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Cell and Molecular Biology (BIOS 20186) Fall 2018 Course Packet

Contents: Section 0: Thinking about Biology, 3 Section 1: Foundations (Ch. 1), 3 1.1: Models, Methodologies, and Universal Features of Life, 3 1.2: Biochemistry, 5 1.3: Thermodynamics and Cellular Disequilibrium, 6 1.4: Kinetics and Enzyme Catalysis, 7 1.5: Exercises, 9 Section 2: Proteins, Metabolism, and Molecular Structure (Ch. 2, 3), 10

2.1: Protein Fundamentals, 10 2.2: Protein Structure, 10 2.3: Protein Function, 12 2.4: Biochemical Pathways and Metabolism, 14 2.5: Exercises, 15 Section 3: DNA and Chromosomes (Ch. 4, 5), 16

3.1: Structure and function of DNA, 16 3.2: Structure and Function of Chromosomes, 17 3.3: DNA Replication and Maintenance, 19 3.4: Exercises, 22 Section 4: The Central Dogma (Ch. 6), 23 4.1: The Central Dogma of Biology, 23 4.2: Structure and Function of RNA, 24 4.3: mRNA and Transcription, 26 4.4: Translation, tRNA, and Ribosomes, 28 4.5: The RNA World, 29 4.6: Exercises, 29 Section 5: Regulation (Ch. 2, 4, 7), 31

5.1: Hierarchical Levels of Biological Regulation, 31 5.2: Regulation of Protein Structure and Activity, 33 5.3: Regulation of Gene Expression, 34 5.4: Epigenetics and Chromatin Modification, 36 5.5: Exercises, 37 Section 6: Membranes and Permeability (Ch. 10, 11), 39

6.1: Compartmentalization in Biology, 39 6.2: The Lipid Bilayer, 40 6.3: Membrane Proteins, 41 6.4: Membrane Transport, 42

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6.5: Excitable Membranes, 45 6.6: Exercises, 46 Section 7: How Cells Extract Energy (Ch. 14), 47 7.1: Eukaryotic Energy Demand and Mitochondria, 47 7.2: Glycolysis and the TCA Cycle, 48 7.3: The Electron Transport Chain, 49 7.4: ATP Synthase, 51 7.5: Chloroplasts and Photosynthesis, 52 7.6: Exercises, 54 Section 8: Cellular Compartmentalization in Eukaryotes (Ch. 12, 13), 56

8.1: Organelles and Mechanisms of Cellular Organization, 56 8.2: Nucleus and Mitochondrial Transport, 57 8.3: The Endoplasmic Reticulum, 58 8.4: Protein Sorting and Vesicular Transport, 59 8.5: The Golgi Apparatus, 61 8.6: Exercises, 63 Section 9: Case Studies (Ch. 11, 16), 64

9.1: The Cytoskeleton, 64 9.2: Synaptic Transmission, 68 9.3: Muscular Contraction, 70 9.4: Exercises, 72

Section 10: Cell Division in Eukaryotes (Ch. 17), 73 10.1: The Cell Cycle and Regulation of Cell Division, 73 10.2: Cell Cycle Control Systems and Cyclin-Dependent Kinases, 74 10.3: S-Phase, 75 10.4: Mitosis, 76 10.5: Cytokinesis, 78 10.6: Meiosis, 79 10.7: Cell Death, 81 10.8: Exercises, 81

Section 11: Cell Signaling and Cancer (Ch. 15, 17), 83 11.1: Principles of Cell Signaling, 83 11.2: Ion Channel-Coupled Receptors, 84 11.3: G-protein Coupled Receptors, 84 11.4: Enzyme-Coupled Receptors, 87 11.5: Other Signaling Routes, 88 11.6: Misregulation of Signaling and Cancer, 89 11.7: Exercises, 90 References, 91`

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Section 0: Thinking about Biology

Many students of science see biology as a collection of immutable facts across disparate fields, and think it is full of memorization. While it is true that there is a lot of terminology to learn – more, in fact, than in a first-year language course – it is possible to see a bigger picture. In the abstract, life is constantly struggling against an inhospitable universe that tends toward disorder. Every term, mechanism, and cellular system that we will discuss contains within it a strategy used by life to continue its quest to create order out of chaos. As you study biology, focus on how each of the particularities enables life to exist in this inhospitable universe. It does not mean there will not be some memorization, but making conceptual connections such as these makes memorization come more easily, and can help answer the question of why we collect so many seemingly unrelated ideas into a single course on cell biology.

