MCB3020 Exam 2

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Exogenous: you go through respiration

List the three chemoorganotrophic fueling processes

- Energy source- o can come from chemicals, chemotrophs. Organic molecules that are a source of energy. The organic molecules are those polymers- carbs, proteins, etc can be chemotrophs and a source of energy. o Chemolithotrophs- inorganic molecules o Phototrophs- light - Carbon source o Autotrophs can fix CO2 from the air o Heterotrophs can break down organic molecules- the polymers. - Electron source o Can be from organic or inorganic molecules `` Organisms can use electrons from inorganic molecules, can get carbon from CO2, can get electrons either from light or chemicals. From these sources, you make your precursors (fatty acids, simple sugars- building blocks), then you turn them into more complex molecules, then you make your macromolecules (like proteins and polymers). That's how growth happens.

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- Free energy change defined at standard conditions of concentration, pressure, temperature, and pH. Free energy is energy available to do work.

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- Redox: two half reactions One is electron donating (oxidizing reaction) One is electron accepting reaction (reducing reaction) Acceptor and donor are conjugate redox pair. Acceptor + e- donor

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- Regulation of Metabolism o Metabolism- the total reactions that happen in the cell that include anabolism and catabolism. o Important in conservation of energy and materials o Maintenance of metabolic balance despite changes in environment. o Three major mechanisms § Metabolic channeling · Differential localization of enzymes and metabolites · Compartmentation o Differential distribution of enzymes and metabolites among separate cell structures or organelles o Can generate marked variations in metabolite concentrations § Regulation of the synthesis of a particular enzyme (transcriptional and translational) § Direct stimulation or inhibition of the activity of a critical enzyme · Post-translational regulation of enzyme activity o Important reversible control measures- allosteric and covalent modification

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- Role in ATP metabolism: exergonic breakdown of ATP is coupled with endergonic reactions to make them more favorable. Every time you couple a reaction with ATP, that reaction goes forward because ATP becomes ADP (you extract a high energy phosphate, which is used to run the other reaction).

EXTRA

-Biosynthetic enzymes are associated with repressible operons. Product determines if operon is on or off. Example: amino acids. If the product is available, the gene is off. If its not available, the gene is on. Tryptophan operon is also controlled by attenuation or binding to DNA. Translation stem loop etc.

Chemical, Transport, and Mechanical work / Entropy

-Chemical work- Synthesis of complex molecules. Need energy to synthesize, to build polymers. -Transport work- take up of nutrients, elimination of wastes, and maintenance of ion balances. Need energy to pass molecules through the cell membrane. Also moving molecules within the cell requires energy. -Mechanical Work- cell motility and movement of structures within cells. Cell needs flagella for motility, which need energy to move. Splitting the cell in half. Need energy to do work. Energy is the capacity to do work. Energy is needed to run reactions.

- Electron Transport Chain, examples of components / Electron carriers

-Electron carriers- bunch of electron carriers that pass them along. Series of molecules that take electrons and pass it on to the next until they finally give it to the electron acceptor. Located in the plasma membranes of chemoorganotrophs in bacteria and archaeal cells. -located in internal and mitochondrial membranes in eukaryotic cells -examples of electron carriers include NAD, NADP, FAD, coenzyme Q (CoQ), cytochromes (use iron to transfer electrons- iron is part of a heme group. First three are diffusible. Last 2 are part of the membrane. -imagine electrons as waste. And the dump trucks are NAD and FAD and they pick it up and take it away. But the trucks get full so they need to go dump them off. This processing center is the electron transport system. This is a place with a bunch of electron carriers. They carry electrons temporarily (acceptors accept for good). -They are walking around and pick up electrons around the cell. Once they pick them up, there must be a place to dump them -terminal electron acceptor is totally reversible. -in prokaryotes, electron acceptors are in the cell membrane. -in eukaryotes, it's in the inner membrane of the mitochondria. -the carrier gets it, becomes reduced, and then passes it on to the next. -in the ETC the first acceptor is NADH dehydrogenase. This accepts from NADH. NADH comes here, becomes NAD, and the electron is accepted by the dehydrogenase. The cytochrome becomes reoxidized and passes on electrons to oxygen. - Electron Transport chain (new slide): -Series of electron carriers that operate together to transfer electrons from NADH and FADH2 to a terminal electron acceptor -electrons flow from carriers with more negative Eo to carriers with more positive Eo -as electrons transferred, energy released -in eukaryotes, the ETC carriers are within the inner mitochondrial membrane. -NADH is a reduced molecule. It donates its electron to the first in line, which is NADH dehydrogenase. NADH becomes NAD, which goes back to glycolysis and krebs cycle again. Everytime an electron carrier accepts an electron, it becomes reduced. It passes on electrons to the next one in line, itself becomes oxidized, next one becomes reduced. Passed on and on. Electron is moved through the chain. Some push proteins outside and some do not (first and third do). The last acceptor is cytochrome. The first (NADH dehydrogenase) is the most negative Eo, it accepts electron. The last (cytochrome) is the least negative Eo, it donates electron to final electron acceptor- oxygen). Now protons outside of cell generate PMF- energy to make ATP. ATP synthase acts as a turbine that turns and generates electricity. The force that runs this turbine is the water that runs through it. As the proton pushes through the pump back into the cell (through ATP synthase) it turns it and gives it energy to produce ATP (produced by ADP to ATP). This is oxidative phosphorylation. Energy isn't generated from scratch, it's from the force of PMF. If you block this process, you die. This is how cyanide kills people.

EXTRA

-Eukaryotic genes -are split or interrupted -have exons (expressed sequences), regions coding for RNA that ends up in the mRNA. The pieces of genes are exons, and then in between are introns, they have to be removed. Initial RNA (hnRNA) has introns and exons. After splicing (which isn't always perfect), only exons left. -exons are separated from one another by introns (intervening sequences) that code for RNA that is never translated into protein -the initial RNA transcript has both intron and exon sequences -introns are removed from the initial RNA transcript by RNA splicing -splicing of the pre-mRNA occurs in a large complex, the spliceosome, that contains the pre-mRNA -alternative splicing -sometimes the pre-mRNA is spliced so different patterns of exons remain. Splicing isn't always perfect, so initial RNA doesn't always give the exact same mRNA. Changes aren't always drastic, just slightly manipulated, which gives a lot of variety, through alternative splicing. In eukaryotes, protein synthesis is in cytosol. Transcription and Replication is in nucleus, but translation is in cytoplasm.

Phosphorylation, Substrate-Level-Phosphorylation, Oxidative phosphorylation, and photophosphorylation

-In substrate level phosphorylation, the pi, high energy level phosphate, comes from another high energy compound. In glycolysis it comes from PE (similar to ATP). -Oxidative phosphorylation: must go through electron transport system. Substrate level phoshorylation: ATP is created by transferring a phosphate group from another high energy compound (adp to atp). One high energy compound to another (ex PEP to ATP). No oxygen needed. After glycolysis and krebs cycle, you have a lot of NADH and FADH (not reduced, they are electron carriers). Regenerate their oxidizing power, so need to go through ele

EXTRA

-Initiation: sigma factor binds to the DNA, flags down the RNA polymerase. Sigma factor recognizes the promoter, flags down the RNA polymerase, RNA polymerase binds and unwinds (opens up) the DNA, and initiates the transcription. Sigma factors come off when it initiates the process and synthesizes few nucleotides. This all depends on the -10 -35 regions. These are the same for every gene. -Elongation: this means you move along the DNA and make more of the mRNA. -Termination: then you finally reach the end of the gene. The end of the gene means RNA polymerase has to stop. Has to have some signal to tell the RNA polymerase it has to get off the DNA. There are 2 types: -intrinsic: when you don't need the rho factor. Here, as the RNA polymerase reaches the end of the gene, it encounters area where a lot of U has to be put in (lots of T at the end of the gene). All these T slow down the RNA polymerase. As it slows down, right before that, mRNA starts making a new formation called stem and loop. It causes a complementary region, which causes a loop formation. Causes a stem with hydrogen bonds between the base pairs. It slows down the RNA polymerase, which results in the stem and loop formation- which pushes the RNA polymerase away from the system, and mRNA is released, and transcription is permeated. This is intrinsic- it is dependent on the stem and loop formation and needs a bunch of U at the end of the gene. -Rho-dependent: depends on the protein called rho, which is a part of the complex called RNA polymerase. Rho also follows the RNA polymerase. When it reaches the end it catches up with it and a stem loop is formed, but it is the push from the rho protein that separates the RNA polymerase from the DNA and ends the process. STEM LOOP IS ON RNA, NOT DNA. RNA is always 5' to 3'

EXTRA

-catabolic enzymes are associated with inducible operons. Substrate controls synthesis of mixed gene expression. Example: carbon source- sugar. With inducible operons, when the substrate is available, it turns on, if not, its off. On the second level of control, when the substrate is available and a preferred carbon source is not available, the gene is on. When the preferred carbon source is available, the gene is off. The preferred carbon source is glucose.

Describe the role of chaperones in polypeptide folding, and molecular chaperones in protein secretion across the membrane

-Molecular chaperones -proteins that aid the folding of nascent polypeptides -these chaperones are proteins themselves that bind to a growing polypeptide. As the polypeptide is coming off of the ribosome, ... idk -protect cells from thermal damage. Heat shock. If you raise the temperature on the bacteria, heat shock damages them, because high temperature melts the hydrogen bond and the protein becomes damages. If heat shock occurs, in order to repair themselves, they use these proteins to properly fold them back to their original shape. Thermal damage leads to denatured proteins, which are not functional. Have to bring them back to the right conformation. Heat shock proteins. -aid in transport of proteins across membranes. Sometimes you have to send the protein through the cell membrane. In order to do this, you must send them single file- so they have to be denatured or open. Because if they are folded too much they are bulky and cannot pass through. The chaperones hold the shape while passing through the membrane. DNAj and DNAk are examples of heatshock/ chaperone proteins. They bind and hold on the protein as it passes through the cell membrane. Energy is consumed in the process.

Autotrophs, phototrophs, chemotrophs, chemolithotrophs,

-chemolithoautotrophs- can rely on inorganic carbon source, inorganic energy source, and inorganic electron source. Can make biomass from no sunlight, no organic matter, and from all inorganic compounds. -chemotrophs use chemicals as an energy source. Can be organic or inorganic. Generate the PMF -Phototrophs- energy comes from sunlight and excites electrons, and then electrons can enter the electron transport system. Both eukaryotes and prokaryotes can be phototrophs. eukaryotic/plants are always oxygenic (made oxygen in process). Algae included. In the case oh phototrophic bacteria, some produce oxygen (like cyanobacteria- important group of archaea that produce oxygen). Other bacteria may not produce oxygen (anoxygenic) -photosynthesis- harvests energy from the sunlight and converts it into chemicals to make ATP. This is a two part process

What are the important features of genetic code?

