Rene Fester Kratz, PhD, teaches us microbiology without using jargon. This book was intended to help 2 audiences: students and self-learners. It's organized into 7 parts: intro to the cell, molecules, working cell, genetics, molecular genetics, tools of molecular biology, and "part of tens".
Molecular/cellular biology is the study of cell structure/function down to the individual molecules that make it up. This chapter is an overview of the book. Everything will be explained in greater detail in later chapters.
Cells are the smallest unit of life. All living things are made up of cells, including us. Besides the cells that make us up, there are also prokaryotic cells:
- Bacteria - they make us sick but do good things too
- Archaea - just as common but less known, recently discovered
Viruses are not cells. They're smaller than bacteria and our eukaryotic cells. They attack other cells and are parasitic. Attacked cells may end up reproducing more viruses. Viruses are the cause of sore throats and HIV.
Cells require a lot of the same things we need:
- eating food or taking in a source of energy for cellular metabolism
- breathing oxygen or cellular respiration for energy
- using food/energy to grow and reproduce
You started life as a single cell, a combination of a sperm/egg. Each of these germ cells donated half of your genetic information (23 chromosomes each).
The instructions for each cell to work is encoded in DNA. Every cell has a copy of your DNA. They're constantly reading it to build new molecules like proteins. Proteins are created via transcription and translation. Signals, like hormones, can tell cells to change behavior. Gene regulation allows cells to turn off some genes and turn others on.
New technologies have been discovered which allow scientists to extract, read, or modify DNA. New branches of biology are opening up:
- Bioinformatics - blending computing, biology, and IT to organize/analyze large amounts of biological information
- Genomics - study of entire genomes of organisms to learn/discover new proteins and genes
- Proteomics - study of proteins for different cells
All cells fall in one of three groups:
- Eucarya: plants, animals, fungi, and protists
- Bacteria: single-celled microorganisms
- Archaea: single-celled similar to bacteria
Cells of Eucarya are called eukaryotic. They have a nucleus containing DNA and organelles. Bacteria/archaea cells are prokaryotic. They don't have a nucleus, their DNA floats within the cytoplasm. They're typically smaller.
All living things are made up of cells. Some are multicelluar - body is made up of multiple cells. Others are unicellular.
All cells contain:
- Plasma Membrane or Cytoplasmic Membrane - the boundary of a cell
- Cytoplasm - area inside cell
- DNA or Deoxyribonucleic Acid - plans for how cell is built and how it functions
- Ribosomes - structures where protein is built
The plasma membrane is selectively permeable and have proteins on it. The proteins consist of receptors, which detect signals in the cell's environment, and transport proteins.
The cytoplasm is crowded. Most of the metabolism of a cell occurs here, including the production of proteins. The organelles move around in here.
In eukaryotic cells, DNA is separated from the cytoplasm via the nucleus. For prokaryotic cells, DNA is in the cytoplasm area called nucleoid.
Ribosomes are the workbenches where proteins are made. They're made up of ribosomal RNA (rRNA) and proteins.
The nucleus contains the cell's DNA, which is the instructions for the cell. The boundary is called a nuclear envelope. DNA is in the form of chromosomes or chromatin (wounded up vs unwounded spaghetti-ish). The cell reads the DNA and builds ribosomal subunits in the nucleoli area of the nucleus and then ships it out to the cytoplasm.
Molecules enter in/leave the nucleus via nuclear pores. This includes:
- RNA molecules and ribosomal subunits
- proteins
- nucleotides - building blocks of DNA and RNA
The endomembrane system is the post office of the cell - it builds proteins and lipids then ships them off. There are a few components:
The endoplasmic reticulum is a component that starts at the nucleus and comes in two types: rough endoplasmic reticulum and smooth endoplasmic reticulum. The rough one has attached ribosomes used to deliver protein to a special destination.
The Golgi apparatus will tag proteins before shipping them off. The middle of the ER is called a lumen.
Vesicles transport things around the cells. Transport vesicles carry molecules, lysosomes dispose of garbage, secretory vesicles bring materials to the plasma membrane.
Peroxisomes help break down lipids. This ends the endomembrane system...
The mitochondrion is the fireplace where cells burn their food via cellular respiration. Mitochondria are the power plants of the cell which transfer energy from food to ATP (stored energy for the cell).
Chloroplasts make food molecules via photosynthesis and are only found in plant cells.
The structure of the cell is supported by the cytoskeleton - kind of a scaffold which holds the cell together. They run though the cell providing a network for vesicles/organelles to travel. Cells flick with cilia and eukaryotic flagella to move about. Cytoskeletal proteins come in three groups:
- Microfilaments - make muscle cells contract and act as railroad tracks to a cell's organelles
- Microtubules - protein inside cilia and flagella
- Intermediate Filaments - reinforcing proteins for cells
The plasma membrane provides a selective boundary for the cell. Most cells have additional layers outside of it. The layer around the animal cell is called the extracellular matrix.
Viruses are microscopic particles of nucleic acid (DNA or RNA) and protein that attack cells and turn them into virus factories. They're responsible for significant diseases: measles, influenza, polio, and AIDS.
Whereas bacteria are living cells, viruses are not. They cannot do anything without hijacking a cell. Scientists don't consider viruses to be alive.
The simplest viruses have two components: a nucleic acid core and a protein capsid. The DNA or RNA contains instructions for taking over cells to produce more virions (viral particles) and the capsid is an outer shell.
Some viruses also have an outer envelope that they steal from the cell's membrane. They might also have proteins that stick out called spikes, which help attach them to cells.
Viruses come in three shapes:
- helical - form of twisting helix around nucleic acid core
- polyhedral - regular geometric shape
- complex - separate patches of protein forming unique structures
Viruses attach to cells if they have the right viral protein that's accepted by the cell's receptors. They then dig a hole through the cell wall, slip in by fusing with the cell membrane, or trick the cell into bringing it inside. Host range is used to denote the type of cells a virus can infect.
Bacteriophage (phage = eat, so bacteria eater) are a type of virus which attack bacteria. In the lytic cycle, bacteriophage will use the cell to create more phage and destroy it. In the lysogenic cycle, bacteriophage will enter during a dormant stage. The host cell will reproduce - creating cells which are infected.
Example steps of the lytic cycle:
- Attachment - proteins of virus attach to receptors of bacteria
- Penetration - genetic material of virus enters
- Biosynthesis - virus uses host cell's nucleotides/amino acids to make more viral parts. ATP from host cell supplies energy
- Maturation - viral particles assemble into new virus
- Release - virus leaves host cell, lysing or destroying as they leave
Bacteriophage that enter the lysogenic cycle remain inactive/dormant in the bacteria for a while. During this time, their DNA is integrated with the host cell's DNA (now called prophage). The steps are then: attachment, penetration, recombination, replication. Environmental signals will trigger the virus to go into lytic cycle.
Viruses that attack eukaryotic cells are the same, but some terms are different:
- Latent instead of lysogenic
- Provirus instead of prophage
- Acute disease instead of lytic cycle
After an enveloped virus attaches to its eukaryotic host cell, it enters via one of two ways:
- Fusion - envelope of virus fuses with host cell's membrane, like two soap bubbles merging together
- Receptor-mediated endocytosis - the host cell is tricked, wraps the virus in a vesicle and brings it in
After entering, the virus removes its capsid (called uncoating) and releases its genetic material into the host cell. Viruses have different reproductive strategies which are dependent on the class of virus:
- Double-stranded DNA viruses: ie smallpox, inserts its genetic material into the host's nucleus. It gets copied whenever the host divides
- Retroviruses: ie HIV, single stranded RNA enters the host cell and reverse transcribes a double stranded DNA to insert into the nucleus
- Double-stranded RNA viruses: introduces their RNA into the cytoplasm to make viral proteins
- Single-stranded RNA viruses: negative-sense or positive-sense. Negative like influenza, insert their RNA into cytoplasm to create new RNA and make proteins. Positive insert their RNA directly, doesn't need to create new RNA.
Viruses may use their own enzymes or host enzymes to copy genetic material. Viral enzymes make more mistakes in host enzymes, causing mutations in viruses. The variation makes it harder to defeat.
After the genetic material is copied, the viruses leave the host cell. Sometimes they leave via budding, pushing up against the membrane and taking a chunk of it while leaving. This helps disguise them as cells.
Putting it all together, the steps are: attachment, penetration, uncoating, biosynthesis, maturation, release.
Organisms are made of cells. Cells are made of molecules. Molecules are made of atoms. Atoms are made of subatomic particles.
Subatomic particles are protons (positive charge), electrons (negative charge), and neutrons (no charge). The nucleus of an atom is made of protons/neutrons and the electrons orbit it. The number of protons determine what element an atom is. It's called the atomic number.