Key terms throughout the course packet are colored purple.

Section 1: Foundations (Ch. 1) 1.1: Models, Methodologies, and Universal Features of Life

Model organisms are a group of organisms frequently used as models for broader questions about how life works. Common model organisms include E. coli (bacteria), S. cerevisiae (yeast), D. melanogaster (fruit flies), zebrafish, laboratory mice, rats, nonhuman primates such as Rhesus monkeys, and humans. In choosing a model organism for an experiment, it is important to understand how that organism can be analogized to humans or to life in general, and what its limitations are.

Some Important Methods in Biology

Microscopy allows us to observe objects with higher resolution, enabling us to see cells and the things that occur inside them. Many types of microscopy are used in biology – fluorescence microscopy enables identification of cellular components, electron microscopy and super-resolution microscopy can observe much smaller differences in cells, and confocal microscopy can scan up and down cells in the z-direction. Microscopy is limited by its resolution – most standard microscopes can observe large organelles and answer broad questions about subcellular localization, but cannot resolve two molecules or even small organelles.

Biochemistry removes cellular molecules such as proteins from their biological context to study them in isolation. This decomplexifies their study, and can be informative as to their behavior in cells, but it removes a lot of the contextual information that can be extremely important to the molecule’s biological function.

Genetic methods involve the manipulation of genetic information or the expression of that information in cells or organisms. The ability to genetically perturb life has revolutionized biology. Studying a gene or a system of genes can now be accomplished

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routinely by simply turning off the gene of interest or lowering its abundance. Genes from other organisms can be transferred and expressed in heterologous hosts. However, cells sometimes compensate for alterations in their genome, and not all hosts can successfully express all genes that one might want to transform them with, demonstrating that limitations still exist even with genetic perturbation as an experimental strategy. Universal Features of Life

All life is based on cells. Cells are membrane-bound sacks of highly concentrated, aqueous solution. The most fundamental feature of cells is their ability to create copies of themselves through growth and division.

All cells store hereditary information. This is the set of instructions the cell needs in order to be able to grow and proliferate. All cells use deoxyribonucleic acid (DNA) to store these instructions.

All cells replicate their hereditary information and ensure that one complete copy is passed to each daughter cell upon division. All cells use the same overall mechanism, that of templated synthesis based on the complementary nature of the DNA molecule. See Section 3.3 for more information on templated synthesis.

All cells express ribonucleic acid (RNA) and protein from DNA. RNA is a similar polymer to DNA that also shares its ability to form complementary base pairing, which forms the basis for its synthesis. See Section 4.3 for more on transcription. Certain RNA transcripts can then be translated into a protein sequence. Nearly all modern cells use the same protein code for translation of nuclear mRNA. For more on translation, see Section 4.4.

All life actively extracts matter and free energy from its environment and converts them to building blocks for its own growth and reproduction. All cells also maintain their internal order by emitting waste matter and entropy.

All cells are enclosed by a membrane across which they selectively exchange matter, energy, and information.

All life responds to its environment in order to maintain its internal conditions (this is known as homeostasis).

All life changes, or evolves, and adapts to the conditions of its environment. Over successive generations, species become more effective at extracting resources from their environments. The mechanism of environmental adaptation used by all life is known as natural selection. Under the paradigm of natural selection, individuals within a species that possess heritable traits that confer an advantage in their environment are more likely to survive and reproduce, which causes the responsible genes to become enriched in a population over a series of generations.

