Cell Biology Final Exam
sphingolipid biosyntehsis
sphingosine made in ER polar head added in golgi some of these are made in mitochondria, too.
Osmium tetroxide is commonly used to a. stain specimens for light microscopy. b. coat specimens for metal shadowing electron microscopy. c. stain specimens for transmission electron microscopy. d. measure the Ca2+ concentration inside living cells.
c
Parietal cells acidify the stomach contents while maintaining a neural cytosolic pH by a. exporting "excess" cytosolic OH- as HCO3-. b. exchanging HCO3- for Cl-. c. preserving electroneutrality by accompanying the movement of each Cl- ion into the stomach lumen by a K+. d. all of the above.
d
Peripheral membrane proteins a. contain many hydrophobic amino acid residues. b. contain membrane spanning domains. c. have covalently attached lipid or fatty acid anchors. d. may noncovalently interact with phospholipid heads.
d
Why do you think that RNA, rather than DNA, primers are employed in the DNA replication process?
DNA polymerase lays each base tightly as it synthesizes the growing strand. If there were no primer, there would be no 3' -OH group for DNA polymerase to "see," and therefore no indication to begin attaching the next nucleotide to replicate the DNA. RNA polymerase, on the other hand, has no 3' -OH requirement to begin adding bases;
Describe down regulation of GPCRs
-decreased affinity of receptor for its signal -enhanced GTPase activity of G protein -phosphodiesterase activity -homologous/heterologous feedback repression -intracellular sequestration of receptors
What is the meaning of "quality control in the ER?"
"Quality control within the ER" refers to the need for proteins to be properly modified and folded before they can exit from the ER to the Golgi apparatus. Improperly modified and folded proteins are typically translocated into the cytosol for degradation.
southern blot northern blot in situ hybridization microarray
- DNA - RNA - detection of DNA/RNA in permeablized cells - allows for evaluation of many genes at the same time (green = express down; red = hot! up!)
four types of protein modifications are:
1) glycosylation - uses dolichol phosphate. starts in cytosol and flips into ER. 2) disulfide bond 3) folding 4) proteolytic cleavages (can happing in ER, golgi, or secretory vesicles)
sodium-linked glucose transporter
2 na+ in with 1 glucose in. once all three are docked, conformational change. see saw action. spit out. reset.
The H+/K+ ATPase in the apical surface of parietal cells exports H+ and imports K+. How is the buildup of excess K+ ions in the parietal cell cytosol prevented?
A K+ channel in the apical surface of the parietal cell removes the excess K+. The outcome of transepithelial transport is the summation of processes mediated by a number of transport proteins.
Describe in general terms how the muscle Ca2+ ATPase pumps Ca2+ ions from the cytosol into the sarcoplasmic reticulum.
A P-class Ca2+ ATP located in the sarcoplasmic reticulum (SR) membrane of the skeletal muscle pumps Ca2+ from the cytosol into the lumen of the SR. The protein is the major integral membrane protein in SR membranes and hence can be readily purified. Ca2+ pumping from the cytosol to the SR involves a series of ordered steps and two conformational states of the pump. These are termed E1 and E2. In the E1 state, the pump binds two cytosolic Ca2+ ions and ATP. The ATP is cleaved to ADP with phosphorylation of an aspartic acid with a high-energy acyl phosphate. Next, a reduction in the energy state of the aspartate acyl phosphate produces a conformational change in the protein from the E1 to E2 state. In the E2 state the affinity of the pump for Ca2+ is 1000-fold less and both calcium ions are released into the SR lumen. With dephophorylation of the pump, there is a second conformational change and the pump is again in the E1 state.
Describe how chloride-bicarbonate antiporter facilitates carbon dioxide transport by blood.
A chloride-bicarbonate antiporter is needed to facilitate carbon dioxide transport in the blood. As erythrocytes drop off oxygen to systemic tissues, they pick up carbon dioxide via passive diffusion. Due to the presence of carbonic anhydrase in erythrocytes, CO2 gets combined with OH- to form water soluble HCO3-. The AE1 antiporter, activated by a rise in cellular pH, exchanges the rising quantity of HCO3- (equivalent to OH- and CO2) for extracellular Cl- (thereby preserving electroneutrality). The movement of Cl- down its gradient from the exterior of the cell into the interior facilitates the expulsion of HCO3- down its own concentration gradient;
An enhancesome is a large nucleoprotein complex bound to an enhancer element. This complex is formed by the cooperative assembly of transcription factors to their multiple binding sites in an enhancer.
A zinc finger is a structural motif found in DNA binding domains; it consists of a short length of the polypeptide chain folded around a Zn2+ ion. The two basic classes of zinc finger domains are the C2H2 and C4 structures. The C2H2 zinc finger domain consists of two cysteine (C) and two histidine (H) residues bound to one Zn2+ ion. The C4 zinc finger contains four cysteines bound to one Zn2+ ion. The three-dimensional structure of the zinc finger forms a compact domain, which can insert its helix into the major groove of DNA.
What is the basic structural organization of an ABC superfamily transport protein?
All members of the ABC superfamily of proteins contain two transmembrane domains and two cytosolic ATP-binding domains, which couple ATP-hydrolysis to solute movement. The transmembrane domains associate with each other. A transmembrane domain and associated cytosolic domain together form what may be thought of as a monomer. In nature, these two domains may be present in one polypeptide and the overall ABC transport protein then consists of two polypeptides. Alternatively, the four domains may be present as separate subunits or in some cases fused into a single protein.