An isotope has different numbers of neutrons than protons. Carbon usually has 6 protons/neutrons. Carbon-5 is an isotope variation that has 5 neutrons.
Periodic tables often include the atomic mass of elements, which is the average mass in atomic mass units of all atoms on earth for each element. It's the number of protons plus the number of neutrons.
Atoms form larger structures called molecules via chemical bonds. This will happen via:
- Atoms share an electron to form a more stable structure
- There is an attraction between opposite electrical charges
Each element has an electron configuration, which is useful in determining how stable that element is. Noble gases have an outer shell full of electrons, so they're pretty stable. Valence electrons are the electrons in the outermost energy levels. They're the electrons which are usually given/shared.
The Periodic Table will tell you how many valence electrons an element has. Most atoms follow the octet rule, most of them want 8 electrons in its outer shell. Elements on the left side tend to want to give away electrons. Elements on the right side really want more electrons. The tendency for an atom to attract electrons is called electronegativity.
If one atom takes an electron, an oxidation reduction reaction or redox reaction has occurred. The atom giving up an electron was oxidized and the atom gaining an electron was reduced (think in term of charges).
For more information on electron configuration and chemical bonds, see notes on Khan Academy's Chemistry lectures.
Whereas an isotope had differing numbers of neutrons, ions have different number of electrons causing a charge. Ionic bonds form due to electrical attractions from opposite charges. They're strong in solid form, but weak in liquid form and in water.
Covalent bonds occur when atoms share an electron. Two oxygen elements are equally electronegative and they both benefit by sharing two electrons by becoming more stable (rule of eights).
In a polar covalent bond, the electron spends more time orbiting the nucleus of the more electronegative atom. After polar covalent bonds form, they setup the formation of hydrogen bonds. Hydrogen bonds are weak electrical forces between opposite ends of polar bonds. They're like velcro. Hydrogen bonds hold the two helixes of your DNA together.
Hydrophobic molecules don't mix well with water. Water has many polar and hydrogen bonds. When a molecule with polar bond is mixed in, it dissolves well. When a covalent bonded molecule is mixed in, it's rejected - like water/oil.
Your body is 60-75% water. Water is essential to living things:
- Water is a solvent. Ions and polar molecules dissolve in water. Water prevents them from forming solids. All chemical reactions in cells occur in water.
- Water helps moves things across membranes.
- Water causes hydrophobic interactions.
- Water helps maintain the structure of important molecules.
Water molecules reach a equilibrium where H(2)O splits and forms hydrogen and hydroxide ion:
H(2)O <--> H^+ + OH^-
The concentration of hydrogen ions is measured in pH (-log of the molarity). The concentration of hydroxide ions is measured in pOH. A pH of 7 is neutral on the acid/base scale.
Cells can only survive within a narrow range of pH - they prefer neutral. Some chemicals can add hydrogen ions and increase the pH of water. These are acids. Chemicals that remove hydrogen ions are bases.
Cells employ buffers to maintain the pH of water. These buffers can release hydrogen ions in a solution or remove them. Your blood has the acid/base pair of carbonic acid and bicarbonate to maintain your blood pH.
Cells often recycle and rebuild structures using existing molecules. When a structure is no longer needed, cells will break it apart to form a new one from the pieces (different shape and recombining with different atoms).
There are four groups of macromolecules (big molecules) important to the cell: carbohydrates, proteins, nucleic acids, and lipids. All of them but lipids are made up of the same repeating type of building block. A long repetitive molecule is called a polymer. The building block of a polymer is a monomer.
Whenever a monomer is added to the polymer, a water molecules formed. The reaction is called condensation or dehydration synthesis. Polymers can be broken down into individual monomers via hydrolysis - a water molecule is inserted into the bond between two monomers to break it apart.
Remember hydro means water and lysis means breaking (as in when a virus lyces from the cell - breaks out of).
Carbohydrates are made of carbons, hydrogen, and oxygen in the ratio of:
CH(2)O
They're divided into two groups: monosaccharides and polysaccharides. They're also known as simple sugars and complex carbohydrates.
Monosaccharides have a backbone of 3-7 carbons and are called trioses, tetroses, pentoses, hexoses, and heptoses. They have Hydroxyl groups (OH) attached to every carbon but one which make up sugars polar (and dissolves in water).
They have one double-bonded oxygen attached to the carbon backbone called a carbonyl group. If it's at the end, the sugar is an aldose. If it's within the backbone, it's a ketose. It's not just the number of atoms which determine what kind of simple sugar it is - the structure is important too.
Polysaccharides are multiple monosaccharides joined together by condensation reactions (removal of water). The bonds between them are glycosidic linkages. They're classified by how many monosaccharides there are: disaccharides like lactose, oligosaccharides (short chains), or polysaccharides (long chains).
Carbohydrates are an important source of energy for cells. They provide structural molecules for cells like the walls. Cells are marked with glycoproteins (proteins with sugar) to identify them. They make a sticky matrix around cells to help bacteria stick.
Most jobs in the cell are done by proteins. The shape of a protein is essential for its job. They control metabolism, transport, communication, structure, division, and more. They can specifically target a particular molecule or process.
Proteins start as polymers of amino acids, also called a polypeptide. Once that folds and becomes functional, it's a protein. Some protein are made of multiple polypeptides.
If a protein denatures (unfolds), it can no longer function. Protein structure or conformation is organized into 4 categories:
- Primary structure - sequence of amino acids
- Secondary structure - small areas folded into alpha helices and pleated sheets
- Tertiary structure - final 3D shape of one folded amino acid chain
- Quarternary structure - found in proteins consisting multiple folded chains
The cell contains 20 variants of amino acids. The choice and ordering of these amino acids makes up the primary structure of a protein. Each amino acid has the same structure, a central carbon atom is bonded to 4 different chemicals:
- a nitrogen-containing amino group (-NH(2) or NH(3)+)
- a carboxyl group (-COOH or -COO-)
- a hydrogen atom (-H)
- A side chain (-R) that varies between the 20 amino acids
The N-C-C backbone pattern is useful in identifying amino acids. You can use it to distinguish proteins/carbohydrates. The bonds between amino acids are peptide bonds and are covalent formed via condensation.
The folded patterns/areas of a protein make up the secondary structure. There are two possible fold patterns:
- Alpha helix - similar to a smooth spiral
- Pleated sheets - sharp turns/twist in the opposite direction
Some proteins only have the alpha helix, some only have pleated sheets, some have both. They're held together with hydrogen bonds in the nitrogen/hydrogen atoms and carbon/oxygen atoms.
The tertiary structure is the final 3D shape of the protein. There are also two categories:
- Globular proteins: have an overall rounded or irregular shape
- Fibrous proteins: are long and cable-like
The tertiary structure is held together with various bonds in the R groups:
- Covalent bonds: the amino acid cysteine has a sulfhydrl group (-SH) in its R group. When two are next to each other, they form a disulfide bridge which is covalent and strong. It doesn't break when protein denatures.
- Ionic bonds: some R groups ionize in water and form ionic bonds. They're weak in watery solution and will be lost if the protein denatures.
- Hydrogen bonds: if a polar covalent bond forms, so do hydrogen bonds which are weak and will be lost if the protein denatures.
- Hydrophobic interactions: some R groups are hydrophobic. Water will push them together to form a bond. These are weak and will also be lost if the protein denatures.
Some proteins will start to function as soon as they form their tertiary structure. Others require a quarternary structure. These are large and complex. They form multiple polypeptide chains. The bonds that hold the polypeptides together are the same bonds holding tertiary structures together, but only with the R groups. The hemogoblin protein in your blood is quarternary.
Proteins carry out many tasks:
- they're enzymes
- they reinforce structure like the cytoskeleton and extracellular matrix
- they transport materials in/out of the cell in the plasma membrane
- they identify cells with glycoproteins on the membrane
- they help cells move (flagella)
- they help communicate via receptors or insulin
- they organize molecules. Chaperone proteins help other amino acids fold into proteins.
- they defend the body against bacteria/viruses (antibodies)
- they regulate which parts of the DNA is used
Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are both polymers of nucleotides. DNA specifies which proteins and RNA to construct.
Nucleotides have 3 different components and make up nucleic acids. Nucleic acids can be single or double-stranded. Chained together, they're called polynucleotide chains. The components of nucleotides are:
- A pentose (5-carbon) sugar - deoxyribose or ribose
- A nitrogenous base
- A phosphate group - a phosphorous atom surrounded by oxygen atoms
To navigate the nucleotides the carbon atoms are numbered from 1 to 5. They're also marked as prime: 1', 2', 3', 4', 5' to denote the difference between the sugar carbons and nitrogenous base carbons. The first carbon (1') is attached to the nitrogenous base. Numbers increase as we move away.