Life is complex, and heterogeneous at every scale, from the different amino acid sequences that make up a cell’s proteins, to the cells that make up our bodies and the

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individuals that make up a species. This characteristic is not shared by non-life, which tends to be disordered and to become molecularly homogeneous at macroscopic scales.

Life can be hard to define. Viruses, for example. require the machinery of living cells in order to replicate, but still display many characteristics associated with life such as replication and evolution.

Despite these ambiguities, the above characteristics have been shown to be at least nearly universal features of life, as far as we have been able to observe, but the border between life and non-life will never be perfectly resolved.

1.2: Biochemistry

Cells and life obey the laws of physics and chemistry. Conservation of energy, laws of chemical equilibrium, and the second law of thermodynamics, that entropy (or disorder) in the universe always increases all still hold inside cells. In this section, we will discuss the mechanisms cells employ to stay alive within the framework of physics and chemistry. The properties of water are incredibly important to biology. It is a liquid at biological temperatures, which allows it to act as a solvent. It can solvate organic molecules and ions. It forms hydrogen bonds, bonding-like interactions between oxygen on one water molecule and hydrogen on another, that can form interactions with functional groups on organic molecules or proteins, such as hydroxyls.

Figure 1.1: Two water molecules engaging in hydrogen bonding.

Molecules that can form strong hydrogen bonds with water or are otherwise polar are referred to as hydrophilic, or water-loving, while non-polar molecules such as hydrocarbons are considered hydrophobic, or water-fearing.

Life is possible due to the activity of a highly diverse group of polymeric macromolecules. These molecules are polymeric because they are formed from a series of repeating, similar units. In the case of DNA or RNA, these monomer units are called nucleotides. For proteins, they are amino acids.

The order in which these units are assembled can have drastic effects on the properties of a macromolecule. Different proteins, for example, act as enzymes, binders, channels through membranes, signaling molecules, motors, or perform one of dozens of other important functions the cell is constantly carrying out, and all are synthesized from the same set of 20 amino acids.

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The power of macromolecules lies in probability. Given that there are 20 protein-coding amino acids in modern cells, there are 20100 or 1.27x10130 possible 100-mer sequences that could be synthesized. If we were to synthesize just one molecule of each of these possible 100-mers, we would run out of matter in the universe, and still have orders of magnitude more to go. Yet the average protein encoded by a mammalian cell is 450 amino acids long. Given this combinatorial argument, it can be easily understood that the vast majority of sequences are functionally useless to biology. But if you’re looking for a protein that can catalyze an important reaction, it seems reasonable that at least one of these 1.27x10130 possible sequences could accomplish this goal. 1.3: Thermodynamics and Cellular Disequilibrium

The Second Law of Thermodynamics states that the total entropy of the universe must always increase. Entropy is a chemical quantity that can be intuitively understood as similar to disorder.

Creation of order requires an input of free energy and export of entropy. Entropy is an important consideration in the spontaneity of chemical reactions. Cells

drive non-spontaneous processes by coupling them to spontaneous processes of higher energy, using the free energy released to drive the nonspontaneous process.

Figure 1.2, adapted from textbook Fig. 2.29: Biology can use the energy of a spontaneous process to drive a non-spontaneous one. However, no process is perfectly efficient, so it must use a larger energy spontaneous process to drive a

smaller energy non-spontaneous one

Gibbs free energy is a quantity in chemistry that defines the direction of a spontaneous process. Spontaneous processes have a negative change in free energy, meaning they release free energy, while non-spontaneous processes have a positive change in free energy, meaning they absorb it.

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Energy Sources and Carriers Cells derive energy from the oxidation of carbon, which releases chemical potential energy. Oxidation involves the removal of electrons from a molecule. The reverse of this process, the transfer electrons to a molecule, is known as reduction.

Cells use the majority of this energy to synthesize adenosine triphosphate (ATP). ATP has three negatively charged phosphates attached to each other, so the hydrolysis of this molecule to ADP and one free inorganic phosphate releases some of this strain, resulting in an increase in free energy. The conversion of one molecule of ATP into two molecules, ADP and phosphate, also increases entropy, which further drives the spontaneity of this process.

