence in a nucleic acid to a specific amino acid in a polypeptide chain

The enzyme binds to multiple sites on any tRNA that has an anticodon corresponding to the amino acid, and it catalyzes formation of the covalent bond between the amino acid and tRNA

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* initiation factors

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* Initiation of translation requires a number of protein initiation factors that bind to the mRNA. In eukaryotes, one group of initiation factors binds to the 5′ cap that is added to the mRNA during processing. These recruit a small subunit of the ribosome, and bring up a transfer RNA charged with methionine. The initiation complex then moves along the mRNA until it encounters the first AUG triplet.

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* elongation factors.

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* In elongation: Ribosome movement along the mRNA and formation of the peptide bonds require energy, which is obtained by breaking the high-energy bonds of the molecule GTP bound with proteins called elongation factors.

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* release factor

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* when a stop codon is encountered, a protein release factor binds to the A site of the ribosome. The release factor causes the bond connecting the polypeptide to the tRNA to break, which creates the carboxyl terminus of the polypeptide and completes the chain.

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* four essential elements involved in communication between all cells,

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* These elements are a signaling cell, a signaling molecule, a receptor molecule, and a responding cell.
* The signaling cell is the source of the signaling molecule, which binds to a receptor molecule on or in the responding cel

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Signaling involves receptor activation, signal transduction, response, and termination

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* Once activated, the receptor transmits the message through the cytoplasm, often by intracellular signaling pathways or cascades, in a process called signal transduction.. The message is carried from outside the cell into the cytosol or nucleus and is amplified
* Next, there is a cellular response, Finally, the signal is terminated. Termination allows the cell to respond to new signals.
* Steps in cell signaling: receptor activation, signal transduction, response, and termination.