What is an enhancesome?
An enhancesome is a large nucleoprotein complex bound to an enhancer element. This complex is formed by the cooperative assembly of transcription factors to their multiple binding sites in an enhancer.
Describe how aquaporins facilitate the movement of water across the plasma membrane.
Aquaporins are water-channel proteins that specifically increase the permeability of biomembranes to water. The level of aquaporin 2 is rate-limiting for water transport by the kidney and is essential for resorption of water in the kidney.
How does the wavelength of the light used to illuminate a specimen affect the ability to resolve objects within the specimen?
Because the limit of resolution is given by D = (0.61(/(N sin (), shorter wavelength light (e.g., blue) will provide better resolution than longer wavelength light (e.g., red).
Describe the patch-clamping technique. Why do investigators often use frog oocytes in their patch-clamping investigations?
By pressing a special electrode against a "patch" of plasma membrane and forming a tight seal, an investigator can "clamp" the voltage (or current) at a constant value and study the opening, closing, regulation, and ion conductance of a single ion channel. The technique can be used on whole cells or isolated membrane patches and there are advantages to using one over the other. Frog oocytes are often used in patch-clamping experiments because they are readily available and large enough to microinject with in vitro transcribed mRNAs encoding channel proteins, which will be expressed on the cell surface. Since frog oocytes normally do not express any channel proteins of their own, only the channel protein expressed from the microinjected mRNA will be present, allowing it to be studied in isolation.
two transporters that are activated at low pH help maintain the cytosolic pH in animal cells close to 7.4 despite metabolic production of carbonic acid and lactic acid. explain
Cl-/HCO3- antiporter is activated at high pH. it exports excess OH- inthe form of carbonic acid.
Rough endoplasmic reticulum can be separated from smooth endoplasmic reticulum by differential centrifugation. What is the basis for this fractionation?
During cell disruption, the endoplasmic reticulum fragments and reseals into small vesicle-like compartments termed microsomes. Microsomes derived from the rough endoplasmic reticulum contain ribosomes and these ribosomes provide additional mass, which allows separation from microsomes derived from the smooth endoplasmic reticulum.
What is the role of substrate-level phosphorylation in glycolysis?
During glycolysis, substrate level phosphorylation is used to synthesize ATP. This process occurs twice during glycolysis and involves the transfer of a high-energy phosphate group (from 1,3-bisphosphate or phosphoenolpyruvate) to ADP.
What is the functional difference between enhancers and promoter proximal elements?
Enhancers can stimulate transcription from a promoter tens of thousands of base pairs away. In contrast, promoter-proximal elements are located 100 to 200 base pairs upstream of the start site and usually lose the ability to stimulate transcription from a promoter when moved only several tens of base pairs away.
Having misfolded soluble or secretory proteins in the RER contributes to what investigators call the "traffic jam," a scenario associated with a number of human diseases where the normal transport of proteins is blocked by these abnormal proteins and protein complexes not getting to their correct site and being able to function properly. Briefly describe how the cell overcomes this particular traffic jam by exporting the misfolded proteins out of the RER into the cytosol, where they are degraded in the proteasome.
Essentially, the misfolded proteins have N-linked carbohydrate chains that are trimmed by the enzyme a-mannosidase. Once trimmed, these proteins are recognized by the lectin-like protein EDEM and/or OS-9, which targets the protein to an ER-associated degradation or ERAD complex, which serves as a type of channel needed to export the protein into the cytosol. Once in the cytsol, these proteins are subjected to enzymes that eventually target them to the proteasome for degradation.
Both ethanol and glycine are small molecules of approximately equal molecular weight. However, ethanol is much more membrane permeable than glycine. What accounts for the large difference in membrane permeability between ethanol and glycine?
Ethanol is a small alcohol; glycine is a small amino acid. Ethanol is hydrophobic. Glycine is hydrophobic. Glycine, like all amino acids, is a zwitterion. At neutral pH, the amino group of glycine is positively charged and the carboxyl group is negatively charged. Charged groups are impermeable to lipid bilayers.
nuclear export
Export cargo proteins must contain a nuclear export signal, which is leucine-rich. The protein exportin binds to the nuclear export signal, and also binds to RanGTP. This triplex may diffuse through the nuclear pore channel by interacting the FG repeats in the pore. T The GAP (GTPase activating protein) found along with the cytoplasmic pore filaments causes the hydrolysis of GTP on Ran, causing Ran to dissociate. The cargo also releases from exportin1. Exportin1 and RanGDP both independently diffuse back into the nucleoplasm through the nuclear pore.
ATP synthase is composed of two oligomeric proteins, F0 and F1. What is the function of each protein complex and where is each found in mitochondria?
F0 is a proton-channel protein and the F1 complex is an ATPase running in reverse. F0 in found in the mitochondrial inner membrane and F1 is associated with F0 on the matrix face of the inner membrane.
Describe how unesterified fatty acids unlinked to CoA are able to move through the aqueous environment of the cytoplasm. Also, describe how are cholesterol and phospholipids transported between organelles.