Figure 1.3, adapted from textbook Fig. 2.33: ATP hydrolysis to ADP and inorganic phosphate releases energy by breaking a phosphoanhydride bond

Cells also synthesize reducing equivalents, electron carrier molecules like NADH. Since the oxidation of carbon releases energy, the reduction of carbon, which allows cells to create the material building blocks they need to grow and divide, requires energy. Molecules like NADH store high-energy electrons and furnish them to enzymes that can catalyze reactions that transfer them to other molecules.

Cells continuously drive themselves away from equilibrium using these stored forms of free energy; "death" constitutes a restoration of equilibrium within the biological system.

1.4: Kinetics and Enzyme Catalysis

In chemistry, kinetics and energetics are unrelated. This implies that a reaction can be accelerated or slowed without any effect on the overall energy change involved in the reaction, or its final equilibrium position – in other words, knowing the start and end points of a system tells us nothing of the path that system took to get there.

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Figure 1.4, adapted from textbook Fig. 2.21: Reactant Y and product X are at the same total energy in panels A and B, but the energy barrier Y needs to overcome to be able to get to X is smaller in panel B. As a result, B corresponds to

a faster reaction, such as one catalyzed by an enzyme

In order to maintain disequilibrium and order, cells use enzymes, which allow them to selectively accelerate chemical changes that would be incredibly slow, or perhaps not occur at all, at their internal temperature and pH.

Enzymes reduce the activation barrier and accelerate the rate of a reaction by stabilizing its transition state.

To expand the range of reactions they can catalyze, enzymes sometimes employ cofactors or coenzymes, organic molecules that can aid in catalysis.

Enzymes, in general, are remarkably selective, binding only one substrate or a small group. This allows cells to control which reactions they execute, and at what times.

Figure 1.5: An enzyme binds its substrate in a highly specific manner and then catalyzes its conversion to a product

Due to their complexity, in most cases they operate in a narrow window of optimal temperature and pH.

pH refers to the concentration of hydrogen ions (protons) in a solution. A lower pH indicates a higher hydrogen concentration, and the pH scale is logarithmic.

𝑝𝐻 = − log([𝐻*]) where [H+] is the concentration of protons. Neutral water has a pH of around 7.

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1.5: Exercises Methodologies:

1. Describe how microscopy, biochemistry, and genetic tools enable the study of biological systems, and explain the limitations of these tools.

Universal features of life: 2. All life uses ribosomes to synthesize proteins, but the structure and sequence of

ribosomes has been relatively conserved throughout the entire history of evolution. Given the importance of proteins to cell function, speculate as to why ribosome structure might be so similar in all the different kinds of species alive today.

3. Why is it important for life to replicate itself? Use entropy and natural selection in your reasoning.

Macromolecules: 4. How many possible 83 amino acid peptides can be made from the set of protein-

coding amino acids?

Hydrogen bonding and solubility: 5. Determine whether the following statements are true and explain why they are true

or false: - Water has a lower boiling point than chemically similar molecules or molecules

of similar molecular weight as a result of hydrogen bonding. - Molecules or regions of molecules that hydrogen bond will generally be

hydrophilic. - Nonpolar molecules are highly hydrophilic.

Thermodynamics and Bioenergetics:

6. DNA has a much lower entropy than free nucleotides, since they are all restrained to a single linear molecule and cannot freely move about their solution. If entropy always has to increase, how can biology synthesize a large biopolymer like DNA?

7. The free energy change associated with ATP hydrolysis is DG = ~-50 kJ/mol. What is the free energy change required to synthesize ATP from ADP and inorganic phosphate? Is free energy absorbed or released by the synthesis of ATP?

Enzymes and cofactors:

8. Determine whether the following statements are true and explain why they are true or false:

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- Enzymes can facilitate reactions that would not otherwise happen in cells by making them energetically favorable.

- Enzymes have a narrow range of temperature and pH in which they can operate. - NADH is a cofactor that stores energy that can be released by hydrolysis.