The pentose is different for DNA and RNA. DNA has deoxyribose as its sugar whereas RNA has ribose - the difference is just one oxygen atom. Ribose has a hydroxyl group (-OH) attached to its 2' carbon while deoxyribose only has a hydrogen (-H). Nucleotides are either ribonucleotide or deoxyribonucleotide. DNA is double stranded while RNA is single stranded.
There are five different nitrogenous bases found in nucleotides:
- Adenine (A)
- Guanine (G)
- Cytosine (C)
- Thymine (T) - only in DNA
- Uracil (U) - only in RNA
These bases fall into two groups. Pyrimidines with a single ring structure and two nitrogen atoms within the ring. Purines have two fused rings with two nitrogen atoms in each ring. Cytosine, thymine, and uracil are pyrimidines. Adenine and guanine are purines.
Polynucleotide chains give DNA its second structure: a double helix. The sides of the ladder are made of the sugar phosphate backbone. The rungs are made of nitrogenous base pairs with hydrogen bonds. The sides are antiparallel, if one side has its 5' carbon pointing up - the other has it pointing down.
Adenine forms hydrogen bonds with thymine. Cytosine forms hydrogen bonds with guanine. The complementary base pairs are A=T, C=G.
For RNA, it's A=U. RNA can also have a 3d secondary structure. They fold upon themselves into complex shapes.
Our genetic code is encoded in those base pairs (A, T, C, and G). Cells decode DNA for many tasks:
- Protein structure: determined via transcription/translation. Code in DNA is copied and used to specify amino acids sequence
- RNA structure: through the process of transcription
- Messenger RNA (mRNA): goes from DNA to ribosomes to produce proteins
- Transfer RNA (tRNA): decodes message from mRNA to match amino acids
- Ribosomal RNA (rRNA): part of the structure of the ribosome
Lipids are molecules that don't mix well with water. Some lipids made by cells include: fats, phospholipids, waxes, cholesterol, steroids, and sterols.
Hydrocarbons are molecules that have long chains of carbon atom bonded to hydrogen atoms. Lipids are hydrophobic because they contain non-polar covalent bonds between carbon/hydrogen.
They're more diverse than the other three macromolecules (carbohydrates, proteins, and nucleic acids). They're not polymers. Their subcategories:
- Fats and oils: made of fatty acids attached to glycerol backbone
- Phospholipids: same as above but with unique hydrophilic group
- Waxes: fatty acids attached to long-chain alcohols
- Sterols: contain four fused carbon rings
Fatty acids are long chains of carbon and hydrogen. Their 2 categories:
- Saturated fats: carbon atoms bonded with single bonds. Carbon has 4 available bonds - this leaves 2 for hydrogens.
- Unsaturated fats: carbon atoms bonded with more double bonds. These carbons can only bond with 1 hydrogen atom.
When three fatty acids join a glycerol backbone (three-carbon alcohol with attached hydroxyl radicals) it's called triglycerides.
When you're trying to recognize lipid molecules, just remember that they're hydrocarbons - they have chains of hydrogen/carbon molecules.
Triglycerides with high levels of saturated fat are called fats, while those with unsaturated bonds are oils. Fats are solid at room temp because the bonds are more organized. Oils are liquid at room temp due to unsaturated bonds.
Phospholipids are similar to triglycerides but only have two fatty acid tails.
Lipids pack a lot of energy into a small space due to the carbon/hydrogen bonds. They have 9 calories per gram while protein and carbohydrates only have 5 calories per gram. Energy storage is the major function of lipids in cells.
Besides energy storage, lipids are also important for:
- insulation
- structure of plasma membranes
- water-proofing
- signaling - ie steroid hormones trigger changes in human development
The plasma membrane of the cell is selectively permeable - molecules can only pass through it the membrane allows it. The major factors determining if a molecule can/cannot pass:
- size: smaller molecules pass through easier
- attraction to water: hydrophobic molecules cross easier than hydrophilic. There's a hydrophobic interior of the phosopholipid bilayer. Hydrophilic molecules require help from proteins to pass.
Transport proteins help control what can enter/exit a cell. Most molecules are too large or too attracted to water. They can't cross without the help of the transport proteins.
Molecules will evenly distribute between the interior/exterior of the cell. Diffusion is the movement of molecules from higher concentration to lower concentration to create an equilibrium. It's called simple diffusion without the help of transport proteins. It's called facilitated diffusion with help.
There are two types of transport proteins that facilitate diffusion:
- Channel proteins: they're like soda straws. Polypeptide chains build straw walls and create an open tunnel down the middle of the protein. They form tunnels through the membrane filled with water for molecules to pass through. Some are always open. Gated channels open/close in response to signals.
- Carrier proteins: Each carrier protein has a binding site made for a specific molecule type. When it's binded, it changes shape and moves across.
Water also diffuses - it's called osmosis. It's passive and requires no energy from the cell. Greater concentrations of water mean pure water and less solutes.
- Hypertonic - solutions have a greater concentration of solutes
- Hypotonic - solutions have a lesser concentration of solutes
- Isotonic - solutions have equal concentration of solutes
Cells need to move molecules from where they're less concentrated to where they're more concentrated
- going against the flow. They do this via active transport. It requires energy from the cell, usually ATP. For example, carrier proteins change shapes using ATP from cells to carry molecules inside. These active transport carrier proteins are called pumps.
Multicellular organisms are made of a community of cells and it's important for them to communicate, act distributed, request/respond. Attachment of cells form tissues. Communication allows cells to coordinate signals/responses.
There are many types of attachments:
- tight junctions: so tight that water cannot pass through. Proteins pass through membranes of both cells. Important for tissue like skin so foreign molecules cannot pass through.
- anchoring junctions: hold cells tightly but allow molecules to pass through the space in between. Important for tissue with structure but requires demanding work like heart tissue.
Anchoring junctions have subtypes:
- desmosomes: use cadherins in the membrane of both cells. Cadherins proteins also with intermediate filaments in the cytoplasm of cells
- hemidesmosomes: anchor cells to extracellular matrix using integrins proteins.
- adherens junctions: anchor cells to each other and extracellular matrix
Between cells in almost all animal tissue are gap junctions formed by connexins proteins. They form rings on cell membranes called pores. Two cells may align the pores and form tunnels between each cell for ion and small molecules to pass through. Plant cells have similar tunnels called plasmodesmata.
Signals from one cell will trigger a response in another far away - the signals are called hormones. The hormone insulin is produced by pancreas cells to tell blood cells to take glucose out of the blood as a food source.
A cell can recognize signals only if it has a receptor for it. Kind of like the observer pattern. They've got receptors in two locations: inside the cells for signaling molecules which can cross the membrane, in the plasma membrane for other molecules.
Receptor proteins have binding sites for signals. When ligands - signaling molecules - bind to these proteins, they change shape and set off a series of events in the cell. This process is called signal transduction.
A signal may be amplified via a signal transduction pathway. This is similar to the series of chain reactions of signal transduction. They main difference is the pathway can activate many secondary messengers at once.
Deactivating a signal happens when the primary messenger stops sending it. Some molecules in the cell are used to shut the messenger down: phosphatases, activated G proteins, Ras proteins, and second messenger molecules.
Cells constantly convert energy/matter into different forms to get what they need. They store energy in food molecules and break it down to get it out.
Metabolism is all of a cell's chemical reactions - like breaking down food molecules or building muscle proteins. Your body breaks down food and converts it into molecules you need. Catabolism are reactions breaking larger molecules into smaller one. Anabolism is the opposite.
Let's review thermodynamics. Work is moving something over a distance. Energy is something you need to do work. The first two laws are important for molecular biology:
- Energy can't be created or destroyed
- Chemical reactions that occur spontaneously increase entropy (disorder) of an isolated system
Energy can be organized into potential or kinetic - from an object's position/arrangement or energy from motion of molecules.
In cellular biology, spontaneous reactions are those that occur on their own without the need for energy. An example of this is diffusion, which follows the second law. Particles will flow from a concentrated area to less concentrated without energy - because it increases the disorder.
Cells maintain their order by constantly using their stored energy to do nonspontaneous reactions.
Kratz gives us a brick wall/pile analogy. The brick pile is disorder and spontaneous. A brick wall is organized and requires work and energy. Over time, the brick wall will become a pile of brick unless energy is used to maintain it. Reactions that build large, complex molecules require energy. Reactions that break down to smaller molecules outputs energy.
Exergonic reactions release energy. Endergonic reactions absorbs it.