-every 3 base pairs is for one amino acid (AUG, CCA, ACU, UAC, CCU). (an RNA is ACUG, a DNA is ACGT). You have 64 base pairs, and out of them all, 61 code for amino acid. 3 code for no amino acid. These are stop codons. -universal- whether you are virus, bacteria, human, plant, elephant, etc. -remained unchanged -first two letters of each codon -initiation codon- initial codon is always the same, it is always AUG. -termination codons (non-sense codons). There are 3 of them. UGA, UAA, UAG. Multiple codons can code for the same amino acid. But two amino acids cannot have the same codon. But can find one amino acid with multiple codons. -code degeneracy -wobble hypothesis- the first two base pairs in the codon are most important. If the first two code match, the last one doesn't have to match. This allows for the same amino acid to have multiple codons. These codons have to match with the anticodon that you find on the tRNA. tRNA brings the amino acid to the area. So when the ribosome complex forms between the mRNA and sandwiches the mRNA, the tRNA brings the amino acid, and when the anticodon matches, the tRNA stays and brings the amino acid.

Global gene regulation by alternate sigma factor

-global regulatory systems often use many types of regulation such as: -regulatory proteins - alternate sigma factors -two component signal transduction (regulatory) systems- this means the molecule is made of two components- one acts like a binding, like control, and basically send the message from the outside to inside. Usually involves and activity of molecule of some sort. Something in membrane, something binds to the outside of it, and either opens or closes the activity inside. -phosphorelay systems- adding or removing phosphate molecules. Kinase or phosphorylase activity- can turn on genes this way. Sigma factors can guide RNA polymerase to different set of genes. There are multiple sigma factors in E. coli. Usher/chaperone that takes you to specific places in the DNA. Each sigma factor recognizes different set of genes and is under different sets of conditions. (table 13.3)- sigma F sigma 28. Genes involved in flagella assembly. Chemotaxis, more flagella. Sigma 60 metabolizes nitrogen. Normally we have sigma factor 70 which is for normal exponential growth. Normal log phase growth. Sigma S for genes needed during the general stress response and during stationary phase. Sigma H (sigma 32)- genes needed to protect against heat shock and other stresses, including genes encoding chaperones that help maintain or restore proper folding of cytoplasmic proteins and proteases that degrade damaged proteins.

Describe different forms of protein secretion mechanisms; Describe Sec-dependent mechanism, ABC secretion pathway, and type I-V secretion mechanisms in Gram-negative bacteria.

-gram-positive and gram-negative bacteria have different problems secreting proteins based on the differences between the structure of their walls -Both G+ and G- use the Sec-dependent pathway for transporting proteins across the membrane -Other secretion pathways also exist, but all systems require energy -Sec-dependent pathway; general secretion pathway -translocate proteins from cytoplasm across or into plasma membrane -attached to pre-protein is signal peptide which: -delays the folding (makes sure the protein is still unfolded, still primary structure). -chaperone proteins keep preproteins unfolded -removed once pre-protein emerges from plasma membrane -Protein Secretion in G-: -Type I,II,III, IV, and V -Transport across the OM -Transport across Periplasmic space (space between cell wall and outer membrane in G-) Protein Secretion Systems of Gram-Negative Bacteria ---Type I (ABC) Protein Secretion Pathway- Type one takes the protein, passes through the exit of the transmembrane protein TolC , and sends it to outside the cell. SecD and Tat send it to periplasmic space (energy from tat comes from PMF generated during electron transport system). They don't go through cell membrane like Type I does- goes through cell membrane, periplasmic space, and outer membrane and sends it to outside. -ubiquitous in prokaryotes -transports proteins from cytoplasm across both plasma membrane and outer membrane ---Type III Pathway- goes though everything Type I does, but it has an extension. The extension goes through the cell membrane of the host cell, another cell. It's like a syringe, it injects toxin into the host. Molecular syringe. Virulence factor- means and structures possessed by microbes that cause diseases. This way (with the syringe) the host cannot remove or block the toxin with the immune system, it just goes straight into the cell. -Type III pathway secretes virulence factors of Gram-negative bacteria from cytoplasm, across both plasma membrane and outer membrane, and into host cell. -Type III is a virulence factor that contributes to the pathogenicity or disease causing of the microbe. ---Type 4 pathways are unique because they secrete proteins and transfer DNA during conjugation -genetic exchange: bacteria can exchange genetic material between each other. One way is called conjugation. Here bacteria dock against each other, the pili from one attaches to the other (pili is hollow structure that DNA can be copied and travel through and go to the host and other bacteria). Type 4 transfers proteins and DNA during conjugation. Type IV pili is like a syringe that injects genes into another bacteria. Makes copy of genes and sends the copies through in forms of plasmids.

EXTRA

-oxidation reduction reactions. Source of Energy is highly reduced. Ex glucose: when you're reduced it means you were rich in electrons. It gives up electrons, and you make ATP from that. For every oxidation, ther is a reduction. When glucose comes and becomes oxidized (it loses electrons), the electron has to be picked up by someone else. Gaining electrons means reduction, you become reduced. Electron carriers pick up electrons, ex NAD, becomes NADH. NAD is oxidized form, NADH is reduced form.

What is meant by DNA repair? List different forms of DNA repair mechanisms. Why DNA needs to be repaired?

-proofreading- DNA polymerase sits on the DNA, zips through the DNA, and reads the DNA. There is distance between the base pairs, it measures this. If there is a mistake between base pairs, it can see that, or see if it's too narrow or too bulgy. It is very quick and makes mistakes -correction of errors in base pairing made during replication -errors corrected by DNA polymerases -other DNA repair mechanisms also exist -direct repair: photoreactivation -used to directly repair thymine dimers -thymines separated by photochemical reaction using visible light catalyzed by photolyase. Pen up the two T's and separate them. -direct repair of alkylated bases -catalyzed by alkyltransferase or methylguanine methyltransferase -Mismatch Repair: type of excision repair. -ex: mismatch repair system in E. coli -mismatch correction enzyme scans newly synthesized DNA for mismatched pairs. -mismatched pairs removed and replaced by DNA polymerase and DNA ligase (DNA polymerase recognizes, for example that a T G base pair is a mistake, goes back and removes it and resynthesizes it. Removes multiple bases around it, resynthesizes it, and finishes it up. -the removal is called 3' 5' exonuclease activity. (exonuclease is removing, endonuclease is synthesizing) -DNA Methylation: used by E. coli mismatch repair system to distinguish old DNA strands from new DNA strands. The DNA, as it is synthesized, becomes methylated. Adds a methyl group. There is an enzyme that methylates the DNA. The new group does not have the methyl group because it is not added immediately. This one with no methyl group is the problem strand- it is the new strand.

Denitrification and Nitrogen Fixation

80% of the air we breathe in is nitrogen, but we cannot use it. There are microorganisms in the soil whose job it is to fix nitrogen gas into polymer nitrogen that people can use. They take nitrogen and can turn it into nitrite or ammonia (NH2) this is called nitrogen fixation. Nitrogen is fixed into ammonia and can turn back into the other forms. Nitrogen from ammonia, you reduce it. But when you have ammonia and go to nitrite you oxidize it, and nitrate you oxidize it further. Microorganisms are involved in all these processes. They help us with it and use it as a source for electron acceptors. Same thing can happen with sulfate (SO4) and others.

List possible molecules that are used as final electron acceptor in anaerobic respiration.

A final electron acceptor is different exogenous acceptor such as NO3-, SO42-, CO2, FE3+, or SeO42-. Organic acceptors may also be used.

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ATP comes from the metabolic activity of the cell.

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Aerobic respiration, anaerobic respiration, fermentation, photosynthesis extract energy from the source and convert them into ATP, which is then used for cellular activity, chemical work, transport work, mechanical work. You grow, get rid of the waste, and multiply.

Explain how attenuation regulatory mechanism works for tryptophan operon.

Attenuation means you slow down. Attenuation is alternate stem loop formation of the transcription, RNA, that is produced. There is DNA and the RNA polymerase travels through the DNA and makes RNA and there's a stem loop formation. When the RNA polymerase hesitates, when it reaches the end of the line, stem loop formation happens before the RNA is finished. The stem group pushes the RNA polymerase away and transcription terminates. The stem loop formation also controls attenuation. Alternate stem loop formation controls trp operon through attenuation. When there is low transcription, low activity, no protein synthesis, the stem loop is between region 1 and 2, and 3 and 4. When the formation is between 1 and 2, and 3 and 4, this is a stem loop formation- there is no transcription. When everything is good and protein synthesis is going on (this is the case of the coupling of transcription and translation). Tryptophan translation means making proteins, and coupling of the transcription means the coupling of the gene and then translating it at the same time coupled together. In this case, there is no tryptophan, the thing moves on, and there is a loop between 2 and 3. When the loop is between 2 and 3, this allows RNA polymerase to continue, because transcription was already initiated, some RNA has already been made and turning into protein. When protein synthesis is going on and you need tryptophan but it is low, this process goes on. Also if too much tryptophan the operon is off because the loop between 3 and 4 is off- this loop terminated the transcription. 1. Low activity, operon is off, low attenuation 2. High activity, like protein synthesis is going on, the coupling between the transcription translation, operon is on, making tryptophan 3. Too much tryptophan, you don't need to make it. When the transcription and translation are coupled. As the mRNA is produced, at the same time a ribosome sits on it and starts protein synthesis. this is unique to prokaryotes. Through the stem loop formation of mRNA- mRNA, as it is produced, sometimes forms in a stem loop- the 2 complementary can form a double strand at the base of the stem loop. The complementary fomr hydrogen bond. This is a good way of pushing the RNA polymerase off of the track and off of the DNA. 1:2 and 3:4 stem loop formations. If 1:2, the process is terminated- no transcription. When the stem loop formation between 2 and 3 happens, then the transcription goes on. This is when the activity is high. There is low tryptophan, and you need the tryptophan made. When tryptophan is too high, stem loop occurs between 3 and 4, which prevents transcription from happening. Alternate stem loop formation allows RNA polymerase either to continue or to fall off of the DNA molecule. At low level of attenuation it makes tryptophan, at high levels of tryptophan it stops and doesn't make tryptophan.

EXTRA

Catabolic Repression: The effect of glucose, the preferred sugar, the preferred carbon source. When you have glucose and other energy sources, glucose is used first. Only time operon is on for the sugars is when the sugar is present. -diauxic growth (two lag phase, two log phase, for each sugar) -a biphasic growth pattern in which there is preferential use of one carbon source over another when both are available in environment (glucose is preferred). -second lag occurs after preferred substrate is exhausted followed by the resumption of growth using the second source. Operon for the second sugar is expressed in the second log phase. -catabolite repression plays a role in this pattern of growth. The lactose operon was off as long as the glucose was available. That's catabolic repression. Affects the glucose availability and glucose present. This happens with lactose and other sugars also

How anabolism and catabolism intertwined in glycolysis and TCA cycle. What is the significance of glycolysis and TCA cycle in providing skeleton carbon to the cell?