Section 2: Proteins, Metabolism, and Molecular Structure (Ch. 2, 3) 2.1: Protein Fundamentals

Proteins are synthesized from a specific sequence of amino acids encoded in the genome.

Cells use 20 amino acids (AAs) to make proteins.

Figure 2.1: The general structure of an amino acid. R represents the side chain that differs for each of the 20 different AAs. The amino and carboxy termini are positively and negatively charged, respectively, at neutral pH.

Proteins have an amino, or N, terminus, and a carboxy, or C terminus. They are synthesized in the N to C direction.

Proteins vary widely in size, shape, and properties. Proteins never branch, unless multiple fully synthesized proteins are chemically

joined. Cells maintain a state of dynamic proteostasis: the majority of proteins are

continuously synthesized and degraded, depending on cell state, and the cell can increase or decrease their synthesis or degradation in response to environmental cues.

2.2: Protein Structure

Protein sequence is the order of the amino acids from N to C terminus, as encoded by the genome. The physical interactions that result from this sequence – the hydrogen bonds, hydrophobic contacts, and ionic interactions between amino acids – cause the protein to fold into a three-dimensional structure. This structure determines the protein’s function.

Structural information is often invaluable to understanding protein function, and almost always more valuable than simple sequence information. The most common method biologists use to obtain structural information is X-ray crystallography, but other methods, such as nuclear magnetic resonance and cryo-electron microscopy are increasing in their use.

Protein structure is exclusively defined by amino acid sequence, and represents the lowest energy conformation of that specific sequence. However, structural information is

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essentially impossible to predict from amino acid sequence (with the current state of computing) due to the complex nature of interactions that dictate protein folding.

Four major groups of intermolecular forces influence protein folding: o Hydrogen bonds are attractive interactions between oxygen or nitrogen and the

hydrogens attached to similar atoms on other molecules o Salt bridges are attractive electrostatic interactions between positively and

negatively charged groups o Disulfide bonds are covalent bonds between two sulfur atoms between residues of

the AA cysteine o The hydrophobic effect results from the fact that interfaces between water and

nonpolar parts of molecules are energetically unfavored, so hydrophobic groups tend to collapse together to minimize the area of interaction with water. As a result, hydrophobic groups often behave as though they are attracted to each other, much as oil drops in water tend to coalesce.

Figure 2.2: Four levels of protein structure (https://cnx.org/contents/GFy_h8cu@11.5:2zzm1QG9@8/Proteins)

Protein structure is organized into four levels (Fig. 2.2): o Primary: The amino acid sequence of the protein o Secondary: Hydrogen bonds formed between backbone carbonyl (C=O) lone

pairs and amide (NH) hydrogens o Tertiary: All other interactions within a single molecule. The dominant type in

determining overall protein structure is usually the hydrophobic effect.

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o Quaternary: Multi-subunit structures that form from interactions between multiple protein molecules

Protein secondary structure is formed form hydrogen bonds. Protein secondary

structure includes a helices, b sheets, and loops, also called unstructured regions. Many proteins are only soluble in their folded state. If a cell is stressed, such as by

elevated temperature, proteins can become misfolded, leading to aggregation – essentially desolvation of the proteins, resulting in solid precipitate within cells. The critical role of protein aggregation is becoming increasingly understood in certain neurodegenerative diseases.

Figure 2.3, adapted from textbook Fig. 3.5: How protein sequence can influence protein folding

2.3: Protein Function Proteins perform the majority of functions within cells and constitute the majority of

their dry weight. Proteins make physical interactions with small organic molecules. Enzymes catalyze chemical reactions. Strategies of enzyme catalysis can include

conformational restraint, desolvation of substrates, acid or base catalysis, redox catalysis, charge stabilization, or energetic coupling (i.e. to ATP hydrolysis). Enzymes often employ a combination of these strategies to catalyze a reaction

Proteins can also form protein-macromolecule interactions with other biomolecules, such as protein-protein, protein-DNA, or protein-RNA interactions.