You can determine if a chemical reaction is spontaneous or nonspontaneous by looking at the changes in free energy. Gibbs free energy (G) is used to measure entropy/heat in a reaction. A negative free energy (nG or deltaG) is a spontaneous reaction:
nG = G products - G reactants
Some of the work cells do:
- synthesis: build large/complex/structured molecules like DNA, proteins, membranes, or ribosomes
- organization: eukaryotic cells create compartments
- creation of electrochemical gradients: cells organize ions to cause differences in electrical charge which helps other reactions
- transport and movement
Cells break down food molecules for energy. They use adenosine triphosphate, better known as ATP:
- Energy from food is transferred to ATP
- ATP provides energy for cellular work
ATP is a nucleotide with the same building blocks as DNA/RNA: sugar ribose, nitrogenous base, three phosphate groups. Cells continually build/break down ATP in ATP/ADP cycles. They store energy by building ATP out of adenosine diphosphate (ADP) and phosphate groups. They obtain energy by breaking ATP back into those components (breaking it down requires water).
Metabolic changes are broken down into small steps, each a chemical reaction. Several reactions in a series make up a metabolic pathway. Enzymes speed up chemical reactions by lowering the energy of activation. Electrons are transferred between molecules during metabolic reactions. Energy is also transferred during metabolic reactions.
Chemical reactions usually happen in many small steps instead of one quick change so cells can control energy changes:
A -> B -> C -> D -> E -> F
The starting molecule A is called the substrate. The ending molecule F is the product. B, C, D, E are called intermediates.
Spontaneous reactions are just waiting to occur. All they need is the right molecules to collide with just enough kinetic energy. To initialize it, the amount of energy needed to trigger is called energy of activation (Ea).
Enzymes lower the energy of activation, making it easier/quicker for reactions to occur. Every cell reaction is catalyzed by an enzyme. Enzymes are very specific for substrates. In the formula above (A->F), five enzymes are used - one for each reaction.
Productive collisions are those with enough energy of activation for the reaction to occur. Reactants must: collide, be oriented correctly, and have enough kinetic energy to overcome energy of activation.
Enzymes will bind substrates in their active sites and move them into the correct orientation. They can alter chemical bonds making reactions more likely.
Electrons are usually transferred during metabolic reactions. Oxidation is when a molecule gives up an electron. Reduction is when a molecule gains an electron (reduced in charge).
Metabolism is all about getting what you need:
- building blocks for growth/repair
- energy for cellular work
- reducing power
When you eat food, your digestive system breaks it down into small molecules. Your cells break it down further for energy. The metabolic pathway to break food or capture stored energy for cells is called cellular respiration. It breaks down 3 of the important macromolecules: carbohydrates, proteins, and fats.
- During respiration, electrons are removed from food molecules and transferred to an electron carrier NADH, then to oxygen (O2)
- At the end, the energy from the food is transferred to ATP
- Through chemical reactions, food is rearranged to waste products - carbon dioxide and water
Glucose is the starting point:
C(6)H(12)O(6) + 6O(2) -> 6CO(2) + 6H(2)O + energy # ignores intermediaries
Burning a marshmallow or paper shows the stored energy. You're converting the sugars into carbon dioxide and water. The energy becomes heat and light.
During respiration, glucose is oxidized and oxygen is reduced. When this electron is moved, it's part of a hydrogen atom. The products are in a more stable state - so it's an exergonic reaction. The energy gets transferred to ATP, a storable form.
Cells actually go through many steps for this reaction. Here's a rough overview:
- Glycolysis - oxidation of glucose begins, electrons transferred to NAD+. Small amount of energy transferred to ATP. Occurs in cytoplasm.
- Krebs Cycle - continues oxidation of intermediaries resulting in CO(2). Occurs in the mitochondrion of the cell.
- Oxidative Phosphorylation (Electron Transport Chain) - transfers electrons from carriers to electron transport chain. Reduction of oxygen to produce water. Also occurs in mitochondrion of cell.
Glycolysis is a series of chemical reactions resulting in:
- oxidation of glucose
- transfer of small amount of energy to ATP
- transfer of electrons to carrier NAD+
The major events are:
- Bonds in glucose break and reform creating two molecules of pyruvate
- Enzymes transfer energy from ATP to intermediaries as the energy investment phase. Then more energy gets transferred back as the energy payoff phase. For every molecule of glucose, 2 ATPs used and 4 comes back.
- Enzymes oxidize glucose and reduce NAD+ -> NADH+H+
Some cells rely on glycolysis without the krebs cycle and electron transport chain. They usually have a 2nd fermentation step to recycle the NADH. Lactic acid fermentation occurs if the hydrogen atoms is transferred to pyruvate, causing the NADH to become NAD again. Alcohol fermentation removes a carbon and two oxygens from pyruvate and releases CO(2), resulting in acetaldehyde - which the NADH gives its hydrogen to. Reduced acetaldehyde is ethanol (or alcohol).
The krebs cycle picks up after glycolysis, continues to oxidize the intermediates, and transfers energy.
The major events are:
- Enzymes oxidize intermediates and reduce electron carriers
- Enzymes break bonds and rearrange atoms in intermediates, removing carbon and oxygen atoms and releasing them as CO(2)
- Enzymes transfer energy to ATP
This produces a total of 2 ATPs per glucose.
Chemiosmotic theory of oxidative phosphorylation - a big name to explain how energy and electrons from food help make ATP. By the end of glycolysis a cell hasn't made much ATP but has made many reduced electron carriers: NADH+H+ and FADH(2). These molecules can cash those electrons to make ATP.
Electron transport chains are made of large protein complexes embedded in a membrane. They accept electrons and pass them from complex to complex. They have an enzyme called ATP synthase. During oxidative phosphorylation, protons enter a small channel in ATP synthase and exits on the matrix side. The synthase has binding sites for ADP and P. Every three molecules provides enough energy for the synthesis of one ATP from ADP and P.
The most electronegative molecule in the electron transport chain is O(2). The proton ends up there, which is why H(2)O is a waste product of respiration.
Food molecules besides glucose need to change into intermediates in glycolysis or the Krebs cycle before being oxidized.
Of all the food molecules, carbohydrates are the easiest to digest because many of the intermediates in glycolysis are carbohydrates. Simple sugars, like glucose, don't need an extra step. Complex carbs are just polymers of simple sugars, so it's also easy to break down.
Enzymes called lipases break fats/oils down into glycerol and fatty acids. These two are then fed to cellular respiration. Glycerol is a 3-carbon compound that's an intermediate to glycolysis. Fatty acids are broken into 2-carbon fragments, then acetyl-CoA - an intermediate of the Krebs cycle.
Proteins require the most work. They're polymers, so they get broken down into their building blocks. Then enzymes make major alterations to the amino acids so they become intermediates to glycolysis or the Krebs cycle.
Cellular respiration must break down many molecules before building new ones. It's a two way street. Catabolism breaks down food molecules, but anabolism builds new molecules from the intermediates.
If a cell needs to build a macromolecule, the intermediates can be used from cellular respiration to build it.
We all know "eat less, exercise more" leads to losing weight. This is partly due to cellular respiration.
Exercising causes cells to use more ATP and increase the amount of ADP. ADP stimulates enzymes in cellular respiration, triggering an increase in the breakdown of food molecules.
If you combine this with the decreased food, cells will have to use the currently stored food molecules to fuel respiration. They turn to stored fat.
Plants, algae, and some bacteria do photosynthesis. Photosynthesis uses energy from the sun, matter from CO(2), H(2)O to form carbohydrates. With these carbohydrates and some minerals, living things can build all the molecules needed for life: proteins, carbohydrates, lipids, and nucleic acids.
Photosynthesis summarized is this chemical reaction:
6CO(2) + 6H(2)O + light energy -> C(6)H(12)O(6) + 6O(2)
Carbon, hydrogen, and oxygen atoms rearrange to form glucose and oxygen. The reactants come from the air and soil. The products end up in the plant body (sugars) or waste back into the atmosphere (oxygen).
Light energy from the sun becomes chemical energy stored in the bonds of sugar.
Plants are autotrophs, meaning self-feeding, they can make their own food. Plant structures are specialized to take in materials:
- Roots take water from the soil
- Xylem, tubes of cells for water transport, moves water from roots throughout the plant
- Phloem, tubes of cells for sugar transport, moves sugars from green leaves to the rest of the plant
- Stomates, pores on leaves, let carbon dioxide into leaves
- Chlorophyll, green pigment on leaves, absorb light
Plants don't need the soil. It's useful for them to stand in and get water from. Also, minerals from the soil sometime help with metabolism - but they're not necessary.
They just need the sunlight, water, and carbon dioxide.
Plants capture the electromagnetic radiation within the visible light band from the sun for photosynthesis.
Photosynthesis is made up of multiple small reactions.
In light reactions, plants capture light energy from the sun and transform it into chemical energy in the form of ATP. They also take electrons from H(2)O and transfer them to electron carrier NADPH.
In the Calvin cycle, plants capture CO(2) and convert it into carbohydrates. To do this conversion, they need the ATP and NADPH's electrons from the light reactions.