Catabolism: -fueling reactions -energy-conserving reactions- means you put them in a higher energy compound like ATP. Energy extracted from a molecule is put into another molecule and has now become an energy currency. Can go to other reactions in the cell and help them. -provide ready source or reducing power (electrons) -generate precursors for biosynthesis. Precursors are building blocks or molecules that end with the building blocks. Anabolism: -the synthesis of complex organic molecules -requires energy from fueling reactions. Large molecules (polymers) get broken up and go to small molecules (monomers). Initial oxidation and degradation to pyruvate. Oxidation and degradation of pyruvate by the TCA cycle. Purpose is to go from highly reduced to oxidized. Example: reduced C6H12)6, convert to fully oxidized CO2 + H2O. NAD and FAD pick up the electrons and become the reduced NADH and FADH. TCA cycle: after pyruvate is produced, it's then turned into Acetyl CoA. 2 carbon enter acetyl CoA, 2 come out as CO2 on other end. TCA aka citric acid cycle aka krebs cycle. Krebs cycle is amphibolic pathway. A key source of carbon skeletons for use in biosynthesis Most glucose goes through and we can draw from that for energy. We can take it and make amino acids with them for example. Not everything comes off as CO2. 2 carbon enter as Acetyl CoA, 2 come off as CO2, amphibolic pathway, molecules are used for synthetic process, and can be broken down to extract energy from them. For each acetyl CoA molecule oxidized, TCA cycle generates: - 2 molecules CO2 - 3 molecules of NADH - 1 FADH2 - 1 GTP (ATP) (GDP to GTP is a substrate level phosphorylation). In both glycolysis and krebs cycle ATP is produced in substrate level phosphorylation (transfer energy from one high energy level compound to another) Moral of story: you have glucose coming in, it goes through glycolysis, makes pyruvate, pyruvate enters the krebs cycle as a form of acetyl CoA, etc Doesn't always go all the way through, sometimes molecules come out and make other precursors (fatty acid, amino acid, sugar, polysaccharide, etc). Anabolism: -energy from catabolism is used for biosynthetic pathways -using a carbon source and inorganic molecules, organisms synthesize new organelles and cells. -antibiotics inhibit anabolic pathways. (ex sulfa drugs inhibit synthesis of folic acid in bacteria, that's how they kill bacteria, since humans don't make folic acid it's a good target since it won't affect us, only bacteria) -a great deal of energy is needed for anabolism -turnover -continual degradation and resynthesis of cellular constituents by nongrowing cells -metabolism is carefully regulated. - for rate of turnover to be balanced by rate of biosynthesis

Compare respiration and fermentation; a. ATP synthesis, pathways involved b. Role of ETS c. Electron carriers, electron acceptors d. Number of ATP produced

Central pathway to metabolism- glycolysis (6 C) to pyruvate (3C, co2 produced) to acetyl coA (2 C) to krebs cycle (2 CO2 come off).

List the laws of thermodynamic and describe their relevance in the chemical reactions

Chemical reactions obeys the laws of chemistry and physics. Thermodynamics analyzes energy changes in a collection of matter called a system (a cell for example). All other matter in the universe is called the surroundings First law of thermodynamics: -energy can neither be created nor destroyed. -total energy in universe remains constant. Within a system energy moves from one form to another. It's not created or destroyed. -"however energy may be distributed either within a system or between the system and its surroundings. Second law of thermodynamics: -entropy: amount of randomness (disorder in a system). Image that the universe is moving toward randomness. Randomness requires the least amount of energy. Molecules want to be away from each other as much as possible, the least amount of energy possible. -physical and chemical processes proceed in such a way that the disorder of the universe increases to the maximum possible.

What are the environmental impacts of Chemolithotrophs activities? ("Eating Rocks")

Chemolithotrophy: -three major groups -electrons released from energy source which is an inorganic molecule transferred to terminal electron acceptor by ETC, make ATP via oxidative phosphorylation. -three major groups -Have ecological importance- can affect nitrogen availability in the soil, metabolize nitrogen and sulfur, etc. -several bacteria and archaea oxidize hydrogen -sulfur-oxidizing microbes -hydrogen sulfide (H2S), sulfur (SO), thiosulfate (S2O32-) -nitrifying bacteria oxidize ammonia to nitrate. They are primary producers. Inorganic energy and electron source. use CO2 as a carbon source, using different chemicals, inorganic chemicals, as electron donors and acceptors. Can also produce oxygen. Dissimilatory nitrate reduction -use of nitrate as terminal electron acceptor -the anaerobic reduction of nitrate makes it unavailable to cell for assimilation or uptake. -some bacteria, because of their metabolic activity remove nitrogen from the soil, and t hen plant cells cannot pick it up. Denitrification: -reduction of nitrate to nitrogen gas. Remove it from soil and release it into environment (N2 to N). as you add your fertilizer to soil, some bacteria have the ability to remove it (causes loss of soil fertility). Nitrifying bacteria -oxidize ammonia to nitrate -take organic, go to ammonia, go to nitrate. This is good for the soil- nitrate is put into the soil- plants use nitrate. Sulfur-oxidizing bacteria -ATP can be synthesized by both oxidative phosphorylation and substrate-level phosphorylation.

EXTRA

Control of Transcription Initiation by Regulatory Proteins -induction and repression occur because of the regulatory proteins -these proteins either inhibit transcription (negative control) or promote transcription (positive control) -their activity is modulated by inducers, corepressors, and inhibitors. Most of the control of gene expression is exhibited on the level of transcription- up to 70%. Bacteria control multiple genes that are or aren't required at the same time under one control- this is operon system. Operons involve multiple genes involved in one activity under one control. Operons are controlled by one system that turn them on or off. This is usually associated by metabolic activities, for example sugar consumption. Under control by local control that controls these enzymes. 2nd level of control controls the gene- lactose operon.

Telomers

DNA at the tips of chromosomes nongenetic material at the end of chromosomes protect against gene loss. cap ends of chromosome during replication to preserve DNA

EXTRA

DNA in archaea and most bacteria is supercoiled-ds-circular Bacteria DNA is organized with help of chromatin-like protein DNA is more highly organized in eukaryotic chromatin where it is associated with histones, small basic proteins- only in eukaryotes. The combination of DNA and proteins is called a nucleosome Archaeal DNA organization is similar to that of eukaryotic cells, has some similarities to bacteria too though In prokaryotes DNA is in nucleoid- it's a region not organelle, no membrane Replication machinery- DNA gyrase, ssb proteins, dna primase etc, table 13.1 lecture 4 slides.

EXTRA

DNA is a polymer of nucleotides. Each nucleotide is made of purine (G,A) and pyrimidine (C,T,U), ribose or deoxyribose, and then you have a phosphate attached to it. DNA has AGCT, sugar is deoxyribose- 5 carbon sugar. Double stranded. Hydrogen bonds hold together A and T, and C and G. A and T have 2 hydrogen bonds, G and C have 3. Hydrogen bonds are weak, so they can separate and reconnect.

Compare DNA polymerase vs. RNA polymerase

DNA polymerase interacts with DNA. It synthesizes DNA. It had primer (a piece of RNA) to initiate it. (always go 3 prime to 5 prime). Continuous strand keeps going with one primer- this is the top strand, the leading strand. The bottom strand has to be done in pieces, done with multiple primers. This is the lagging strand. It is done in multiple fragments called okazaki fragments. The primers are eventually removed so then the DNA can be complete and sealed when the primers are removed. DNA always adds nucleotide to the 3 prime end of the molecule, so we can only go 5 prime 3 prime for replication. The other strand is always used as a template (complementary strand), that's how you know what other base pair to add. The 3 prime to 5 prime is continuous, the 5 prime to 3 prime is in fragments. RNA polymerase synthesizes RNA. There are reducers and suppressors that are capable of binding to the protein, to the DNA.

What is the role of Dnaj and Dnak in protein secretion process? List and describe protein secretion systems in G+ and G-.

DNAj and DNAk are examples of heatshock/ chaperone proteins. They bind and hold on the protein as it passes through the cell membrane. Energy is consumed in the process. DNAJ and DNAK consume ATP to properly fold and also protect the protein if there is damage due to high temperature.

List and describe effects of environmental factors on enzymatic activities. How?

Enzyme activity is significantly impacted by substrate concentration, pH, and temperature. Denaturation of proteins occurs- this is the loss of enzyme's structure and activity when temperature and pH rise too high above optima. Temperature effects hydrogen bonds. Proteins become denatured when they lose their structure. As you increase the substrate level, enzyme activity will go up until it hits the saturation level. When it becomes saturated it cannot do anymore. As substrate increases, activity increases until a certain point,

Define apoenzyme, cofactor, and prosthetic group. What is the significance of cofactors? How enzymatic activities are regulated or inhibited. What is the role of prosthetic groups?

Enzyme inhibition -competitive inhibitor- directly competes with binding of substrate to active site. Substrate level. If you add more substrate it can bind easier again. Ex sulfa drugs can inhibit bacteria by blocking the site for PABA (substrate), one of its enzymes, so now bacteria cannot replicate. This is a treatment for disease. -noncompetitive inhibitor- binds enzyme at site other than active site. Changes enzyme's shape so that it becomes less active. Changes conformation. Cannot be overcome by the substrate level. Prosthetic groups (permanent groups) are attached to enzymes. They are made of proteins. Feedback inhibition -also called end-product inhibition -inhibition of one or more critical enzymes in a pathway regulates entire pathway -pacemaker enzyme -catalyzes the slowest or rate-limiting reaction in the pathway. The end product controls the pathway. This is feedback inhibition.

Riboswitches

Folded RNAs that act as switches regulating protein synthesis in response to environmental conditions

EXTRA

From ATP to ADP is an exergonic reaction. It has excess energy to give.

EXTRA

GC content of the cell- the more GC you have, the harder that region is to open because you have 3 hydrogen bonds instead of 2.

Briefly discuss the regulation of gene expression in eukaryotes and archaea

Gene expression means making RNA molecule. This costs energy and time and must be used for some purpose. Bacteria are very efficient in gene regulation, because they don't want to express genes when they don't need them. -Regulation occurs at many levels -transcription initiation (most regulation occurs here. About 70 %. making RNA) every gene has a beginning and an end. Upstream from the gene is the promoter region, where RNA polymerase initially binds to initiate the process. So if you can manipulate the initiation, you can manipulate the expression of that gene. -transcription elongation. The system starts but stops somewhere after the promoter binds -translation- can manipulate the protein synthesis (assembling the ribosome on the mRNA and start the process). -posttranslation- after you make the protein you can manipulate that as well, add something to it, modify it like allosteric, or modify it through covalent modification, which means you can add something to it or change the conformation of the protein or change the protein action. -Three domains of life differ in genome structure and regulatory mechanisms used -Archaea are also prokaryotes, but in many ways archaea are different, they have one chromosome, no compartment, but they conduct their business more like eukaryote. Translation and transcription in a way is similar to that of eukaryotes, but they work in an environment like prokaryotic environment. -No archaea cause any diseases.