Fatty acid binding proteins (FABPs)--small cytosolic proteins--facilitate the movement of free fatty acids in the cell. These FABPs contain a hydrophobic pocket lined by beta sheets, where the fatty acid can interact. Cholesterol biosynthesis begins in the cytosol, and is completed by enzymes in the ER membrane. Cholesterol, like free fatty acids, is a hydrophobic molecule that would not be able to move efficiently in the aqueous cytosol. Cholesterol, when not bound for the secretory pathway, relies on lipid transfer proteins to carry it from the endoplasmic reticulum (where it is synthesized) to other membranes. Phospholipids biosynthesized by cells can incorporate free fatty acids, and the biosynthesis relies on enzymes attached to the cytosolic membrane of the ER, similar to cholesterol.
Describe how unesterified fatty acids unlinked to CoA are able to move through the aqueous environment of the cytoplasm.
Fatty acid-binding proteins contain a hydrophobic pocket lined by sheets. The ability of unesterified fatty acids to fit inside this pocket and interact non-covalently with the surrounding protein facilitates their intracellular movement.
What are the primary functions of the plasma membrane in all cells?
In both bacteria and higher eukaryotic cells, the plasma membrane provides similar functions. These include regulation of nutrient transport into the cell and release of metabolic waste to the extracellular environment. By allowing certain material to pass in and out of the cell, and preventing other material from passing in and out of the cell, the plasma membrane acts to set up a molecular environment inside the cell that is different from the extracellular environment.
Separation of most blood cells is difficult, if not impossible, to achieve because they have similar properties and/or densities. What procedure is used to separate T-cells of the immune system from the many other different types of white blood cells or spleen cells? What feature of the T-cell facilitates the isolation protocol?
Flow cytometry and fluorescence-activated cell sorting is used to select and isolate T cells away from the excess number of other cell types. Briefly, T cells, unlike other cells, express CD3 and Thy1.2 proteins on their cell surface. Antibodies specific to these markers are linked to a fluorescent dye and incubated with the pool of cells. The antibodies bind the cell-surface markers on the T-cell surface and when all cells are placed in the FACS machine, a laser is used to excite the dye causing it to fluoresce. Fluorescing T cells, selectively sorted from the non-fluorescing cells, can be cultured in vitro.
How are proteins imported into the thylakoids of chloroplasts?
For cytosolically synthesized proteins targeted to chloroplast thylakoids, multiple N-terminal uptake-targeting sequences are required. These act sequentially with the N-terminal, most targeting sequence being removed in the chloroplast stroma to expose the next targeting sequence. Four different pathways are known for the import of proteins from the chloroplast stroma into thylakoids. Three are for proteins imported from the cytosol and one is for proteins made in the chloroplast stroma. All pathways are variations on those used for export of proteins by bacteria. Examples of proteins homologous between bacteria and chloroplasts have been identified.
How can a uniporter, GLUT2, be sufficient to support the entry of glucose from intestinal epithelial cells into the bloodstream?
Glucose is actually present in a higher concentration inside the intestinal epithelial cell than in the bloodstream. Hence, a uniport in the basolateral surface of the intestinal epithelial cell can be effective in facilitating the entry of glucose into the blood stream. It is the two-Na+/one-glucose symporter located in the apical surface, facing the intestinal lumen, that as an example of secondary active transport generates the high glucose concentration inside the epithelial cell.
There are several enzymes involved in cholesterol biosynthetic pathway. Which of these is subject to feedback regulation? How does this enzyme sense cholesterol levels?
HMG-CoA reductase catalyzes the key rate-controlling step in cholesterol biosynthesis, the conversion of HMG CoA to mevalonate. The activity of this enzyme is tightly regulated. The enzyme is an endoplasmic reticulum (ER) membrane protein and has a number of transmembrane segments. Five of the transmembrane segments compose the sterol-binding domain. The sterol-binding domain senses the level of cholesterol in the ER membrane.
the type of restriction enzymes
I - cleave at random sites hella far (1000bp) from its recognition seq. II - cleave rec sequences themselves and these are about 4-6 bp long. III - cleave about 25 bp from recog seq.
Which of the following statement(s) regarding the transcription initiation and RNA Pol III is (are) true? a. ATP hydrolysis is not required for initiation. b. Pol III is responsible for synthesizing tRNAs and 5S-rRNA. c. The promoter elements of tRNA genes lie entirely within the transcribed sequence. d. all of the above
In prokaryotes, there is only one RNA polymerase, which consists of five subunits. In eukaryotes, there are three RNA polymerases. RNA polymerase I synthesizes ribosomal RNA; RNA polymerase II synthesizes messenger RNA; and RNA polymerase III synthesizes tRNA and other small RNAs. The eukaryotic RNA polymerases are more complex than the bacterial RNA polymerase. All three contain two large subunits and 12-15 smaller subunits. All three eukaryotic RNA polymerases contain subunits with some sequence homology to the E. coli RNA polymerase subunits (, , and ´).
Describe the role of sensor and response regulator in bacteria's two-component regulatory system.
In summary: Signal is detected by sensing histidine kinase receptor>> conformational change Histidine kinase auto-phosphorylates (transfers γ-phosphate from ATP to the histidine transmitter domain) Aspartic acid in the response regulator's receiver domain gets phosphorylated by histidine kinase >> activity of response regulator effector domain is altered. The response regulator can now act differently on DNA
How does Ran·GTP participate in the nuclear export of the HIV Rev protein?