Carbohydrates made by plants, algae, and bacteria serve as the food for all life on Earth. Plants are on the bottom of every food chain. Autotrophs make their own food, heterotrophs eat autotrophs:
Sun -> Grass -> Cow -> Human
You can see how the energy from the sun eventually makes its way to you.
Eukaryote division is a little more complicated than prokaryote division. This chapter goes over the steps of a cell cycle and cell division.
All cells come from pre-existing cells. They divide for:
- Growth - most living things started as a single cell (zygote). That single cell kept dividing to grow into you
- Repair - if a multicellular organism is wounded, cells surrounding the wound will reproduce to repair the tissue
- Reproduction - single cell organisms reproduce by making clones
Bacteria uses a simple process of copying their cells called binary fission:
- Bacterial cell makes a copy of its chromosomes
- The cell gets larger as it makes copies of ribosomes and molecules in the cytoplasm
- New plasma membrane and a cell wall are built to divide the cell in two
Some bacteria can finish this in two minutes. That's one to a thousand cells in less than 2 hours.
Some cells divide all the time, like cells on the skin and mucous membrane. They're constantly being shed from the body and need to be replaced.
Some cells divide when signaled, like liver cells which only divide if the liver has been damaged.
Some cells don't usually divide, like cells in nervous tissue. That's why nerve damage cannot be repaired.
The dividing phase of eukaryotic cells is called mitosis. The nondividing phase is called interphase which is made up of three subphases: G1, S, and G2. The alternating cycle of mitosis/interphase is the cell cycle.
Cells spend most of their time in interphase G1 (Gap 1). They're active and functioning. Mature cells that don't divide stay in G0. In order to get to the next phase for division, cells must pass a checkpoint. If they fail, they commit cell suicide - apoptosis. The checks are:
- Signals that tell cell to divide
- Must have plenty of nutrients
- DNA must be in good condition
- Must be large enough to divide
At S (synthesize) phase, cells copy their DNA via DNA replication. After the DNA is copied, cells proceed to G2 (Gap 2). At G2, cells check the work they did during S as another checkpoint:
- The DNA can't be damaged
- Cell copied all chromosomes
- Signals tell cell to proceed into mitosis
If all checks are valid, the cell goes into mitosis.
At this stage, cells have duplicated DNAs and are ready to divide into two. Each cell needs a complete set with one of every chromosome.
You have 46 chromosomes (23 pairs) in each cell of your body. During S phase, they're duplicated. Each one has an identical copy (sister chromatid) that remains attached at the centromere - forming an X shape.
The mitotic spindle is a cell structure responsible for separating the chromatid pairs. They're made of microtubules which are cytoskeletal proteins.
Mitosis consists of four phases:
- Prophase - chromosomes coil up (condense), nuclear membrane breaks down so the mitotic spindle can reach into the center, mitotic spindle attaches to the chromosomes, nucleoli break down
- Metaphase - cells organize chromosomes by lining them up in the middle of the mitotic spindle. There's a metaphase checkpoint to make sure it's attached.
- Anaphase - replicated chromosomes separate and each sister chromatid goes to opposite sides of the cell
- Telophase - reverses the events of prophase. Chromosomes uncoil, nuclear membrane reforms, mitotic spindle brakes down, nucleoli reforms
After the chromosomes separate and nuclear membrane reforms, the cytoplasm separates and forms two distinct cells. The separation of cytoplasm is called cytokinesis.
Organisms that reproduce sexually produce eggs and sperms that join to make a new individual. Eggs and sperm are sex cells or germ cells. To produce them, cells need to go through a special dividing process called Meiosis.
Body cells called somatic cells have twice the genetic information of the gamete cells (eggs/sperm). Somatic cells produce gametes via meiosis.
The life cycle is:
- Somatic cells undergo meiosis to produce gamete cells
- Sperm and eggs join to create the first cell called a zygote
- Zygote divides to form a multicellular organism
Gametes have half the number of chromosomes as somatic cells. One complete set of chromosomes is called the haploid number and is usually represented with the letter N. Two complete sets are 2N or diploid number. Humans have 23 pairs - so our haploid number is 23. Gamete cells are haploid, whereas zygotes are diploid.
Cells have matching pairs of chromosomes called homologous chromosomes. Each human gamete contributes one set of 23 chromosomes.
Scientists can differentiate chromosomes based on a few characteristics:
- Chromosome length
- Position of centromere during DNA synthesis
- Staining patterns
22 pairs are exactly the same and are called autosomal chromosomes. The 23rd pair is different between men and women. It's called the sex chromosomes. Men have a large X and small Y, women have two large X's.
Meiosis is a special type of cell division which separates homologous pairs of chromosomes so that gametes receive one of each type of chromosome.
Meiosis is similar to mitosis in many ways:
- Cells undergoing meiosis will receive a signal causing them to leave G1 of interphase and enter S1
- At S phase, cells copy all chromosomes creating identical sister chromatids attached at the centromere
- Cells proceed through G2 and enter into meiosis
- Chromosomes are moved in a similar way to mitosis and have the four phases just like mitosis
The purpose of meiosis I is to separate pairs of homologous chromosomes. Meiosis II separates sister chromatids.
Meiosis 1:
- Prophase 1: nuclear membrane breaks down, nucleoli disappear, homologous chromosomes find each other and pair up. The chromosomes coil up and spindle attaches to chromosomes
- Metaphase 1: chromosome pairs line up in middle of cell
- Anaphase 1: chromosome pairs separate, one from each goes to opposite ends of cell
- Telophase 1: nuclear membrane forms to make two nuclei, they're haploid
- Cytokinesis: formation of two cells
After meiosis 1, both cells skip interphase and go straight to meiosis 2:
- Prophase 2: spindle forms and attaches to chromosomes, breaks nuclear membrane
- Metaphase 2: chromosomes are lined up in the middle of the cell
- Anaphase 2: sister chromatids are separated and move to opposite sides of the cell
- Telophase 2: chromosomes uncoil, spindle breaks down, nuclear membrane reforms, nucleoli reappear
- Cytokinesis: formation of two cells each - total of four cells
During prophase 1 of meiosis 1, sometimes bits of DNA are swapped between two homologous chromosomes via a process called crossing-over. It actually swaps pieces of chromosomes from Mom with Dad and vice versa.
Crossing over helps increase the genetic diversity of gamete cells. The steps are:
- Homologous chromosomes are attached all long their length
- Proteins make small cuts into the DNA backbone of chromosomes
- Proteins reseal the breaks in the DNA backbone, attaching pieces of one homologous chromosome to the other
Genetics is the study of how parents pass traits on to their children. Gregor Mendel began work on genetics and is credited to starting the field. He made a few laws known as Mendel's Laws:
- Law of Dominance: one form of a trait can hide another form. The hidden form is said to be recessive and the other is dominant
- Law of Segregation: gametes (sperm, eggs) only receive one of two messages for each trait from the adult
- Law of Independent Assortment: each pair of genetic messages sort itself independently from other pairs of genetic messages - roundness and color are segregated independently
Here are some keywords used in genetics:
- Gene: a message for a particular trait
- Allele: different variations of a gene
For example, pea plants have a gene for flower color. One allele is purple flowers while another is white flowers.
- Genotype: total of all genetic messages in an organism
- Phenotype: the way an organism appears and functions because of its genes
- Homozygous: when an organism has two identical alleles for a gene
- Heterozygous: when an organism has two different alleles for a gene, sometimes called hybrid
When analyzing breeding, scientists have some common notations:
- P1 is the parental generation and always pure-breeds
- F1 is the first generation resulting from two P1s, stands for first filial generation
- F2 is the second generation resulting from two members of F1
Punnett squares are grids used to figure out the probability of alleles. The edges are symbols representing alleles, the squares are possible combos, capital letters for dominant, lower case for recessive:
r r R r
+----+----+ +----+----+
R | Rr | Rr | R | RR | Rr |
+----+----+ +----+----+
R | Rr | Rr | r | Rr | rr |
+----+----+ +----+----+
The left punnett square represents members of P1. RR is the round pea, rr is the recessive wrinkled pea. No matter what, offspring will always end up Rr - round.
The right punnett square represents members of F1. Here you have a 3:1 ratio of round peas to wrinkled peas.
Scientists today have discovered the factors of inheritance that Mendel proposed are located on chromosomes made of DNA. Parents have two copies of each gene, they segregate when given to the offspring. The segregation occurs during anaphase 1 of meiosis.
Traits don't always behave the way Mendel described. Scientists have discovered types of inheritance that are complicated by other factors:
- Incomplete Dominance: phenotype is somewhere between the dominant and recessive
- Codominance: two different alleles contribute equally to phenotype
- Pleiotropy: one gene has an effect on many different phenotypes
- Polygenic Traits: many genes effect one phenotype
- Linked Genes: close together on chromosome and break law of indie assortment
- Sex-linked Traits: located on the sex chromosomes, X or Y
The dominant allele only partially masks the presence of the recessive allele. A good example is the color of snapdragons. The red (R) allele is incompletely dominant to the white (W) allele. RR is red, WW is white, RW is pink.