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Gene structure Gene -the basic unit of genetic information -also defined as the nucleic acid sequence that codes for a polypeptide, tRNA, or rRNA -linear sequence of nucleotides with a fixed start point and end point -Codons are found in mRNA and code for single amino acids Reading frame -every gene has a reading frame- this means the start and the end. -organization of codons such that they can be read to give rise to a gene product. -importance of reading frame- only 1 strand is usually the gene, the other is a complementary strand. Gene always has start and end. Gene always goes from 5' to 3' direction. The term coding strand, this is where the gene is. Template is the complementary strand to the coding strand. mRNA switches U for a T (example: DNA 5' ACTGCCCATGA... 3', then 3' TGACGGGTACT... 5', then mRNA 5' ACUGCCCAUGA 3'.) Have to use the template as the complementary strand. When we make mRNA, it's the same as the DNA 5' to 3', but there is a U in mRNA instead of the T in DNA. mRNA is exact copy of the coding strand, except there is a U for the T, and ribose for deoxyribose. So, the coding strand is the gene. mRNA is the exact copy of it. Can only make another gene from the coding strand. Cannot have another gene made from the 3' to 5' DNA (template strand), can only make it from the coding strand. The template is the complementary strand to use as a template, it is not another gene. One is always coding strand while the other is template.

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Genes That Code for tRNA and rRNA: -DNA sequences that code for tRNA and rRNA are considered genes -genes coding for tRNA may code for more than a single tRNA molecule or type of tRNA -spacers between the coding regions are removed after transcription, some by the use of special ribonucleases called ribozymes.

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Genes that code for proteins- the coding region: -template strand- directs RNA synthesis -read in the 3' to 5' direction -promoter.. -leader sequence- transcribed into mRNA but is not translated into amino acids -shine-delgarno sequence important for initiation of translation. This is where ribosome is going to recognize and bind to. rRNA is going to recognize these shine- sequences in mRNA -Begins with the DNA sequene 3'-TAC-5' -produces codon AUG- mRNA always starts with AUG -codes for N-formylmethionine, a modified amino acid use to initiate protein synthesis in bacteria -coding region ends with a stop codon -immediately followed by the trailer sequence , which contains a terminator sequence used to stop transcription. This is not part of the gene, but it added to the mRNA that ends the transcription

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Genetic info is organized in chromosomes, the section of chromosomes that contain info are called genes, but not all chromosome is gene, just segments. Every gene has a start and an end. These sequences code for polypeptides. Some genes don't code for proteins, they are just used for what they are, like for rRNA and tRNA- these are genes too. Genes can code for mRNA, which codes for polypeptide, codes for tRNA, codes for rRNA. mRNA codes for protein, contains a codon. tRNA has the anticodon. During translation, which is the protein synthesis, the whole units are assembled together- ribsome, mRNA hold on together, and tRNA brings the amino acids. These 3 are involved in protein synthesis. We know which ones are genes because there are some base pairs that the RNA polymerase knows these are the gene.

Define and give examples of global regulatory mechanisms and SOS response.

Global Regulatory Systems: -regulatory systems that affect many genes and pathways simultaneously. Multiple genes can have one control. multiple operons can have one control. There are levels of control. (multiple counties under one state (example of operon)). (operons control multiple genes). -important for bacteria since they must respond rapidly to a wide variety of changing environmental conditions -regulon -genes or operons controlled by a common regulatory protein. So there are multiple operons controlled by common regulatory protein. So there are multiple operons controlled by a common thing- it is a collection of genes under once control by one protein. Under direct control here. Can exert local control over their own operon as well. -modulon -operon network under control of a common global regulatory protein but individual operons are controlled separately by their own regulators. -example is control of glucose over other sugars. CAP protein (catabolite repression) -global regulatory systems often use many types of regulation such as: -regulatory proteins - alternate sigma factors -two component signal transduction (regulatory) systems- this means the molecule is made of two components- one acts like a binding, like control, and basically send the message from the outside to inside. Usually involves and activity of molecule of some sort. Something in membrane, something binds to the outside of it, and either opens or closes the activity inside. -phosphorelay systems- adding or removing phosphate molecules. Kinase or phosphorylase activity- can turn on genes this way. SOS response Bacteria are very efficient and quickly respond to their environment- carbon source, energy source, electron donor, temp changes, pH change- bacteria can respond to these quickly because they have sigma factor- which can immediately turn normal growth- for example one of the sigma factors allows you to produce multiple flagella- which is for motility. This results in chemotaxis. It lets you go faster towards or away from something. SOS response -inducible repair system -you don't use SOS system on a regular basis, you use your other structured system. This SOS is to just quickly (sloppily) but something together. SOS for emergency cases when under a tremendous amount of danger. -controls multiple genes and activities. Basically when you have something of a problem. -a global control network -used to repair excessive damage that halts replication, leaving many gaps. But still, the bacteria overall survives. -RecA protein initiates recombination repair. -RecA protein also acts as a protease, destroying a repressor protein and thereby increasing production of excision repair enzymes. When you destroy repressor proteins, you can make an excessive amounts of excision repair enzymes. Excision repair enzymes are enzymes that actually cut pieces and repair them with the new ones. -a highly error-prone repair used in a life-or-death situation. Right now we just want the function restored to some extent. -for example like the heat shock, you can have SOS response to repair the damage

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Glucose splits into an electron and ATP and CO2.

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Hallmark of cell genetic info in the form of DNA DNA is organized in the form of chromosomes. You may have one or multiple chromosomes There are also extra chromosomal DNA found in some cells, in bacteria you have plasmids. In eukaryotes you have chromosomes in mitochondria and chloroplasts. They're still double stranded, still contain genes, they are just smaller. Go to DNA, make copies in the form of RNA through transcription and then close up and don't touch DNA again. Now RNA can go through translation, be used for RNA synthesis or whatever you want to do with it.

Rolling circle replication

Happens with circular DNA, and some viruses use this. Also use this for plasmids. Plasmids are small circular DNA. Some viruses can make their DNA into small circular DNA. Imagine a double layer like a ferris wheel. Cut one strand and separate it. As you cut this piece and pull it away from the wheel, you start adding nucleotides to it (always 5 prime 3 prime direction). As you pull it away, you synthesize more. What happens, it fills the gaps so it's still always double strand. When every the piece finishes, we cut it and recircularize it. Can have unlimited copies of this DNA. In regular replication we can only have 2 copies. In rolling circle we can have multiple. We do this for the plasmid, viruses, -some small circular genomes (ex viruses and plasmids) -replicated by rolling-circle replication

Define and give examples of horizontal gene transfer (HGT). What are the significances of HGT?

Horizontal (lateral) Gene Transfer (HGT) in Prokaryotes, not Eukaryotes -occurs via three mechanisms (conjugation, transformation, and transduction) evolved by bacteria to create recombinants. -all three mechanisms depend on some type of recombination -genes can be transferred to the same or different species Bacteria can do genetic exchange. We can do vertical genetic exchange. This means there is gene in the parent cell, it is copied, every daughter cell has a copy of the gene and you keep going. Each copy of the chromosome comes from the one parent, sometimes mix and match, and you get combination of the gene from both parents, and then there are two copies of genes- one in each chromosome. Eventually the gene is transferred from the parent cell to the daughter cell in vertical gene transfer. However, bacteria have horizontal gene transfer. Bacteria can do vertical exchange with daughter cells, but across as well. DNA can be copied in one cell and be picked up by another. There is a donor and recipient Three types of horizontal gene transfer: 1- Conjugation- bacterial conjugation: The transfer of genes between bacteria that depends on: -direct cell to cell contact mediated by the F pilus (pili from one bacteria is connected to another. Pili is like a hollow tube that DNA is copied from the donor, travels through the hollow like a tunnel, and goes to the recipient). -a type IV secretion system- G - secretion mechanism. Type IV is via pili. Bacteria can have plasmids travel through the type IV secretion mechanism. Travels from donor to host, and in new host, through rolling circle replication: -rolling circle replication of plasmid: makes multiple copies of the plasmid in the new host. Now have bacteria with the new DNA. Plasmids carry antibiotic resistant genes!! They can spread resistance to each other. Rolling circle makes multiple copies of the plasmid, not just 1 or 2 copies like regular replication. Plasmids are double stranded circular DNA. They are extrachromosomal DNA. Additional DNA in the cell. Pathogen (disease causing) genes are also carried by plasmids. Genes code for toxins are carried by these plasmids. Microbes become more virulent, more problematic when they have plasmids. 2- Transformation- In the environment, some cells die, and their DNA is released in the open, it is picked up by others- they like swallow it, breathe it in, pick it up, and they integrate it into their system. They can pick up DNA fragments. It is a rare but natural phenomenon. In the lab we can mix those receptive to the outside DNA. We can make cells competent by treating them with calcium chloride and subject them to heat shock and cold shock and see what they pick up. Transformation happens in the lab. Can make genes, put them in plasmids, and have the cell pick up the plasmids for you. It is a laboratory and natural phenomenon. 3- Transduction The DNA then combines with the DNA of the host, the recipient. Recombination is mixture of DNA from different sources in one cell. -via viruses. Viruses attack the host and pack DNA from the host in their system, go attack another host and take the DNA from the old host into the new one. Phage affects bacterial cells. Host DNA is hydrolyzed into pieces, and phage DNA and proteins are made. Phage assembles and it occasionally carries a piece of the host cell chromosome and package them into the system- this is transducing phage with host DNA. Then it goes and attacks a second host, and carries the DNA into second host- this is transduction. Remember, vertical gene transfer is what everyone does- from parent cells to daughter cells. Then there is horizontal gene transfer: happens only in bacteria, in prokaryotes, 3 versions- conjugation (direct contact), transformation (picks up segments of DNA), transduction (virus). All 3 used in genetic engineering. Donor DNA à conjugation, transformation or transduction à partially diploid recipient cell à (all of these : go to recombinant cell) -Integration of donor DNA à reproduction à population of stable recombinants -Donor DNA self replicates (ex a plasmid) à reproduction à population of stable recombinants -Donor DNA cannot self replicate à recipient reproduces; donor DNA does not à no stable recombinants -Host-restriction à reproduction à no stable recombinants

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In fermentation: One of the end products of metabolism is used as electron acceptor. You accumulate the compound in the cell. Examples: lactic acid bacteria (streptococcus, lactobacillus), bacillus; yeast, zymomonas; propionic acid bacteria (Propionibacterium); Enterobacter, serratia, bacillus; enteric bacteria (Escherichia, Enterobacter, salmonella, proteus); clostridium.