In the nucleus, Ran·GTP binds to the nuclear export receptor exportin 1 and then to the leucine-rich nuclear export sequence (NES) in Rev. Exportin 1, in this trimolecular cargo complex, interacts transiently with FG repeats in FG-nucleoporins, allowing it to traverse the nuclear pore complex (NPC). In the NPC, the cargo complex encounters Ran·GAP, stimulating Ran to hydrolyze GTP, which causes it to reduce its affinity for exportin 1. Exportin 1 now loses its affinity for the NES, releasing Rev to the cytoplasm.
TEM
In transmission electron microscopy, resolution is around 2,000 times better than it is in light microscopy. The difference is that instead of using visible light, which has a longer wavelength range, electron microscopy can focus an electron beam emitted from a filament and accelerated by an electric field. In order to view the specimen, it must be stained with a heavy metal. During the staining process, the specimen is absorbed to an electron microscope grid that is then coated by a plastic. Then, heavy metal is applied to the sample and surrounding grid. The metal stain coats only the grid, leaving a "negative" exposed imprint of the sample. When the specimen is viewed, one can only see the lack of heavy metal stain, as the metal stain scatters incident electrons from the beam. This is why TEM is an indirect method of visualizing a sample. Additional methods can use TEM to visualize not just the shape of the sample, but also the topography with the metal shadowing technique. The sample is absorbed to a silicate mineral sheet (mica) and then coated when a heavy metal is evaporated from a source at an indirect angle to the specimen. Metal atoms accumulate on surfaces facing the metal source directly, leaving indirect "shadows" of deposited metal on areas affected less directly. [1] Finally, the biological sample is dissolved by acid and bleach before the metal replica is view by TEM.
How do integral proteins travel from the ER to the Golgi and from the Golgi to the plasma membrane?
Integral proteins travel in the membrane of transport vesicles that move from the ER to the Golgi and from the Golgi to the plasma membrane.
Ca2+ ATPase in SR
It involves conformational states: E1 and E2. The E1 state presents two cytosol-facing binding sites for Ca2+ located the membrane-spanning domain. The E2 state is the conformation in which those two binding sites face towards the lumen of the SR. When calcium and ATP bind in the E1 phase, an aspartate residue on the cytosolic face gets phosphorylated via the Mg2+ dependent hydrolysis of ATP. This causes the protein to conform to the E2 state, whereby calcium may be released into the lumen due to lowered affinity for its binding site. Once the aspartyl-phosphate bond is hydrolyzed, the protein reverts back to the E1 phase and more Ca2+ can bind to stabilize the protein in the E1 state.
During prolonged exercise, oxygen is scarce in muscle tissue. Under these conditions, muscle cells convert pyruvate to two molecules of lactic acid. What happens to the lactic acid that is generated in this way?
Lactic acid is secreted from muscle cells into the blood stream. Some is taken up from the bloodstream by the liver, where it is either reoxidized to pyruvate and then metabolized to generate energy and CO2 or it is converted back to glucose and stored as glycogen in the liver. Some of the lactic acid is metabolized to CO2 by the heart.
How do animal cells maintain membrane fluidity, and hence membrane function, in response to decreased temperature?
Membrane fluidity normally decreases with decreasing temperature and the bilayer becomes more gel-like. To maintain sufficient fluidity and hence function, animal cells could increase the ratio of unsaturated to saturated phospholipids in the membrane.
Fixatives such as formaldehyde are routinely used in certain types of electron microscopy and light microscopy. However, fixatives may introduce complications in analysis of the resulting images. What problems may result from using fixatives?
Most fixatives are cross-linking agents that immobilize proteins and other cellular molecules. As part of the fixation process, samples are often also dehydrated. Such chemical changes may alter the normal structure and spatial relationship of cellular components, and this must be taken into account when interpreting images of fixed specimens.
muscarinic acetylcholine receptors
Muscarinic acetylcholine receptors in heart muscle are a type of GPCR responsible for slowing heart rate upon activation. The GPCR is coupled to a Gai protein--inhibiting its effector protein--through the action of the GBy subunit, which is the subunit responsible for interacting with the effector protein--a K+ channel. The following occurs: -GPCR is bound by acetylcholine, =G protein to interact with it. GDP dissociates from the Gai subunit and gets replaced by GTP-- this allows the Gai subunit to dissociate from by the GPCR and the GBy subunit. GBy subunit stimates effector K+ channel--and in doing so, it opens the K+ channel. This channel remains open until the GTP on Gai-GTP is hydrolyzed to GDP.
What are the general features of an N-terminal signal sequence that targets secretory proteins to the ER?
N-terminal signal sequences targeting proteins to the ER are 16 to 30 amino acids in length and have a hydrophobic core of 6 to 12 amino acids. Preceding the core is one or more positively charged amino acids. Otherwise, N-terminal signal sequences have little in common.
Most fixatives are cross-linking agents that immobilize proteins and other cellular molecules. As part of the fixation process, samples are often also dehydrated. Such chemical changes may alter the normal structure and spatial relationship of cellular components, and this must be taken into account when interpreting images of fixed specimens.
Normally, antibody labeling can be used in tandem with electron microscopy to visualize proteins in thin sections. Cells are lightly fixed to avoid denaturing the epitopes on the desired protein and, after freezing, the tissue is sectioned at very low temperature. The tissue is thawed and a specific antibody to the desired protein is applied as in immunofluorescence microscopy. To detect the antibody, however, the tissue is incubated with electron-dense gold particles attached to protein A, a bacterial protein that binds the Fc segment of all antibodies. These gold particles are seen at the level of the electron microscope, however, you would not be able to see lamins in E. coli because bacteria do not contain membrane-bounded organelles and lamins are a component of the nuclear envelope.
basic components of a plasmid vector?