Codominance is similar to incomplete dominance, except there's no blending. Both traits will show up in the offspring. For the snapdragon example, both red and white colors will appear in the flower.
A real world example is blood type. There are three alleles: IA, IB, and i. They represent the blueprint of enzymes that attach carbohydrates to your blood cells. IA attaches carbohydrate A. IB attaches carbohydrate B. Allele i does not attach a carbohydrate.
IAIA or IAi offspring have A blood type. IBIB or IBi offspring have B blood type. IAIB have AB blood type - both carbohydrates will be present. ii will have O blood type.
Genes that affect more than one trait are called pleiotropy. A good example is the gene that causes sickle cell anemia. The hemogoblin gene controls the structure and function of hemogoblin. When two recessive, defective alleles are present they also:
- Cause anemia and extreme fatigue due to destruction of red blood cells
- Cause sickled cells that plug capillaries
- Cause the kidney and spleen to fail
When many genes effect one phenotype, the trait is polygenic. For example, eye color is polygenic.
Eye color is determined by the amount of melanin in your eye. There are many genes which control the melanin deposit in your eye/face/skin. People with dark eyes have a lot of melanin, people with light eyes have fewer melanin. The alleles which deposits a lof of melanin are dominant to the alleles which deposit fewer.
Since there are many genes for the eye color trait, there's a wide range of eye colors. From dark brown to light blue, there's green and even purple.
Some traits are linked together. A person who is heterozygous for two traits, controlled by genes A and B, will make different gametes depending on if the genes are unlinked or linked:
- if independent, a heterozygote (AaBb) will make gametes of all possible combinations: AB, Ab, aB, and ab
- if linked, a heterozygote (AaBb) will only make AB or ab
Traits are linked because the genes are located on the same chromosome. The tendency of linking is correlated with how close they are together.
Genes that are very close together are said to be completely linked - they always appear linked. The only time they aren't is if cross-over occurs.
Some traits break Mendel's law because they aren't equally inherited by males and females. These are known as sex-linked traits. This happens when the gene is on the X or Y chromosomes.
X-linked dominant traits affect both males and females. Y-linked traits affect only men.
The human X chromosome contains more than a thousand genes, while the human Y chromosome only has about 200 genes.
Cells copy their DNA by a process called DNA Replication. Before a cell divides, it needs to copy its DNA which contains the blueprints of functioning. Any mistake in the DNA code is called a mutation, which could mess up the cell's functioning.
To make an exact copy of DNA, cells use semiconservative replication. The two halves separate and each half serves as a template to synthesize another half. Each old strand gets a new partner.
The enzymes involved in DNA replication:
- Helicase breaks or melts the hydrogen bonds that hold the two halves of the double helix together
- Topoisomerase breaks and reseals the backbone to release tension
- Primase (RNA polymerase) puts down small pieces of RNA called primers, serving as starting points for copying the DNA
- DNA polymerase makes new strands of DNA. DNA polymerase III builds new strand using complements of the template. DNA polymerase I replaces RNA nucleotides in the primer with DNA nucleotides
- DNA ligase seals up breaks in the DNA backbone by forming covalent bonds
The enzymes work together in a crew, called enzyme complex. They slide along the DNA, copying as they go.
DNA replication starts at specific locations of chromosomes called origins of replication. Proteins recognize the origin, opens the helix there, and the enzyme complexes begins copying the DNA. Two enzyme complexes move in opposite directions, copying DNA in both directions.
As replication occurs, a replication bubble is formed. On both sides are V-shaped areas where DNA is being opened upcalled replication forks. This happens because the enzyme complexes are moving in opposite directions. The forks are where copying is currently occurring.
After DNA has been opened at the origin, the enzyme helicase catalyze the breaking of hydrogen bonds. Helicase is like the metal piece that slides along zippers. Once separated, proteins called single-strand DNA-binding proteins will hold the DNA open so the strands don't re-bond.
As the strands get separated, the double helix backbone gets tension. Topoisomerase catalyzes the breaking of covalent bonds in the backbone. This untwists DNA and relieves the backbone of tension.
Primase creates short pieces of RNA (primers) that are complementary to the DNA. Once the primers are made, DNA polymerase III starts attaching DNA nucleotides to the 3' ends of the primers. It uses the original strand of DNA as a template and continues synthesizing DNA down the line.
DNA polymerase I slides along the new DNA strand and replaces the RNA nucleotides with their DNA counterpart.
Some covalent bonds are missing in the backbone now. There are gaps between the primers and the DNA synthesized by DNA polymerase III. The ligase enzyme goes along the strand and seals those gaps in the backbone.
The two strands of the double helix are antiparallel to each other. The 5' and 3' ends are always pointing in opposite directions.
DNA polymerase III always adds nucleotides to the 3' ends. This presents a problem to the enzyme complex, which moves as one unit.
To solve it, DNA polymerase creates short pieces of DNA and then skips to the next primer. These pieces are called Okasaki fragments.
The new strand made in fragments is called the lagging strand. This strand must be started over and over again. RNA polymerase keeps putting down primers so the DNA polymerase III can keep working.
The opposite strand doesn't have this problem and is called the leading strand. Remember, the leading/lagging strand always refer to the new strands of DNA. Also remember, DNA always have nucleotides added to the 3' ends.
Cells with linear chromosomes (including humans) the lagging strand gets shorter every time the DNA is copied. This happens at the very end of chromosomes. Once the primer is removed, there's no room for a new primer and DNA can't copy without a primer. The ends of chromosomes are capped with repetitive DNA sequences called telomeres. They act like buffers which can be shortened because they don't contain useful information for the cell. Once it becomes too short, though, the cell can no longer divide without losing critical information.
DNA contains the genetic code of the cell which controls the cell's structure and function. This chapter explains how the cell uses those instructions to make worker molecules - which actually does the cell's function.
The worker molecules are:
- RNA molecules - information carriers and enzymes (called ribozymes)
- Proteins - main worker molecules performing wide range of functions
A gene is the blueprint for a single worker molecule. If your genome is a file cabinet, chromosomes are drawers, and genes are files. Humans have 46 chromosomes and ~22,500 genes.
A high level view of how we go from DNA to proteins:
- Gene in DNA is copied into a messenger molecule called messenger RNA or mRNA via a process called transcription.
- mRNA goes to ribosomes where it goes through translation to build a protein. The protein is made from amino acids attached to translation RNA or tRNA.
The flow of information to build a protein (DNA -> RNA -> protein) is called the central dogma of molecular biology.
During transcription, the double helix of DNA is opened and one strand of DNA is used as a template for construction of an RNA molecule. Transcription can make several types of RNA, not just mRNA.
Genes only make up 2% of DNA, we're still trying to figure out what the other 98% do.
Cells recognize genes for proteins by their promoters - unique sequences of DNA located at beginning of genes. Going back to the file cabinet analogy, promoters are like those tabs on files that let you quickly find a file.
RNA polymerase is the enzyme that reads genes and copies the information into RNA. It also catalyzes the covalent bonds between nucleotides. The DNA strand being read is called the template strand. The other is called nontemplate strand (also called coding strand).
RNA polymerase need helpers to recognize promoters. In bacteria, sigma proteins attach to the polymerase and form a holoenzyme. The holoenzyme attaches to promoters and the polymerase can begin transcription. In eukaryotes, the regulary proteins are transcription factors.
Transcription terminators are the opposite of promoters, they mark the end of genes. It contains code that makes the RNA to fold back on itself, forming a hairpin loop and knocking the RNA polymerase off the DNA.
The transcribed RNA is still not ready to be translated. Right now it's called pre-mRNA or primary RNA transcript. The RNA code contains introns - short stretches of nucleotides that don't code for the protein. These introns need to be removed. The portions which contain code for proteins are called exons, which remain in the final mRNA.
Exons are expressed; introns are interrupt
Particles called snRPs ("snurps") made from RNA/proteins remove the introns. This method is called splicing. After splicing, the cell has two more steps:
- A protective cap - 5' cap - is added to the 5' end. It identifies finished mRNA for the cell
- Enzymes clip off the 3' end of the pre-mRNA and replace it with between 100 and 250 adenine nucleotides called the poly-A tail which protects the finished mRNA
The mRNA is shipped out to the ribosomes in the cell's cytoplasm. The tRNAs use the mRNA to build the protein. The cell reads mRNA in blocks of three nucleotides called codons. Each codon tells the cell which amino acid to attach to the polypeptide chain.
Twenty different amino acids exist and there are 64 possible combinations for codons (4^3). The genetic code is redundant, some amino acids are represented by more than one codon.