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In protein synthesis we also have initiation, elongation, and termination (but not in replication) -the goal- position ribosome properly at 5 prime end of mRNA. Shine-delgarno sequence plays a role here. There are 3 seats on the large part of ribosome- E P A. first that gets put is AUG. first amino acid sits on P site. E is just exit site. Elongation is when the next amino acid comes and keeps going, and every time it comes ad sits on the A site, the whole thing translocates, which means the whole complex moves one triplet, one codon at a time. -Elongation cycle: -sequential addition of amino acids to growing polypeptide -consists of three phases -aminoacyl-tRNA binding (to A site) -transpeptidation reaction- happens between the amino acids at the P site to add in A site. The whole things moves and the tRNA comes off of the amino acid on the P site, and on the exit site, E site, the vacant tRNA comes off. The A site of now available, the next tRNA comes and sits on the A site, and it continues. This is elongation. Transpeptidation is an enzymatic reaction, the 23S RNA forms a peptide bond between the incoming amino acid and the growing polypeptide -translocation- the ribosome complex moves one codon at a time. -EF- elongation factors that guide the process. These become important when treating patients with the antibiotics -Peptidyl (donor; P) site -(the tRNA is formed between the A site and T site?) -binds initiator tRNA or tRNA attached to growing polypeptide (peptidyl-tRNA) -Aminoacyl (acceptor; A) site- incoming amino acid tRNA binds to A site. -Exit (E) site- the empty tRNA exits from this sit -briefly binds empty tRNA before it leaves ribosome.

Describe how proton motive force (PMF) is generated. How this force (energy) is utilized

In respiration, as electrons pass through the ETC to the final electron acceptor, a proton motive force (PMF) is generated to synthesize ATP. By creating holes in the cell membrane and allowing protons to flow back in, you are creating PMF. This PMF can be used to synthesize ATP.

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Microbial Fermentations: - Oxidation of NADH produced by glycolysis - Pyruvate or derivative used as endogenous electron acceptor - Substrate only partially oxidized - Oxygen not needed - Oxidative phosphorylation does not occur o ATP formed by substrate-level phosphorylation.

Polysome

Multiple ribosomes can get on and mRNA at the same time. This is possible because everything moves 5 to 3. Nothing runs into eachother. Multiple proteins simultaneously

Define point mutation and frame-shift mutation

Mutations: -stable, heritable changes in sequence of bases in DNA -in prokaryotes usually produce phenotypic changes (phenotypically expressed, you can see results in the offspring the cell produced). -can occur spontaneously or be induced by chemical mutagens or radiation. Without intervention from outside. DNA Polymerase makes mistakes naturally. Or it can happen from something inducing it. Chemicals or radiation. Mutagens are the things (agents or reagents) that induce mutation. The mutations go to daughters etc. on and on. DNA can change in places where there's not even a gene, it doesn't matter, not required a mutagen. -there are some molecules called base analog. This means it looks like a G C A or T and fits as so, fits into system, but doesn't pair, doesn't form hydrogen bond with the one across from it. Mutagens can act in these ways -analog- looks alike -modifying- chemically modifying the base pair -acting like the base pair Intercalating agents can insert into DNA and insert into the stack of DNA and separate the base pairs from one another. This stretch that it forms is a problem for DNA polymerase to recognize. -How chemicals can work and modify the DNA- intercalating agents, modifying agents, and analog agents. -table 14.1- examples of mutagens Every gene has so many base pairs, 1000, 2000, whatever the polypeptides can make. When you have ACCATGCAT, every 3 is a codon, RNA would look ACCAUGCAU. Each codes for an amino acid. Can go to codon table and figure out what each set of 3 is. DNA polymerase sits on the DNA ad makes copies of the DNA as it goes through. G for C, A for T, etc. they can make mistakes though- mutations. Only mutations if felt at level of mRNA and continues. 10^-9 mistakes but usually corrected before next base pair. Radiation -UV radiation- not penetrating, affects surface. When there are two Ts together (ex CATTACTTCCA) the two Ts are fused together. Thymine dimer means 2 Ts are fused together so DNA polymerase doesn't know if it is one or 2 Ts or no Ts. Mistake is made. -xray and gamma ray radiation- depenetrating- breaks the chromosome, breaks the DNA into pieces. It's not like it damages one base or another. It actually breaks the DNA- more lethal than UV light. Mutation has to be felt on the codon level. It has to somehow change the codon at the end. That means changing the amino acids. Amino acid can change the protein. Base analogs -structurally similar to normal bases, but don't work like them, just looks like them. -mistakes occur when they are incorporated into growing polynucleotide chain DNA-modifying agents -altering a base causing it to mispair, C to something else Intercalating agents -distort DNA to induce single nucleotide pair insertions and deletions. -when you separate the stacks too far you may miss something or accidentally insert something. These can happen by POINT MUTATION: single base pair changes Point mutation: -Silent mutation- not felt at the codon level. Change nucleoside sequence of codon- but not the encoded amino acid. No amino acid changes. Perhaps one codon for one amino acid changes is changed to another codon for the same amino acid. One amino acid can have multiple codons. No harm done -Missense mutation- a single base substitution that changes a codon for one amino acid into codon for another amino acid. -Nonsense mutation- converts a sense codon to a stop codon. Truncates protein, stops protein halfway. Function is lost, some function may still remain. -Frameshift mutation- results from insertion or deletion of one or two bases pairs in the coding region of the gene. For example you have amino acids, but then there's a shift and a bunch of different amino acids are put in there. This is the most dangerous. Can result in protein disfunction. Frameshift mutations occur as long as it is not a multiple of three that is added or taken away.

- Reducing power / Standard Reduction Potential

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Allosteric Regulation and Covalent Modification of Enzymes

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Compare and contrast ribozymes and enzymes

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Compare mRNA synthesis in prokaryotes vs. eukaryotes (post-transcriptional modification)

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Compare post-transcriptional modification and post-translational modification. Give examples.

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Compare the transcription process in prokaryotes vs. eukaryotes

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Define Quorum sensing (autoinduction). What is the role of AHL in this process?

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Define and list different form of phosphorylation. Which one is associated with glycolysis, Krebs cycle, and electron transport system?

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Define endonuclease/exonuclease activities. The significance of 3'-5' exonuclease activity

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Describe the Calvin-Benson Cycle. List the two enzymes specific to this cycle.

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Do any of the eukaryotes perform fermentation? Give examples

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Electron flow through branched electron transport chain

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Examples of Reserve Polymers

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How riboswitches and small RNAs can control transcription.

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Indicate if the following portions of a gene is located upstream or downstream of the coding region? 1. Promoter, 2. +1 Nucleotide, 2. Terminator, 3. Trailer. Which portions of a gene are transcribed, but not translated?

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Know major nutritional types of microorganisms.

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Know the enzymes discussed in lectures (listed in Table 13.1)

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Know the terms listed in Table 11.1 and the terms discussed in lectures and listed in Table 11.2)

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List and define different forms of mutagen

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List and describe and compare three common routes of glucose conversion to pyruvate

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List different forms of photoautotrophs (oxygenic vs. anoxygenic). What is the significance of bacteriorhodopsin? Where do Cyanobacteria fit in?

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List groups of organisms based on their energy source, carbon source and the source of electron

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Ribozymes (give examples, Table 10.4)

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True or False: Calvin cycle is used by both prokaryotes and eukaryotes. / True or False: Calvin cycle is used by both photosynthetic and non-photosynthetic bacteria

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What are Riboswitches? How attenuation and riboswitches can stop transcription prematurely.

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What are the differences between repressors, co-repressors and inducers?

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What are the environmental impacts of sulfur-oxidation, nitrification, and de-nitrification?

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What are the important features of Chemoorganotrophs? Can they use the same compound as a C source, energy source, and as a source for reducing power? Give an example

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What are the important features of Two-Component Regulatory and Phosphorelay Systems?

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What does coding strand/complementary strand mean? Which one is complementary to mRNA?

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What is meant by CO2 fixation? List three pathways used by microbes to fix CO2

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What is meant by positive and negative control mechanism?

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Where in bacteria, archaea, and eukaryotes the components of ETC are located?

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What is operon? Compare two forms of operons. Compare lactose and tryptophan operons.

Operons are unique to bacteria usually. Several genes under one control. Multiple genes with one control mechanism. You make one big mRNA The operon usually controls sugar metabolism. Need an enzyme to bring the lactose in, need the enzyme to break down the lactose so there are multiple enzymes needed to break down this molecule. You just break it down and use it as part of the system, just like glucose, like a carbon or energy source. -the sequence of bases coding for one or more polypeptides along with the promoter and operator or activator binding sites -the lac operon is an example of negative transcriptional control of inducible genes. Inducible operon- The lac Operon -has two sites where the regulatory molecules bind that affect the RNA polymerase binding. We do all of this to manipulate of the RNA polymerase can bind or not. The CAP site and operon are upstream from the gene in the promoter region. -inducible operon. Usually off unless you turn it on. -contains genes needed to catabolize lactose -positive and negative control -negative control -regulated by lac repressor which binds to operator. Lac repressor binds to the operator and manipulate the RNA Polymerase to bind to the promoter region. -presence of lactose to lac repressor -allolactose can bind and prevent the lac repressor from binding to the operator site. -positive control -regulated by catabolite activator protein (CAP) which binds to CAP site. -controlled by level of glucose. Glucose is sugar of choice. Always use this first. when it's done, then use lactose When lactose is present, the lac repressor is inactive and cannot bind to operator region- this allows RNA polymerase to bind and initiate transcription, when lac is not present, lac repressor is active and binds. Repressible operon- trp operon Repressible operon produces something. It synthesizes a pathway- like amino acid synthesis. You usually need this, repressible operon is usually on unless deliberately turned off. Trp operon by tryptophan and the trp repressor. Tryptophan is amino acid, and is always needed. Low tryptophan levels, transcription of the entire trp operon occurs. When there is no operon, the corepressor is inactive and cannot bind to operator region. So RNA polymerase binds to promoter and initiates the process. So you synthesize that amino acid. No tryptophan (or low), trp operon is on because the activator. When there is plenty of tryptophan, the repressor now binds to the operating region and prevents the RNA polymerase from binding and no transcription happens. In this case, the repressor protein is active and binding to operator region and prevents RNA polymerase to bind to promoter region and initiate the process. Tryptophan operon is also controlled by another mechanism called attenuation. When there is low tryptophan and you need it, the trp repressor is inactive and transcription moves on. When there is plenty of tryptophan, it binds to the trp repressor and tryptophan acts as a corepressor and prevents it from binding to the operator region, and prevents the synthesis- no transcription! This is associated with amino acid synthesis. Normally on, unless you turn it off. You turn it off when you have plenty of the product. When the product of the operon is available, the operon is off. But in the cased of inducible operon, when the substrate is available, it is on. But in this case when the product is available the pathway is off. There are different operons for different sugars Operator region is where the inducible molecules bind. Upstream from here (before this) is the promoter region). The inducible molecule is the lac repressor in this case. When the lac repressor is active, it binds to the operator region and inhibits transcription. When there is no lactose, the lactose repressor is active. When no lactose, lac repressor is active and binds to operator region and prevents RNA polymerase to bind to DNA and initiate transcription. These are all DNA binding molecules. they have unique structures. 2 examples of domains that a DNA binding molecule must have. Domain must have protein. Polypeptides sometimes have unique regions of the molecule which have specific structure. The 2 structures, the 2 domains that DNA binding molecules usually have, an example is called leucine zipper- several amino acids are binding together through the help of multiple number of leucine molecules binded together. Leucine zipper is a domain that some of the DNA binding molecules have. Another possible domain that they have is zinc finger. Here, several amino acids are bonded together through a zinc molecule. This is a privilege to mingle with the DNA. Some of the repressors, CAP molecules, have these in their structure. When there is no lactose, there is a molecule called lac repressor that is active and can bind to the operator region, and prevents the RNA polymerase to bind and initiate transcription. No transcription, no lactose genes. When lactose is present, allolactose is produced. This binds to the lac repressor and inactivates it. Allolactose prevents the lac repressor from binding to the operator region. Here, transcription goes on. No lactose, no genes. Lactose, genes. This is an inducible operon. Normally off unless deliberately turned on in the presence of substrate, usually associated with sugar metabolism (catabolism, not anabolism).