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Figure 2.4, adapted from textbook Fig. 3.18: Two identical proteins form a homodimer via a protein-protein interaction

Proteins can be post-translationally modified. After they are fully synthesized, other cellular enzymes can derivatize them with a variety of chemical alterations, including:

o Phosphorylation (addition of a phosphate group) o Ubiquitination (addition of the small protein ubiquitin) o Glycosylation (addition of sugars) Post-translational modification can affect the structure, activity, function,

localization, or degradation of proteins Nearly all proteins are activity-regulated post-translationally. Some proteins’ entire

function is to regulate other proteins, or to transmit information and signal to other parts of the cell or to other cells.

Recall that structure determines function – many proteins have dynamic structure, and undergo conformational change that can affect its function or activity as a result.

Some proteins exhibit allostery. In allosteric systems, the activity at one binding site on a protein can transmit structural information to other locations on the protein or its binding partners. This can result in cooperativity in binding, where binding of one substrate can increase the affinity of the substrate for other binding sites on the protein.

Figure 2.5: Proteins with multiple binding sites can engage in allostery, where binding of one ligand predisposes the molecule to bind other ligands1.

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Many proteins serve as structural elements for the cell, such as its cytoskeleton. Other proteins such as integrins form adhesive contacts with molecules outside the cell.

Proteins can act as transporters or channels to selectively permit the passage of soluble molecules or ions across biological membranes.

Multiprotein complexes can form molecular machines that execute complex tasks, such as the ribosome, a complex composed of RNA and protein molecules, that is responsible for protein synthesis.

2.4: Biochemical Pathways and Metabolism

Glycolysis is the central axis of metabolism in cells. It breaks down glucose into smaller three carbon units, extracting energy in the form of ATP and reducing equivalents in the process. These three carbon units can then be further oxidized for more energy or serve as material building blocks for nucleic acid, protein, or fatty acid synthesis.

During periods where energy is plentiful, such as after a large meal, fatty acids (carboxy groups with long hydrophobic chains of reduced carbon) are synthesized for energy storage. When energy is needed, they can be oxidized to CO2 to release energy, or partially oxidized into amino acids or other building blocks.

Figure 2.6: General structure of a fatty acid

Excess carbon not needed from glycolysis is sent through pathways known as the citric acid, or tricarboxylic acid (TCA), cycle and oxidative phosphorylation, where it is oxidized to CO2, carbon’s most oxidized state. In the process, energy is extracted into reducing equivalents, which provide energy to phosphorylate ADP to make ATP.

Cells metabolize excess nitrogen, such as from the breakdown of proteins, through the urea cycle, which converts toxic ammonia into lower toxicity urea. Urea is also more water soluble than ammonia, allowing it to be easily exported through the kidneys.

Cells organize their metabolism into biochemical pathways. Essentially all pathways lead into and out of glycolysis, which minimizes the material that the cell wastes and ensures that it can always convert materials it has in excess to materials it is deficient in.

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Figure 2.7: A diagram of metabolic pathways in the cell, with glycolysis and the TCA cycle highlighted in red

2.5: Exercises Protein Structure and Folding:

1. Describe the three major elements of secondary structure.

2. Given what you know about protein tertiary structure, would it be likely to find hydrophobic amino acid residues at the core of a soluble protein? Explain.

3. Determine whether the following statements are true and explain why they are true

or false: - Chaperone proteins help proteins fold by enforcing a particular 3D structure. - Protein structure determines function. - Protein sequence will not vary significantly for two proteins that perform a

similar function. -

Post-translational Modification of Protein: 4. Using what you know about the factors that influence protein structure and function,

give an example of how the addition of a negatively charged phosphate to an amino acid side chain might affect an enzyme’s ability to catalyze a reaction.

Allostery: 5. ****The glycolytic enzyme phosphofructokinase 1 (PFK1) catalyzes the transfer of a

phosphate from ATP to fructose-6-phosphate. This step commits the carbon in fructose-6-phosphate to the glycolytic pathway, the last checkpoint before it is broken