Scientists have made a codon dictionary. Given a strand of RNA, you can figure out which amino acids they represent and in what order. Follow these steps:
- Find large row on left of table with first letter of codon
- Find column that's the second letter of codon, use the entire area
- For the area, find small row on right of table with third letter of codon
- The intersection of all three is the amino acid
Each mRNA molecule has a section near the 5' cap that's recognizable by the ribosome called the ribosomal binding site and isn't part of the code for protein. The protein code starts at the START codon or AUG. It's decoded in the 5' to 3' direction until it hits a STOP codon (UAA, UGA, or UAG). Then just read each nucleotide by groups of three.
tRNA or Transfer RNA is the decoder. tRNA molecules carry in the amino acids for the protein and figure out where to place them in the polypeptide chain.
tRNA molecules have an anticodon structure made up of three nucleotides. These three nucleotides will pair with the codon of mRNA, binding with hydrogen bonds just like the two strands of DNA. For example, the tRNA's anticodon may be 3'UAC5' and will fit correctly with mRNA's 5'AUG3'. 40 tRNAs exist.
The matching arranges the amino acids in the correct order - which mRNA decides. Ribosomes organize the meeting of mRNA and tRNA. Ribosomes have pockets called the A site, P site, and E site. tRNAs enter through the A site, move through the P site, and exit at the E site. The ribosome also catalyzes peptide bonds as they're being brought together.
Translation occurs in three phases:
- Initiation of translation: subunit of ribosome binds to mRNA, tRNA binds its anticodon to the START codon, large subunit of ribosome binds to small subunit and mRNA. Second codon of mRNA is at A site.
- Elongation of translation: amino acids are brought together and joined to form a polypeptide chain
- Termination of translation: stop codon enters A site and is recognized by an enzyme called release factor, which catalyzes the break up
Changes in DNA are called mutations and may result in disease or death. DNA polymerase doesn't often make mistakes copying DNA and it proofreads its own work. It still makes one uncorrected mistake for approximately every billion base pairs of DNA. Mutations that just happen because of the enzyme are called spontaneous mutations.
Cells have a lot of DNA. Every time a cell divides, there will be approximately six mutations.
Certain chemicals and types of radiation in the environment will increase the mutation rate of DNA polymerase. Agents that increase the rate are mutagens and mutations that result from it are induced mutations.
Cells can function fine with mutations due to a few reasons. Not all mutations cause changes in proteins - they may change DNA outside of genes. Even if it does occur in the gene, it may not affect the final protein (AAA and AAG code for the same amino acid). These are silent mutations.
Other types of mutations are:
- Missense mutations change of amino acids in proteins
- Nonsense mutations prematurely encode a stop codon for proteins
- Frameshift mutations causes reading frame of mRNA to be altered
Gene regulation is about cells choosing which genes to use and which to not use. Genes are expressed when cells are using the gene to make a gene product (usually protein or RNA).
Gene regulation is important for specialized cells. Your original cell was a stem cell with the potential to become any type of cell. Descendants are given signals to become specialists. Signals turn on/off genes.
Cells that become specialists are differentiated for a certain task. Cell differentiation is achieved through differential gene expression. Differentiated cells have access only to the genes they need to survive and perform their function.
Some genes are essential for all cells' survival and are always turned on. This includes genes for metabolism and DNA/RNA synthesis.
Cells have two types of genes based on gene expression:
- Constitutive gene: expressed all the time
- Regulated gene: turned on and off as needed by cells
Bacteria has to regulate its gene to take advantage of the current situation. For examples, sometimes there's ample food or a scarcity. The basic steps for regulating genes in bacteria are:
- Cell receives an environmental signal
- Signal activates or de-activates a DNA-binding protein
- DNA-binding proteins either bind or let go of regulatory DNA sequences
- Transcription is turned on or off by DNA binding proteins
Multiple genes are under the control of a promoter - all together they're called an operon for bacteria. The genes in an operon are usually for a single process so all proteins for a process are made during transcription/translation of the operon. Some properties of the operon:
- promoter marks the beginning of the operon
- structural genes are genes for proteins within the operon
- the operator is a regulatory sequence of DNA between the promoter and the structural genes
Escherichia coli or E. coli has a bad rap, but most strands of it are nice. It's bacteria that's great for studying and the nice ones even exist in your stomach. E coli is often used for studying gene expression.
Food sources are unpredictable for bacteria, so E coli will turn on or off the enzymes to break food particles in certain situations. We'll take a look at how E coli breaks down lactose - a sugar found in milk.
The genes for producing enzymes that breakdown lactose are called the lactose operon or lac operon. The lac operon has three components: promoter, operator which is the binding site for DNA-binding protein called lac repressor protein, and three structural genes (two proteins and one protective enzyme). The structural genes are for:
- lacZ gene blueprint for beta-galactosidase enzyme that catalyzes the splitting of disaccharide lactose into two monosaccharides, glucose and galactose, and then glycolysis breaks it down
- lacY gene for membrane protein galactosidase permease which brings lactose into the cell
- lacA gene blueprint for transacetylase, protective enzyme that allows certain sugars to be removed from the cell when sugars are too plentiful
The lac repressor is a DNA-binding protein that regulates the lac operon (it controls the transcription). It has two binding sites critical to its function: a DNA-binding site that binds to the operator of the lac operon, a allosteric site that binds to an isomer of lactose. When lactose is present at the allosteric site, the repressor is de-activated. When it's missing, the repressor is activated. The operator of the lac operon is next to the promoter, so when lac repressor is active - it'll get in the way of RNA polymerase.
So the lac repressor guarantees effeciency. When lactose is present, the cell will use transcribe the lac operon gene. When lactose isn't present, transcription is blocked and the cell won't waste its resources. It all comes down to whether lactose is bound to the allosteric site of the lac repressor.
Gene expression in eukaryotes is a bit more complicated but has some similarities to gene expression in bacteria:
- regulation at the level of transcription called transcription control is the same for both
- DNA-binding proteins regulate the activity of genes by binding to regulatory sequences in the DNA for both
The pathway from genes to proteins is more complicated in eukaryotes, so there are more opportunities for regulation:
- access to genes is affected by compaction of DNA into chromosomes
- transcriptional controls regulate whether RNA polymerase binds to promoters
- control of RNA processing determines if pre-mRNA is processed to mRNA
- control of mRNA stability determines how long it is available for translation
- translational control regulates if mRNA is translated into protein
- control of protein modification determines if polypeptide can become fully functional protein
If you unpack the DNA from a single cell into a straight line, it'd be about 6 feet in length. Eukaryotic cells pack DNA into chromatin, a complex of DNA wound around protein. The steps to packaging DNA are:
- DNA molecules are wounded around positively charged proteins called histones that gather together in groups of eight proteins. Beads of DNA plus histones are called nucleosomes.
- String of nucleosomes and DNA twist until they form coiled fibers that are 30 nanometers wide
- Continued twisting of the DNA and protein fibers packs the DNA even more tightly until DNA is very condensed
Because the DNA is so tightly coiled up, regulatory proteins and RNA polymerase can't get access to the genes. Before transcription can occur, the chromatin of the gene must be decondensed. Histone acetyl transferases (HATs) proteins control the condensation of chromatin (they attach acteyl groups to histone proteins which neutralize their positive charge). When the DNA becomes less attracted it will loosen and decondense.
Regulatory proteins bind to regulatory sequences of DNA located near promoters. Regulatory proteins controls transcription in eukaryotes. They work similar to bacteria's transcription control but are wider in variety:
- Promoter proximal elements: sequences located close to promoters. When regulatory proteins bind to these, transcription is turned on. Each exact sequence is unique so cells know which genes they're switching on/off.
- Enhancers: regulatory sequences located far away from genes they regulate. When regulatory proteins bind to them, transcription is on.
- Silencers: regulatory sequences located far away, but when regulatory proteins bind to these transcription is off.
Regulatory proteins that bind to regulatory sequences in order to turn on transcription are called transcription factors. There are two types:
- General transcription factors: aka basal transcription factors are required for transcription of any gene in all cell types and work to attract RNA polymerase to bind them to promoters
- Regulatory transcription factors: more targeted, bind to regulatory sequences of specific genes
Proteins called coactivators bind to transcription factors and bring them together to form transcription initiation complex. They initiate transcription.
Scientists are able to read, copy, cut, sort, and replace DNA. When DNA comes from two different sources it's called recombinant DNA. Scientists have combined human genes and DNA from E. coli and placed them into E. coli, so it produces human insulin. Doctors use the insulin to treat human.
Restriction endonucleases or restriction enzymes are made by bacteria and cut strands of DNA into smaller pieces. It's used to fight off viruses, chopping off infected DNA so it can't destroy the entire cell. Restriction enzymes make cutting/combining pieces of DNA easy.