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Oxidation-reductions (redox) reactions: o Many metabolic processes involve oxidation-reduction reactions (electron transfers). o Electron carriers are often used to transfer electron donor to electron acceptor. o Transfer of electrons from a donor to an acceptor § Can result in energy release, which can be conserved and used to form ATP § The more electrons a molecule has, the more energy rich it is.

PMF, Proton gradient, Charge gradient, ATP Synthase

PMF: by creating holes in the cell membrane to allow protons back in, you are creating PMF. ATP synthase acts as a turbine that turns and generates electricity. The force that runs this turbine is the water that runs through it. As the proton pushes through the pump back into the cell (through ATP synthase) it turns it and gives it energy to produce ATP (produced by ADP to ATP). This is through oxidative phosphorylation. PMF to make ATP. Also flagella motility, and transporting molecules across membrane. PMF can be generated from energy from the light source or from chemical source. In either case It can be used for multiple purposes.

Define and give examples of amphibolic pathways

Pathway that can be used for catabolism and anabolism. Amphibolic pathway starts with glucose. Glucose to pyruvate, then pyruvate enters the krebs cycle (?? No, acetyl coA). From glucose to pyruvate can be embden-meyerhof pathway, pentose phosphate pathway, tricarboxylic acid (TCA) cycle. (entner-duodoroff pathway also goes from glucose to pyruvate but it is not amphibolic). Pentose phosphate pathway: an amphibolic pathway that can operate aerobically or anaerobically. Oxidation steps produce NADPH, which is needed for biosynthesis. It provides precursors for making nucleic acids. Ex 5 carbon sugars to make RNA or DNA. Doesn't really make ATP. Entner-Doudoroff Pathway- glucose and turns it into pyruvate. Yields 1 ATP, 1 NADPH, and 1 NADH. Example: reduced C6H12)6, convert to fully oxidized CO2 + H2O. NAD and FAD pick up the electrons and become the reduced NADH and FADH.

Compare light vs dark reactions in photosynthesis.

Photosynthesis - Energy from light trapped and converted to chemical energy - A two part process o Light reactions in which light energy is trapped and converted to chemical energy (energy is harvested and ATP is produced, NADPH is produced) o Dark reactions in which the energy produced in the light reactions is used to reduce CO2 and synthesize cell constituents (uses ATP and NADPH to produce glucose). CO2 is fixed (convert gas into solid, usable form) ex CO2 into glucose). N2 (nitrogen ga)s into NO3 in soil is nitrogen fixation. - Oxygenic photosynthesis- eukaryotes and cyanobacteria. Makes oxygen - Anoxygenic photosynthesis- all other bacteria. No oxygen is produced in the process (green sulfur bacteria, etc.) - Chlorophyll based phototrophy- light comes to the chlorophyll and the light is absorbed. The electron enters the ETC like other organisms (chemotrophs) and NADPH and ATP is produced. Then in dark reaction, CO2 is fixed and ATP is produced. - Bacteriorhodopsin-based phototrophy- doesn't use ETC. its like a pump. The pump produces PMF. Only some archaea can do this. o Some archaea use a type of phototrophy that involves bacteriorhodopsin, a membrane protein which functions as a light-driven proton pump o A proton motive force PMF is generated o An electron transport chain is not involved - Rhodopsin-based phototrophy- use light, don't use ETC but still produce ATP. - The light reaction in anoxygenic photosynthesis -H2O is not used as an electron source; therefor O2 is not produced. -only one photosystem involved -uses different pigments and mechanisms to generate reducing power. -carried out by phototrophic green bacteria, phototrophic purple bacteria, and heliobacteria.

Monocistronic/polycistronic transcription

Polycistronic mRNA is often found in bacteria and archaea (found in prokaryotes, not eukaryotes). Usually lots of genes doing the same or similar things are synthesized together. This is the concept of operon. Means several genes under one control all express at the same time or not at all if they are all needed for the same process. -contains directions for >1 polypeptide catalyzed by a single RNA polymerase -reaction similar to that catalyzed by DNA polymerase Monocistronic: in monocistronic, you have one mRNA, one gene, one polypeptide. This shows efficiency. You make one big piece of RNA and then chop it into the pieces that you need. Individual mRNA. This makes things go faster and more efficient. You don't need to produce genes when you don't need them.

Distinguish allosteric regulation and covalent modification

Post translational regulation of enzyme activity -two important reversible control measures -allosteric regulation -by another molecule, you affect the protein activity. When the protein is made and then you add something onto it -covalent modification -permanently modifying the protein Both can be reversed though.

What is meant by post-transcriptional and post-translational modifications? Give examples.

Post-transcriptional modification: so you have the initial RNA, the hnRNA, we have exons and introns and remove the introns by splicing. Add a cap, add a 5 prime cap to it, and a 3 prime end tail. This is a poly A tail. The cap helps the RNA polymerase come out into the cytosol/cytoplasm. Enzyme involved in splicing is the spliceosome- this is the enzyme that removes the intron. So overall, RNA polymerase is an enzyme that recognizes the base pair, etc. this happens in nucleus.

Define promoter, operator, upstream region, downstream region, TATA box, -35 region, and CAP binding site

Promoter is located at the start of the gene- it is upstream, before the gene starts, at the 5 prime end. (upstream is always toward the 5 prime end). -is the recognition/binding site for RNA polymerase. RNA is a big complex of enzymes with multiple polypeptides. Only polypeptide is sigma factor. This decides where the RNA polymerase is going to sit on the gene -functions to orient polymerase. Before the DNA starts there is a promoter region. This is where the RNA polymerase recognizes the gene. This area is not part of the gene, it is before the gene. It is not transcribing to RNA, but it is the beginning of the gene- this is where the RNA polymerase will recognize and bind to. The leader sequence starts right after the promoter region. There is a -35 and -10 region. Gene starts at the +1. 10 and 35 base pairs before the initial base for the gene. The RNA polymerase binds to the -10 -35 region and opens up the DNA (separates the two strands, unwinds). It uses the 3' 5' end as a template and synthesizes the RNA. These -10 -35 regions are the same for every gene, so we call them consensus regions. This means they don't change much. The -10 is sometimes called pribnow box, or TATA box. Control of Transcription Initiation by Regulatory Proteins -induction and repression occur because of the regulatory proteins- regulatory proteins are inducers and repressors. Inducible genes are usually catabolic genes involved in the breakdown of molecules. When the molecule is not available, this gene is off (so it doesn't break down what is not there). Inducers stimulate the transcription on inducible genes. -Some genes are inducible genes- usually off but when needed they turn on. Examples are some sugars, or lactose. -Repressible genes are normally on unless turned off deliberately. Repressible includes anabolic activities, like synthesizing amino acids. For example synthesizing molecules like amino acids. You always need them to make protein, but if that amino acid is coming from outside, is coming from you nutrients, you don't need to make them anymore, so you turn them off. Now they are temporarily off. This is for genes used in synthesizing things. When you need them you keep synthesizing them, but when you have them in your diet you turn them off. -these proteins either inhibit transcription (negative control) or promote transcription (positive control) -their activity is modulated by inducers, corepressors, and inhibitors. Inducers induce the inducible genes Corepressors bind to the repressor and either turn on or off the gene. Inhibitors- molecules that inhibit the synthesis of something Operatory region- an area on the promoter region. Imagine that this is a landing space. This is a part of the DNA that RNA polymerase is supposed to sit. RNA polymerase is a big complex molecule, multisubunits. It needs space to hug the DNA and open it up and initiate the process. Operator region is another region where regulatory molecules bind, and when it is occupied by other molecules, there is no room for the RNA polymerase to bind. Some promoter regions have another area where molecules can bind, called CAP (catabolic activator protein). Catabolic Activator Protein (CAP) -regulation of the lac operon by the lac repressor and CAP. -activated by cAMP- hunger signal. cAMP is when the glucose goes down, the cAMP goes up. Higher cAMP means active CAP protein. Active CAP protein can bind to CAP site on the DNA on the lactose operon, and then genes are expressed. -No glucose, yes lactose, gene/operon is on. -Lactose and glucose yes, lac repressor wants to turn this on, but since, CAP binding is not binded since this is low cAMP, CAP is inactive, lactose operon is off- no transcription. -No glucose and no lactose, the lac repressor binds to the operator and the lactose operon is off. -glucose yes, lactose no, lactose operon is still off because the lac repressor is still binding to the site. CAP molecules binding is a result of glucose, and this is catabolic repression. CAP molecule is a DNA binding molecule. Another example of a DNA binding molecule is helix-turn-helix. You have alpha helix molecules, then turn molecule, then alpha helix molecule again. This is another domain present in binding molecules. CAP binding is a helix-turn-helix molecule. Have to have a molecule that can hold onto the DNA. Double helix create saddle like grooves that DNA molecules can sit on and hug the DNA. Structure dictates function!

Describe proofreading process in replication. List the enzymes involved in this process

Proofreading is carried out by DNA polymerase III Removal of mismatched base from 3 prime end of growing strand by exonuclease activity of enzyme- by going backwards and removing the DNA. This activity is not 100% efficient

EXTRA

Pyruvate can go to lactic acid or alcohol. Products are organic acids or alcohol (ethanol). Also can make for example acetate, propionate, butanol, etc. Overall, take NADH back to NAD and usually only makes 2 ATP. Both prokaryotes and eukaryotes can do fermentation. Final electron acceptor is endogenous. Transfer electron to one of the products. The molecule will accumulate and prevent the cell from growing any further.

EXTRA

RNA in ribosome recognizes the mRNA. Also RNA in ribosome has enzymatic activity that forms a peptide bond between the amino acids. Ribosomes are made of proteins and RNA.

Describe ribosome structure, its role in protein synthesis. Role of different RNA molecules?