ORI - origin of replication AMPr gene - for selection Polylinker - where you can splice in your gene via any of several restriction sequences.
peroxisome import?
Peroxisomes are small organelles with a single membrane that perform oxidation reactions. Most peroxisomal matrix proteins contain a C-terminal PTS1 peroxisomal targeting sequence, recognized by Pex5, which binds to it.
glycolysis regulation
Phosphofructokinase-1 (PFK1) is the rate limiting enzyme of the glycolytic pathway, PFK1 is activated by AMP. Fructose 2,6-bisphosphate is another important activator of PFK1. ATP allosterically inhibits PFK1, and yet remarkably is also the substrate of PFK1. ATP has two binding sites on PFK1, the catalytic site and the allosteric site. ATP has a lower Km (and thus a stronger affinity) for its catalytic site than it does for its allosteric site.
What is the approach that plants use to respond to differences in osmotic pressure between the inside and outside of the cell?
Plants have rigid cell walls. The normal concentration of solutes is higher inside the plant vacuole than in the cytosol. Likewise, solute concentration is higher in the cytosol than in the extracellular space. Hence, the plant cell presses against the cell wall and is retained within the cell wall.
During in vitro translation of mitochondrially targeted proteins, when must mitochondria be added for import of proteins synthesized on cytosolic ribosomes?
Proteins are imported into mitochondria post-translationally. Therefore, although mitochondria can be added during the translation process and import will occur, cotranslational presence is not a requirement as it is for import into the ER. The mitochondria can be added post-transtranslationally.
In the absence of targeting information, what is the default location of proteins synthesized on cytosolic ribosomes?
Proteins synthesized on cytosolic ribosomes that contain no information for targeting to organelles diffuse throughout the cytosol.
how proteins imported into thylakoids?
Proteins to be imported contain two important sequences: a stromal-import sequence on the N-terminus of the protein, and an adjacent thylakoid targeting sequence. can be SRP dependent.
What experimental evidence supports the fluid mosaic model of biomembranes?
Results from fluorescent recovery after photobleaching (FRAP) experiments have demonstrated the two-dimensional movement of membrane components and allow quantitative measurement of the extent of membrane fluidity.
sanger seq
Sanger sequencing of DNA begins with the double stranded DNA of interest. These molecules are first denatured into a single strands that can be selected for sequencing. The DNA is mixed with a radioactively labeled DNA primer complementary to the 3' end of the strand of interest, DNA polymerase, four types of deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) and then a very small amount (about 1% relative to the other nucleotide building blocks) of one of any of the four types of dideoxynucleoside triphosphates (ddNTP), such as ddATP.
In multipass membrane proteins synthesized in association with membrane-bounded ribosomes of the rough ER, signal-anchor and stop-transfer anchor sequences alternate. What do these sequences do?
Signal-anchor sequences cause insertion of internal segments of the protein into the ER membrane; stop-transfer sequences cause the transfer of the protein across the membrane to stop. The alternation of the two produces a protein that loops in and out of the membrane multiple times.
Propose a rationale for why the coupling of the import of amino acids or sugars into cells is typically to Na+ ion import.
The Na+/K+ ATPase establishes and maintains large differences in Na+ and K+ distributions across the plasma membrane. The concentration of Na+ is low inside cells and high outside. The membrane potential is inside-negative. The import of Na+ is both concentration and membrane-potential favorable and can therefore drive the import of amino acids or sugars. The exact opposite is true for K+. Coupling to Na+ movement is accomplished by symports.
Explain why ATP-powered proton pumps cannot by themselves acidify the lumen of the lysosome.
The V-class proton pump responsible for acidifying the lumen of the lysosome, where the net movement of electric charge occurs during transport, is electrogenic. As each proton is pumped into the lumen, it leaves behind a negatively charged ion in the cytosol. Positive and negative ions attract each other across the membrane of the lysosome, which causes an electric potential. Soon, pumping leads to a buildup of protons in the lumen, which repels other protons. At this point, a significant transmembrane proton concentration gradient cannot be established. Thus, to generate an acidic environment necessary to breakdown components imported into the lysosome, proton transport must be accompanied by an equal number of anions, in this case in the form of Cl-, into the lumen.
What evidence suggests that all of the P-class ion pumps evolved from a common ancestor even though they now transport different ions?
The catalytic subunits of all P-class ion pumps share a similar sequence including a conserved aspartate residue that is phosphorylated during transport. The 3-D structures of the catalytic subunits are similar for those P-class ion pump structures that are known.
When examined by fluorescent recovery after photobleaching (FRAP), certain integral membrane proteins are significantly less mobile than others. What accounts for this reduced mobility?
The decreased mobility of certain integral proteins is due to interactions with the cytoskeleton or the extracellular matrix.
Each cytochrome in the electron transport chain has a different reduction potential. What is the importance of these differences for electron transport?
The different reduction potential (or tendency to accept an electron) of the cytochromes in the electron transport chain allows these molecules to establish a unidirectional electron flow along the chain.
To what extent do peroxisomal matrix protein import and peroxisomal membrane protein import share the same machinery?