A plasmid is a DNA molecule separate from chromosomal DNA that can replicate on its own. It's usually in a ring shape and found in bacteria (though sometimes can be found in eukaryotes).
Example of cutting a human gene and placing it into bacteria:
- Choose a restriction enzyme that forms sticky ends when it cuts DNA, sticky ends being pieces of single stranded DNA that are complementary
- Cut the human DNA and bacterial plasmids with restriction enzymes
- Combine human DNA and bacterial plasmids
- Use DNA ligase to seal the backbone of the DNA
Gel electrophoresis separates molecules based on their size and electrical charge. The gel is made of a gelatin-like polysacharrides. Molecules are placed into pockets of the gel (usually DNA or proteins) and will be moved by a charge.
Anode +
+---------+
| o O o | <-- large, low positive charge
| - O O | <-- small, low positive charge
| - - o | <-- large, high positive charge
| - - - | <-- small, high positive charge
+---------+
Cathode -
- Molecules are attracted to electrode with opposite charge
- Small molecules can move more quickly through gel than large molecules, causing molecules to be separated by size. The long fibers of polysacharrides throughout the gel act as obstacles for the molecules - which smaller molecules can move through quicker.
Gel electrophoresis is often used to separate DNA from other molecules. DNA has a negative charge due to its phosphate groups. "DNA runs to red" because the positive side is red. The steps to do it:
- Pour liquid gel and let it solidify. There's a comb on the gel platform which creates little pockets called wells in the gel.
- Place gel into an electrophoresis chamber and fill chamber with buffer. The buffer is a solution that conducts electricity.
- Cut DNA samples with restriction enzymes and mix samples with loading buffer. The buffer settles the DNA into the wells and marks them with dye.
- Load samples into wells using a micropipettor. DNA will sink to bottom.
- Seal the box and start the current to separate DNA samples.
- Stop the current and stain the gel. Stain will stick to DNA and create stripes called bands. Each band represents a collection of DNA molecules of the same size.
Earlier, Kratz mentioned how we add human DNA into bacteria to create human proteins. Human DNA has introns and bacteria doesn't know how to splice pre mRNA. To get around this issue, scientists create intron-free genes in the form of complementary DNA or cDNA.
cDNA is made from eukaryotic mRNA that has already been spliced. The steps to make cDNA are:
- Isolate mRNA from protein you're interested in
- Use enzyme reverse transcriptase to make a single-stranded DNA moelcule that is complementary to the mRNA (it's a viral enzyme that uses RNA as a template to make DNA)
- Use reverse transcriptase or DNA polymerase to make a partner strand
DNA cloning makes many identical copies of the gene. To clone a gene, you put the gene in a vector (plasmid or virus DNA) that helps carry DNA into a cell. The steps are:
- Use restriction enzyme to cut the vector and target DNA containing the gene to have same sticky ends.
- Mix the vector and DNA together and add DNA ligase. Vectors picking up genes are said to be recombinant.
- Introduce the vector into a population of cells. The vector will be reproduced inside the cells and the gene will be cloned
DNA libraries are recombinant vectors that store genes and keep them handy for scientists to use.
Scientists use DNA probes to find vectors containing specific genes in DNA libraries. Probes are single stranded DNA complementary to the sequence you're looking for. It's like fishing - catching means forming hydrogen bonds.
- Prepare DNA sample to be probed for the gene of interest. Treat it with heat or chemicals to make it single-stranded.
- Wash DNA probe over the surface of DNA sample
- Locate probe to find gene of interest
The polymerase chain reaction (PCR) lets you make copies of genes. It can turn a single copy of genes into billions in a few hours. Doing this is also known as gene amplification. The DNA sample that contains the gene to be amplified is combined with thousands of copies of the primers so DNA polymerase creates thousands of strands. The steps are:
- Heat sample for 1 minute to disrupt hydrogen bonds to make DNA single-stranded
- Cool sample for 1 minute so hydrogen bonds can reform. Primers will stick with hydrogen bonds to single-stranded target DNA
- Heat sample to optimum temperature for DNA polymerase
Repeat these three steps 30 times.
DNA sequencing determines the order of nucleotides in a DNA strand. It uses a special nucleotide called dideoxy-ribonucleotide triphosphate (ddNTP) which are like typical nucleotides except instead of the hydroxyl group attached to 3' carbon it's just hydrogen. If ddNTP gets attached to the growing chain of nucleotides while DNA polymerase is copying DNA, it'll be the last one added to the chain. The chain interruption determines the order of nucleotides.
Cycle sequencing combines original methods of DNA sequencing with PCR.
Introducing a gene in order to cure a genetic disease is called gene therapy and it's still far from perfect:
- scientists must discover safe vectors that can transfer genes into human cells
- scientists must develop methods for introducing therapeutic genes into population of targeted cells (ie only affected cells)
- stem cells producing target population of cells must be identified
Genomics is the study of the entire DNA sequence of an organism. The Human Genome Project sequenced the entire genome of a human and developed faster methods for DNA sequencing.
Genomics research is taking several approaches now:
- Comparative genomics compares genomes from different species to better understand evolution and determine function of genes and noncoding regions
- Functional genomics tries to figure out functions of genes
- Pharmacogenomics studies how a person's unique genome affects their response to drugs with a goal of highly effective/individualized drug development
Shotgun sequencing is a fast sequencing method which breaks apart the genome into small fragments, sequence the fragments, then re-assemble it. Here is how to do hierarchical shotgun sequencing:
- Cut genomic DNA into fragments of 150 million base pairs
- Place fragments into vector then clone into bacteria to be replicated
- Large fragments are mapped and organized
- Each large fragments are broken up to set of smaller fragments
- Small fragments are sequenced
- Computer lines up overlapping sequences to determine order of small fragments
Right now pathogens are being sequenced to help develop new strategies to fight diseases. Extremophiles are being studied for industrial use (organisms that can live in extreme conditions). Crop plants and model organisms are also being studied for agriculture and comparison to humans.
Some interesting facts about the human genome:
- We only have about 25,000 genes (before HGP, we expected 100,000)
- Average gene size is about 3,000 base pairs long
- Number of genes vary from 2,968 genes on chromosome 1 to 231 genes on Y
- At least 50% of DNA is noncoding
- Genes are randomly clustered together on chromosomes and separated from other clusters of genes by noncoding DNA
- Areas of DNA with genes contain lots of G-C base pairs. Noncoding regions contain lots of A-T base pairs
- Less than 2% of genome code for proteins and RNA molecules. The other 98% has other functions or we don't know yet. The other functions include: regulatory sequences used for promoters or origins of replication, structuring DNA within the cell, junk DNA (50%, we don't know what it's for), pseudogenes are mutated relics of formerly functioning genes
Scientists use computers to look for regions of DNA that are similar across different organisms. It will help answer questions about evolution, gene function across organisms, genes for unique features, and effects of natural selection.
All life on Earth is very similar. Human genome is 99.9% identical to other humans, 96% identical to chimpanzees, and 40% identical to mice.
More genes or bigger genome doesn't mean a more complicated organism. The size of a genome doesn't correlate with how many genes it has. Mice have 2.5 million base pairs in the genome and 30,000 genes. Humans have 3 million base pairs but less genes (25,000).
Some organisms have evolved to have multiple sets of chromosomes. They're polyploid organisms and usually have larger genomes. Some organisms, like humans, have a lot of repetition in their DNA. Humans have an "Alu" sequence which is 300 base pairs long and appears more than 1.1 million times.
Scientists are trying to answer these questions with functional genomics:
- How much variation is there within a genome?
- When and where does gene expression occur?
- How do genes and the products of different genes interact with each other?
Genes are encoded with open reading frames (sections of DNA that begin with START codon and end with STOP codon). Scientists are searching through these to discover new proteins.
Gene expression is an important part of functional genomics, especially in cancer research. To determine which genes are expressed at any one time, geneticists track the presence of RNA sequences in cells using DNA microarrays.
A DNA microarray is a glass slide with many single-stranded copies of DNA for a single gene stuck to it. To use it:
- Molecules of mRNA are isolated from the two cells to be compared
- Reverse transcription produces labeled cDNA copies of each population of mRNA
- The cDNA molecules from each cell are washed over DNA microarray surface. They'll stick to any complementary molecules on the glass slide
- Use a laser to activate fluorescent tags on the cDNA molecules and observe color patterns to determine which DNA sequences are being expressed
Pharmacogenomics combines pharmacology and genomics to tailor drug therapies to a specific individual. With it, scientists hope to better associate genes and proteins with a disease. They can figure out the best treatment for a person with a particular genotype. They can do pre-emptive check ups using the genome to figure out which disease a person is most likely to get.
A lot of genome information is available on the internet! Scientists all over the world are mining this information to learn and contribute.