RNA is made of similar nucleotides, but there is a G, C, A, and instead of T you have U (uracil). Polymer of nucleotides Sugar is ribose Most RNA molecules are single stranded Three different types which differ from each other in function, site of synthesis (in eukaryotic cells) and in structure. mRNA- messenger RNA. Contains codon for amino acid. carries the codon for making protein. Goes into ribosomes, which is part of the ribosomes that synthesizes protein- protein maker machine rRNA- ribosomal RNA. Part of ribosome. controls interaction between ribosome and mRNA. Some have enzymatic activity.tRNA- transfer RNA. brings amino acid to the ribosome- to the complex so they can be put together to make protein molecule. Contains the anticodon. Codon and anticodon are complements every cell has ribosomes. Ribosomes are the protein maker, the machinery that makes protein. Ribosomes themselves are made of protein and RNA. The size and composition of ribosomes and in eukaryotes and prokaryotes is different. In prokaryotes: ribosomes are 70S = 30S and 50S subunits. In eukaryotes: 80S ribosomes = 40S + 60S -mitochondrial and chloroplast ribosomes resemble prokaryotic ribosomes. These have their own protein synthesis machinery. Theory of Endosymbiosis says that perhaps mitochondria and chloroplast have origin in prokaryotes, because they are like a complete system. They produce their own energy, own protein synthesis, they have their own DNA, and they are very similar to prokaryotes. -this 70S and 80S makes a big difference in terms of antibiotics. There are antibiotics that specifically affect the 70S ribosome, and not the 80S ribosome. Antibiotics is a natural compound made by some bacteria and fungi. They produce it to kill other bacteria and competition. -RNA in ribosome can have enzymatic activity. It can form the peptide bonds in polypeptides between the amino acids. (ribozyme) . the reaction is called transpeptidation. -ribosome not only binds to the mRNA, but also does the actual synthesis of the protein itself as well. The ribosome in RNA has enzymatic activity. They are ribozymes. These rRNA form peptide bonds between amino acids. They form bonding between the 2 amino acids. The difference between prokaryotes and eukaryotes make protein a main target of antibiotics. The cell wall of bacteria is made of peptidoglycan. Penicillin and penicillin-based antibiotics kill bacteria. Human cells don't have peptidoglycan, so penicillin and those antibiotics do not affect human DNA. And human doesn't have cell wall, so it won't affect humans. There are differences between human and bacterial ribosomes, and antibiotics target them differently- against bacteria and not humans. Some of the most important antibiotics are antiprotein synthesis. Initiation, termination, and elongation factors are involved in protein synthesis. Those antibiotics affect that.

EXTRA

RNA polymerase in bacteria recognizes the -10 -35 and that's what it binds to. It does this with the help of sigma factor- it recognizes this and initiates the process. Initiation allows the sigma factor to bind, allows the RNA polymerase to bind, and initiates the process to open up and unwind the DNA and start the process. One strand is 5 prime 3 prime- this is the coding strand. The 3 prime 5 prime is the template strand that we use to synthesize the coding strand, and DNA and RNA are the same except U and T and ribose and deoxyribose.

List the events occurs during initiation of replication with regards to OriC, helicase, topoisomerase, primer, ssDNA binding proteins,

Replication ends with 2 of the same strands, 2 identical copies. Then in transcription you make copies in the form of RNA (tRNA, rRNA, and mRNA) DNA binding proteins can interact with the DNA. All DNA binding proteins have domains- region of the protein that is capable of interacting with the DNA. Some examples of domains found in DNA binding protein are Zn-finger. This is a DNA binding domain that you can find in protein that interact with DNA. Leucine zipper- this is another DNA binding protein domain. We can identify proteins that are capable of binding with DNA. They all have similar regions that are capable of binding with DNA. Helicases, topoisomerases, and DNA polymerase III are part of the replisome. These enzymes are involved in unwinding and rewinding the DNA. Synthesizes DNA by 5 prime 3 prime activity. That's the polymerase activity. It also has 3 prime 5 prime exonuclease activity- you can go back and remove base pairs if you made a mistake. (DNA polymerase does both 3 prime 5 prime and vice versa) Replisome is a complex that, as it goes, they open up the DNA, synthesize the primer, synthesize the DNA, and keeps moving in one direction The lagging strand is synthesized in short fragments called okazaki fragments A new primer is needed for the synthesis of each okazaki fragment. SSB- Single stranded binding protein. They bind to the DNA to hold it, to separate it, because if not they'll go back together and reconnect. Have to hold the DNA open. Since there are 2 forks, and they go in different directions, we call this bidirectional DNA. DNA polymerase synthesizes DNA Termination of replication: -replication stops when replisome reaches termination site (TER) on DNA -topoisomerases temporarily break the DNA molecules so the strands can separate. They unwind the DNA and then wind it back.

Compare respiration and fermentation. Why anaerobic respiration and fermentation make less ATP than aerobic respiration?

Respiration - Can be aerobic or anaerobic. - Here, you take organic molecules (organic energy source) and electron source, and you go through central pathway to metabolism- which is glycolysis followed by krebs cycle. This process extracts electron and proton. These go to electron transport chain. Up to this point aerobic and anaerobic are exactly the same. Then, in aerobic, the final electron acceptor is oxygen. In aerobic it's organic or inorganic compounds such as sulfate, nitrate, carbonate, fumarate, etc. - Have to have a constant supply of final electron acceptor. So person always has to keep breathing. - The electron acceptor is exogenous (constant supply from the outside). - Final electron acceptor is oxygen Fermentation - In fermentation, you only go through glycolysis. - There is still an electron acceptor, but it is an endogenous compound- this means something within. One of the products of fermentation act as the final electron acceptor. You accumulate something. You don't have a supply of it outside. For example you accumulate lactic acid. For example if you put yeast in grape juice, in a few days you have alcohol. The acids and alcohol produced act as a final electron acceptor for you. - Final electron acceptor is different exogenous acceptors such as NO3-, SO42-, CO2, FE3+, or SeO42-

EXTRA

Reverse transcriptase- a DNA polymerase enzyme. It is RNA dependent. Regular DNA polymerase uses DNA as template, but this one uses RNA as template. Some bacteria has reverse transcriptase though, that takes a single stranded RNA (ss RNA), and converts it to a double stranded DNA. This is for replication- DNA is semiconservative- half of the DNA comes from the parent, half of the DNA is brand new DNA. The two double stranded DNA are antiparallel. G, A for one, C, T for other- complimentary. One always goes 5 prime 3 prime, the other 3 prime 5 prime direction.

What is the significance of having alternative sigma factors for Escherichia coli?

Sigma factor is part of RNA polymerase. It moves ahead of the RNA polymerase. It recognizes the promoter region. It goes ahead of the polymerase and guides the RNA polymerase to the promoter region. When it sees the promoter region, it binds to it. As the RNA polymerase comes along, it flags it down and says this is a promoter here, bind here. So first, sigma factor binds to and recognizes the promoter, followed by the rest of the RNA polymerase, the core part. The RNA polymerase binds to the -10 -35 region and opens up the DNA (separates the two strands, unwinds). It uses the 3' 5' end as a template and synthesizes the RNA. As soon as transcription starts, the sigma factors come off and leave. It moves on to the next gene. Replication and Transcription always goes 5' 3', mRNA is always produced 5' 3' direction. Translation (protein synthesis on mRNA) also goes 5' 3'. Transcription and translation are simultaneous in bacteria. Protein synthesis and RNA synthesis happen simultaneously, because they all move in the same direction. Protein synthesis happens in cytoplasm. E.coli sigma factors: Walks ahead of the complex and runs around and finds the promoter region and flags down RNA polymerase. Sigma factors initiate the process by finding the operon, and flags down RNA polymerase. Once the RNA polymerase is bound to the promoter region, the sigma factor takes off and finds the next. Sigma factor is the usher. It can control multiple seats, and multiple ushers work. Heat shock proteins can refold the protein and minimize the damage caused by heat. Each sigma factor controls a group of genes.

Phosphorelay system

Sporulation in Bacillus subtilis is controlled by a mechanism for altering enzyme (or other protein) activity that involves the transfer of phosphate from one molecule to another A set of proteins involved in the transfer of phosphate from one protein in the set to another. It is often used to regulate protein activity or transcription

Define the standard reduction potential. Why aerobic grow generates the highest amount of energy (ATP). How this value plays a role in organization of electron transport system. Compare Eo of aerobic and anaerobic respiration.

Standard reduction potential (Eo)- the difference between the number at the top and the number at the bottom of the electron tower (better electron donors to better electron acceptors). -equilibrium constant for an oxidation-reduction reaction -a measure of the tendency of the reducing agent to lose electrons -more negative E is better electron donor -the greater the difference between the Eo of the donor and the Eo of the acceptor, the more negative the delta G standard- the more free energy available.

EXTRA

TRANSCRIPTION: -RNA synthesis under the direction of DNA -RNA produced has complementary sequence to the template DNA -three types of RNA are produced -mRNA carries the message for protein synthesis -tRNA carries amino acids during protein synthesis -rRNA molecules are composed of ribosomes -catalyzed by a single RNA polymerase -produce a complementary RNA copy of the template DNA sequence -Bacterial RNA polymerases: -the core enzyme (5 polypeptides) catalyzes RNA synthesis -the sigma factor; no catalytic activity, helps the core enzyme recognize the start of genes -holoenzyme = -core enzyme + sigma factor -only a short segment of DNA is transcribed -promoter: site where RNA polymerase binds to initiate transcription (is not transcribed) -has specific, consensus consequences before transcription starting point and called the -10 (pribnow box) and -35 sites. These consensus regions don't change much.

EXTRA

Template to synthesize next strand is 5 prime 3 prime, then next one is 3 prime 5 prime. Example: 5' GCATTACC 3', next is 3' CGTAATGG 5' (complementary to it). Antiparallel. One is 3 to 5, other is the other way.

EXTRA

Termination of Protein Synthesis: UAA, codon that does not code for any amino acid. It is a stop codon. When you reach this codon, your polypeptide is sitting on the P site. On the A site there is nothing. So, the enzyme removes and releases the polypeptide and tRNA, the whole thing dissociates and then let go. When you reach a stop codon no amino acid, no tRNA comes. So, the polypeptide is released into the environment. -takes place at any one of the three codons -nonsense (stop) codons- UAA, UAG, And UGA. Release factors (RFs) -aid in recognition of stop codons -3 RFs function in prokaryotes -only 1 RF active in eukaryotes These factors are like ushers

Describe how Ames test detects carcinogens.

Test by FDA -based on observation that most carcinogens are also mutagens -tests for mutagenicity are used as screen for carcinogenic potential Have a bacteria that is missing something. Ex bacteria cannot produced histamine, have to add histamine for the bacteria to grow. They exposed this bacteria to different chemicals. If they are mutagens, they can cause change in the DNA of this bacteria. If the mutagen results in changes of the DNA, its called carcinogen- which causes cancer in humans. Uncontrolled cell division, which becomes tumor, which then is cancer. Can be benign or metastasized. Oncogene. The point of this is must test products. Makes the bacteria be in its presence for a while in high concentration. They have a culture of salmonella histidine auxotrophs. Plate the culture on 2 agar plates, and see if spontaneous revertants (normal), but need to see if revertants induced by the mutagen (nuch higher, chemicals change DNA of salmonella, and it can now produce histamine and grow. This is on a plate meant for the fact that they cant grow unless the revise and stuff. They were histamine negative before, now histamine positive.