The fact that cells having mutations that give rise to Zellweger syndrome, a defect in peroxisomal matrix protein import, still have peroxisomal membranes (peroxisomal ghosts) with a normal composition of peroxisomal membrane proteins strongly indicates that the import machinery for membrane proteins is very different from that for matrix proteins. This situation is different from that for other organelles such as ER, mitochondria, and chloroplasts.
How are cholesterol and phospholipids transported between organelles?
The final steps in the synthesis of cholesterol and phospholipids take place primarily in the ER. Transport from there to other organelles is by poorly understood mechanisms. The transport is Golgi-independent. It is proposed to be either by membrane-limited vesicles or other protein-lipid complexes, direct contact between membranes, or by transfer by small, soluble lipid-transfer proteins. Some combination of these processes is likely important.
Describe how you would prepare liposomes from a biological membrane.
The first step would be to treat the biological membrane with an organic solvent such as a mixture of chloroform and methanol. This would dissolve phospholipids and cholesterol, but not proteins or carbohydrates. Once the dissolved lipids were separated from the proteins and carbohydrates, you would evaporate the solvent. The final step would be to disperse the residue (which consists of phospholipids and cholesterol) in water to form liposomes.
mitochondrial signal seq?
The general features of a mitochondrial matrix targeting sequence are 20-50 amino acids located at the N-terminus. The sequence is rich in hydrophobic residues, positively charged basic residues (arginine and lysine), and hydroxylated residues (serine and threonine). These sequences do not tend to possess negatively charged acidic residues.
ER signal seq?
The general features of an N-terminal ER signal sequence (for the secretory pathway) are 16-30 residues, 6-12 of which are hydrophobic. They contain one or more positively charged residues adjacent to the 6-12 hydrophobic residues (the hydrophobic core).
Propose a rationale for why the coupling of sucrose import into the plant vacuole is to the export of H+ ion into the plant cytosol.
The plant vacuole is acidified by proton pumps. Moreover, the proton pumps generate an inside-positive vacuolar membrane potential. The export of H+ into the cytosol is both concentration and membrane-potential favorable and can therefore drive the import of sugars (i.e., sucrose) into the vacuole. Coupling of sucrose import to H+ export is accomplished by antiporters.
attenuation of trp operon
The genes needed to create tryptophan are all contained in the Trp operon. In the absence of tryptophan, no repressor binds to the operator, and RNA polymerase is free to copy the transcript. Attenuation of transcription occurs in the leader region (the region preceding the genes codings for tryptophan-synthesizing enzymes) of the operon. In the leader sequence, there is a series of four palindromic segments. [3] Transcription stops if a terminating step loop is formed in the RNA via complementary base pairing occurs between segments 3 and 4. However, if complementary base pairing occurs between segments 2 and 3, then segment 3 is "occupied" and unable to pair with segment 4, so transcription may continue. What determines this? In E. coli, during transcription, a ribosome trails RNA polymerase and translates the growing mRNA strand into a chain of amino acids. If tryptophan is in low supply, the ribosome slows when it encounters two Trp codons in the mRNA, due to the fact that tRNAs tasked with delivering tryptophans (tRNATrp) are not widely available. Because the ribosome has stalled due to lack of materials, the 2-3 stem loop is able to form; thus, the 3-4 stem loop will not form, and transcription may continue.
GPCR for blood sugar?
The glucagon circulates to the liver, where it binds to a GPCR, stimulating the Gas protein, which in turn activates its effector protein adenylyl cyclase. synthesizes cyclic adenosine monophosphate (cAMP). cAMP is a secondary messenger. Once synthesized, cAMP may transduce a variety of physiological signals. When cAMP interacts with protein kinase A (PKA), which possesses two cAMP binding sites: CNB-A and CNB-B. When cAMP binds to both of these sites cooperatively---and the kinase activity of PKA is activated. Activated PKA activates phosphorylase kinase, which in turn activates glycogen phosphorylase, which cleaves glucose off the non-reducing end of glycogen, yielding glucose-1-phosphate. Glucose-1-phosphate may then feed into the glycolytic pathway for ATP production.
What is the function of the malate-aspartate shuttle?
The malate-aspartate shuttle functions to deliver the electrons produced during glycolysis and carried by cytosolic NADH across the mitochondrial inner membrane to the matrix, where the electrons serve to reduce matrix NAD+ to NADH. This matrix NADH can then donate electrons to the electron transport chain.
Each organelle has a specific function in the eukaryotic cell. What determines the function of each organelle?
The specific function of a given organelle is determined by the specific proteins present in that organelle.
What is meant by de novo formation of peroxisomes?
This is the concept that peroxisomes can arise from nonperoxisomal membranes. The peroxisomal proteins, Pex19, Pex3, and Pex16 are involved. The nature of the precursor membrane is unclear. "Peroxisomes are interesting organelles with a single membrane, and possess the talent of being able to import native proteins. Pex5 is one of the proteins the helps guide proteins into the peroxisome. Then it associates which many other Pex proteins so that it releases the import protein and goes out of the peroxisome. The peroxisome has a large complement of Pex proteins that facilitate protein import and insertion. Peroxisomes are formed in two ways: via budding from the endoplasmic reticulum (de novo), and also by dividing themselves. When forming de novo, they are created from immature membranes that lack the full complement of proteins. Once they possess all these proteins, they are "mature" and can divide themselves."