Beta Oxidation pathway

The major pathway of fatty acid oxidation to produce NADH, FADH2, and acetyl coenzyme A. series of biochemical reactions in which the fatty acids are completely oxidized in the mitochondrial matrix; results in significant amounts of ATP being produced means by which fatty acids are degraded

EXTRA

The size and composition of ribosomes in eukaryotes vs prokaryotes is different.

Describe how enzymes are involved in chemical reactions: activation energy, lowering Eo

There are enzymes that catalyze reactions. Enzymes: -carry out reactions at physiological conditions -speed up the rate of a reaction to reach equilibrium quicker -act as protein catalysts -high specificity for the reaction catalyzed and the molecules acted on -increases the rate of a reaction with being permanently altered -substrates = reacting molecules -products = substances formed by reaction -some enzymes are composed solely of one or more polypeptides. -some enzymes may contain nonprotein components. -Apoenzymes- protein component of an enzyme. -cofactor- nonprotein component of an enzyme. Prosthetic group- firmly attached Coenzyme- loosely attached, can act as carriers/shuttles -haloenzyme = apoenzyme + cofactor Examples of enzymes: Hydrolase- in hydrolysis of molecules. Hydrolytic reactions to break down molecules. Lyase- similar to hydrolase, but not like hydrolysis. It means without using water. Ligase- putting things together.

EXTRA

Transcription and translation is coupled in prokaryotes. The significance is that no time is wasted between mRNA being made/synthesized, then processed, then stopped. It results in speedy growth for prokaryotes.

EXTRA

Transcription in bacteria: -polycistronic mRNA often found in bacteria and archaea -contains directions for >1 polypeptide catalyzed by a single RNA polymerase -reaction similar to that catalyzed by DNA polymerase. No overlap of genes. But can have 1 mRNA and multiple genes, multiple polypeptides. This is called polycistronic Transcription in Eukaryotes: -differs from bacterial transcription in several ways, for example, -eukaryotes have 3 major RNA polymerases -promoters differ from those in bacteria by having combinations of many elements -RNA polymerase II is a large aggregate, containing 10 or more subunits -catalyzes production of heterogenous nuclear RNA (hnRNA) which undergoes posttranscriptional modification to generate mRNA (hnRNA is the initial RNA, it has to be processed before it becomes a functional mRNA). This post-transcriptional modification changes hnRNA, processes it, trims it- removes the introns. Does splicing.

Describe and explain the significance of tRNA charging, attachment of an amino acid to a tRNA as a (proof-reading step in translation).

Transfer RNA (tRNA) and amino acid activation. tRNA allows the anticodon to know which amino acid to bring. tRNA has to be activated or charged- which means adding amino acid to tRNA. There are enzymes involved in this process. It is catalyzed by aminoacyl-tRNA synthase. This is the enzyme that adds amino acid to tRNA. It makes sure it adds the right amino acid corresponding to the anticodon. Anticodon is complementary to codon. This is a proofreading step. This means this enzyme is capable of adding the amino acid, makes sure it's the right amino acid, and if it's the wrong amino acid cut it off and replace it with the right one. This enzyme has it's own energy to add and remove, to charge. -at least 20 aminocyl-tRNA synthase. -each specific for single amino acid and for all the tRNAs to which each may be properly attached (cognate tRNAs). Match right tRNA with right amino acid

EXTRA

Translation Translation requires the assembly of the ribosome around the mRNA. Synthesis of polypeptide directed by sequence of nucleotides in mRNA. mRNA contains the codons and always goes 5 prime 3 prime on the mRNA. It has the triplets for making protein (triplets are the 3 base pairs). Direction of synthesis N-terminal to C-terminal. Ribosomes sit on the RNA and move 5 prime to 3 prime. As the polypeptide comes out, the amino acid comes out, the N-terminal comes out first. The process of transcription and translation is simultaneous here. There is no post transcriptional modification in bacteria, so mRNA is put together, is sandwiched by ribosome, immediately as it comes out. Also, can have multiple ribosomes jump in. as one ribosome moves forward, it makes room for another, so you can have multiple proteins made at the same time. This is called polyribosome. This coupled transcription and translation results in fast and speedy growth for bacteria. Bacteria have a much faster generation time than eukaryotes. Ribosome is the site of translation -polyribosome (polysome (where multiple ribosome can work on the same mRNA)- complex of mRNA with several ribosomes. This is all because everything has a direction. Everything moves in the 5 prime 3 prime direction.

Regulation of transcription by Antisense RNA

Translation Regulation by Antisense RNA: we make tRNA, rRNA, and mRNA. sRNA- small RNA molecules (40-50 hundred base pairs, 100 base pairs). These sRNA molecules are involved in regulatory mechanisms. Some of these can act as antisense RNA. In order to synthesize coding strand, you use complementary strand as template, and you make your RNA. It is exact copy of complementary (coding..?) strand, except there is a U for every T and ribose for deoxy ribose. If use coding strand as template, you make antisense RNA- which is complementary to mRNA- may bind to it. Antisense RNA prevents ribosome from binding to mRNA. Prevents shine-delgarno sequence, and 16S RNA of ribosome to find each other and initiate translation. It prevents ribosome from binding to RNA. Regulates translation and binding to mRNA Shine delgarno is in mRNA. 16s RNA is in ribosome. They must form an RNA RNA hybrid to initiate the translation. Small RNA molecules can prevent this from happening and doesn't allow the translation to happen. This is by antisense RNA. It is complementary RNA upstream region. Keeps ribosome from binding to the area.

EXTRA

Viruses have hundreds of genes, bacteria have thousands of genes, eukaryotes have tens of thousands of genes.

EXTRA

We have ADP, ATP, PEP (intermediate of glycolysis, used in PTS, phosphate transport system- used to bring glucose in), 1, 3 bisphosphate glycerate (also from glycolysis. These reactions are exergonic. They have high energy phosphates that can be used to drive other reactions. The basis of all of this is redox reactions. Also some from krebs cycle.

Define: semi-conservative replication, antiparallel, complementary strand, major/minor grooves

When the ladder of DNA twists, major and minor grooves are made. Major and minor grooves form when the two strands twist around each other. The major grooves can actually grab on the DNA. Have to be able to grab and unwind so we can do the replication or transcription. Grooves are important in the interaction between DNA and outside molecules. Proteins are made of polypeptides, which are made of amino acids. Polypeptides have to be built in a way that allow them to interact with DNA- not everything can interact with DNA because it is so important. Proteins have a region of them called domain- there are domains that can interact with the DNA, we call these proteins DNA binding proteins. The job of replication is to be able to separate the DNA, open the DNA, make 2 copies of it, and it goes to daughter cell. DNA is supercoiled. Bacterial DNA is circular, like a double banded rubber band. Something has to sit on the ORI (origin of replication) on the DNA to open it up and replication can occur there. Easily open because held together by hydrogen bonds. It opens up both direction and replication occurs and when the ends meet they are separated. Replication always goes 5 prime 3 prime direction. It starts at the ORI and goes both directions- bidirectionally Antiparallel- one is 5 prime 3 prime, the other is the other way. By knowing one you can tell the other. If the complementary strand is GCAT, then you know the antiparallel. This replication is called semi-conservative. This means when the DNA replicates, half is old DNA and half is new DNA.

Shine-Delgarno sequence/coding region

Where the rRNA in ribosome binds to the mRNA to initiate the process for translation, for protein synthesis, makes sure that ribosomes recognize the first codon on the mRNA. Shine-Delgarno sequence on mRNA is complementary to 16S RNA in 30S ribosome.

Regulon

a set of separate genes or operons that contain the same regulatory sequences and that are controlled by a single type of regulatory protein multiple operons controlled by the same regulatory protein genes or operons controlled by a common regulatory protein

- Endergonic vs. exergonic reaction

ender- products have more Potential energy than reactants (took in energy). exer- potential energy of reactants is released Endergonic: unfavorable. Requires input of energy. Delta G standard is positive. Exergonic: favorable spontaneous reaction. A and B go together to C and D without much of a push from the outside. Delta G is negative. The more negative, the more energy you have available outside this reaction. So in nature we couple these reactions. Put an endergonic with an exergonic, and the excess energy from the exergonic drives the endergonic.

Chemiosmosis theory

holds that ATP is created during cellular respiration when ATP synthase uses the PMF to phosphorylate ADP Says these electron carriers enter the electron transport system, the electron from them are extracted, the proton from them is also extracted, and then put on the system through the electron transport chain, and there are a bunch of molecules and the proton and electron move through the system and are eventually handed to the final electron acceptor. ETC is located in the membrane. As electron passes through ETC to final electron acceptor, proton is pumped out of the membrane, and it generates the force, the proton concentration gradient outside, making molecules want to come back in. charge gradient. Overview: -ETC organized so protons move outward from the mitochondrial matrix as electrons are transported down the chain. -proton expulsion during electron transport results in the formation of a concentration gradient of protons and a charge gradient -the combined chemical and electrical potential difference make up the proton motive force (PMF). Oxidative phosphorylation: This is substrate level phosphorylation Process by which ATP is synthesized as the result of electron transport driven by the oxidation of a chemical energy source -only 2 ATP molecules synthesized -ATP produced from PMF is oxidative phosphorylation. Max of 38 atp through aerobic, only 2 through substrate level

Protoplast fusion

is accomplished by enzymatically removing the cell walls of organisms of two strains and mixing the resulting protoplasts Removing cell walls from two bacteria allows them to fuse The fusing of two protoplasts from different plant species that would otherwise be reproductively incompatible

List several possible end products of fermentation

lactic acid, propionate, isopropanol, acetate, butanol, ethanol, butyrate, and 2,3-butanediol Fermentation uses an endogenous electron acceptor -usually an intermediate of the pathway used to oxidize the organic energy source (ex pyruvate). Fermentation does not involve the use of an electron transport chain nor the generation of a proton motive force. ATP synthesized only by substrate-level phosphorylation (no oxidative phosphorylation, no PMF). Here, energy from a high energy compound is transferred to ADP to make ATP.

EXTRA

mRNA is made as one polypeptide, but can separate from one another can have multiple RNA made and then cut and processed. rRNA is also this same way. rRNA is part of the ribosome. You usually need each piece of ribosome one to one ratio. Need one 16 S, one 23 S, one 5 S. you make all 3 pieces together. Spacers have to be removed between them and processed. have promoters, readers, coding spacers. Spacers were removed during the maturation process. There are some modifications to tRNA as well- mostly just pieces have to be removed. rRNA genes have promoter, leader, coding, spacer, and trailer regions.

Modulon

set of operons or regulons that are collectively regulated in response to changes in environmental conditions but may be under the control of multiple regulatory molecules operon network under control of a common global regulatory protein but individual operons are controlled separately by their own regulators

True or False: A redox pair with more negative reduction potential will spontaneously donate electrons to a pair with more positive potential. TRUE True or False: The more positive the reduction potentials, the greater the affinity for electrons.TRUE

true and true