ubiquitin and proteasome
Three enzymes (E1, E2, and E3) coordinate to tag proteins with ubiquitin. At least four ubiquitin molecules must be added to the target protein. The initial ubiquitin is added to the target protein, and then additional ubiquitins are added to the initial ubiquitin. It is the 19S complex that "recognizes" the ubiquitin tag on the target protein with its Ub-receptors, binds to it and, using deubiquitinase enzyme, removes the ubiquitin proteins for release back to the cytosol. The 19S complex then (using ATP) feeds the protein into the 20S complex, where the protein is hydrolyzed into its component amino acids. A proteasome inhibitor could be used as a cancer-treated agent. Once type of cancer, multiple myeloma, results in the production of exceptionally high antibody-producing cells. Immunoglobulins are produced in such high quantities that the cancerous cell requires a well-functioning proteasome to degrade these immunoglobulins before they accumulate to toxic levels. By inhibiting the proteasome, one would allow the cancer to poison itself with its own product.
Why are bacteria often a poor choice for the production of proteins for therapeutic purposes?
Typically, proteins used for therapeutic purposes are secreted proteins in animals; disulfide bonds stabilize their structures. Disulfide-bond formation occurs spontaneously in the lumen of the ER but not within bacteria. With this realization, animal cells became the preferred choice for the production of such proteins.
Which one of the following is the best technique/approach to allow you to localize catalase in peroxisomes? a. a catalase monoclonal antibody and transmission electron microscopy b. platinum or gold and scanning electron microscopy c. FRAP and FRET d. all of the above
a
. An enhancer a. is a DNA element that stimulates transcription of eukaryotic promoters. b. binds to RNA polymerase and stimulates transcription. c. acts as a binding site for RNA polymerase. d. interacts with repressor proteins to enhance transcriptional repression.
a
. Glycosylation, a post-translational modification to proteins, occurs in the a. Golgi. b. proteasome. c. mitochondria. d. none of the above
a
3. How does binding of the lac repressor to the lac operator block transcription initiation? a. lac repressor binding blocks RNA polymerase from interacting with DNA at the start site. b. lac repressor binding induces a DNase that cleaves the DNA at the transcription start site. c. lac repressor binding causes a conformational change in RNA polymerase. d. lac repressor binding induces a protease that degrades the sigma subunit of RNA polymerase.
a
All of the following statements about the essential carboxy terminal domain (CTD) of RNA polymerase are true except: a. The CTD is present in RNA polymerase I, II, and III. b. The CTD can become phosphorylated. c. The CTD is critical for viability. d. The CTD of mammals contains more than 50 repeats of a heptapeptide.
a
Cholesterol mixes with phospholipids in a biomembrane because cholesterol molecules are a. amphipathic. b. steroid derivatives. c. entirely hydrophobic. d. phospholipid derivatives.
a
During ATP synthesis, protons move "down" their electrochemical gradient through a. the F0 complex of ATP synthase. b. the F1 complex of ATP synthase. c. a proton channel protein. d. CoQH2-cytochrome c reductase.
a
How does inhibition of the Na+/K+ ATPase increase the force of heart muscle contraction? a. It increases cytosolic Na+ and therefore decreases Ca2+ export. b. It increases cytosolic K+ and therefore decreases Ca2+ export. c. It decreases cytosolic Na+ and therefore decreases Ca2+ export. d. It decreases cytosolic K+ and therefore decreases Ca2+ export.
a
Ion channels achieve selectivity in transport through all of the following mechanisms except a. evolution from distinct and different parent proteins. b. divergent evolution from a single type of channel protein. c. low activation energy for selective passage of the dehydrated ion. d. use of P segments to form an ion selectivity filter.
a
Lipid droplets arise from the a. endoplasmic reticulum. b. plasma membrane. c. cytosol. d. exoplasm.
a
NADH-CoQ reductase and CoQH2-cytochrome c reductase each use the energy derived from electron transfer to transport ________ the mitochondrial matrix. a. four protons into b. two protons into c. four protons out of d. two protons out of
a
Post-translational translocation of some secretory proteins in yeast is powered by a. ATP hydrolysis by BiP. b. cAMP hydrolysis by cAMP phosphodiesterase. c. GTP hydrolysis EF-Tu. d. phospholipid hydrolysis by phospholipase C.
a
Which of the following is the correct order of binding of general transcription factors to initiate transcription at RNA polymerase II promoters? a. TFIID, TFIIB, Pol II, TFIIH b. PolII, TFIID, TFIIB, TFIIH c. TFIIB, PolII, TFIIH, TFIID d. TFIID, TFIIH, TFIIB, PolII
a
Which protein domains are found in nuclear-receptor family members? a. variable region, DNA-binding domain, ligand-binding domain b. acetylase domain, DNA-binding domain, ligand-binding domain c. variable region, acetylase domain, ligand-binding domain d. variable region, DNA-binding domain, acetylase domain
a
Which statement describes the mode of action of the ABCB1 transporter (the first eukaryotic ABC transporter to be recognized)? a. During transport, the ligand binding site is alternately exposed to the exoplasmic and the cytoplasmic side of the membrane. b. During transport, a conserved aspart residue is phosphorylated. c. This class of pumps transports only H+ ions. d. This transporter acts as a chloride channel.
a
to clone DNA you need
a vector (plasmid or viral) that carry the DNA> cell can replicate with that DNA.
In mitochondria, the proton-motive force is due largely to a. a voltage gradient across the outer membrane. b. a voltage gradient across the inner membrane. c. a pH gradient across the outer membrane. d. a pH gradient across the inner membrane.
b
resting potassium channel
always open. let K+ leak out. specificity for K+ given by coordination of selected ion with the 8 carbonyl O atoms = selectivity filter ions move through single file.
acidification of stomach lumen
apical mem has H+/K+ ATPase. H+ gets dumped into stomach, and K+ into the cell. then K+ exits on same side through the K+ channel H20 split into H+ and OH- to donate the secreted H+. so the OH- builds up. that gets sent out into the blood through the HCO3 antiporter with Cl- (maintains cells charge). (* CO2 from blood entered the cell and comnbined the OH-_
. __________ is (are) composed of a series of compartments that function to sequentially modify proteins and lipids. a. The endoplasmic reticulum b. The Golgi apparatus c. Peroxisomes d. Vacuoles
b
A leucine zipper motif contains a. a stretch of five leucine residues in a row. b. a leucine residue at every seventh position. c. a leucine residue complexed with a zinc ion. d. an alternating leucine-alanine-proline structure.
b
A myeolma cell is best described as a. a precursor cell that gives rise to gametes. b. an immortal immune cell that cannot produce . c. a self-renewing stem cell. d. a and c
b
All the following proteins interact with exposed amino acids during protein folding in the ER except a. BiP. b. calnexin. c. PDI. d. prolyl isomerase.
b
Although many types of vesicles are similar in size and density, it is possible to isolate specific types of vesicles through the use of a. a fluorescent-activated cell sorting machine. b. antibodies attached to bacterial carriers and low speed centrifugation. c. ultracentrifugation. d. light microscopy.
b
PTS1- and PTS2-bearing matrix proteins target to a. a common cytosolic receptor. b. a common import receptor and translocation machinery on the peroxisomal membrane. c. a common receptor on the nuclear pore that catalyzes entry into the nucleus via pore targeting sequences. d. a common receptor protein within the peroxisomal matrix that activates protein processing for PTS1- and PTS2-bearing proteins.
b
Sorting of protein to mitochondrian and chloroplasts is a. cotranslational. b. post-translational. c. pretranslational. d. quasitranslational.
b
The fluorescent properties of dyes such as SNARF-1 can provide information on the a. location of specific proteins. b. concentration of H+ ions in specific regions of the cell. c. the amount of RNA in a cell. d. volume of a cell.
b
The partition coefficient K, the equilibrium constant for partitioning between oil and water, for butyric acid is about 10-2, and for 1,4-butanediol it is about 10-4. You add liposomes containing only water to a solution with an initial concentration of 1 mM butyric acid and 100 mM 1,4-butanediol outside the liposomes. What is the relative rate of diffusion of the two substances into the liposome interior? a. Butyric acid diffuses into the liposome 100-fold faster than butanediol. b. The two diffuse into the liposome at equal rates. c. Butanediol diffuses into the liposome 100-fold faster than butanediol. d. Not enough information is provided to permit an answer.
b
Transepithelial glucose transport uses a symport to transport glucose up a concentration gradient by a. coupling glucose transport to proton movement. b. coupling glucose transport to Na+ movement. c. coupling glucose transport to Ca2+ movement. d. coupling glucose transport to Cl- movement.
b
Transport of unspliced HIV mRNA from the nucleus to the cytoplasm of host cells is promoted by a virus-encoded protein named a. Tat. b. Rev. c. nucleoplasmin. d. Ran.
b
Type I membrane proteins in the ER have all of the following properties except a. cleavable signal sequence. b. internal signal-anchor sequence. c. internal stop-transfer sequence. d. N-out, C-in topology.
b
What is the expected effect on salt accumulation in the plant vacuole of a mutation in the plant vacuolar ATPase that decreases the H+ concentration in the vacuole? a. increased Na+ accumulation b. decreased Na+ accumulation c. no effect d. complete seed germination failure
b
What is the function of TFIIH in the transcription initiation complex? a. binding to the TATA box b. unwinding the DNA duplex c. catalyzing the synthesis of RNA d. all of the above
b
Which of the following allows one to circumvent the theoretical resolution of the microscope? a. total internal reflection fluorescence microcopy b. photo-activated localization microscopy c. indirect immunofluorescence microscopy d. double-label fluorescence microscopy
b
Which of the following is present in the nuclear export sequence of PKI (an inhibitor of protein kinase A)? a. a proline-rich sequence b. a leucine-rich sequence c. a lysine-rich sequence d. all of the above
b
Which of the following proteins does not "footprint" the lac operon control region? a. lac repressor b. -galactosidase c. RNA polymerase d. cAMP-CAP
b
Which of the following statements regarding the E. coli two-component PhoR/PhoB regulatory system is (are) true? a. PhoB has kinase activity. b. PhoB is a cytosolic protein. c. Phosphorylated PhoB is an inactive transcriptional activator. d. none of the above
b
Which type of RNA participates in nuclear export of mRNA? a. snRNA b. hnRNA c. tRNA d. rRNA
b
. How does uniport transport compare to simple diffusion? a. Similar to simple diffusion, uniport transport is nonspecific. b. Uniport transport is slower but more specific than simple diffusion. c. Uniport transport is much faster and more specific than simple diffusion. d. Simple diffusion is reversible but uniport transport is not.
c
. Operator constitutive mutants of the lac operon would a. express the lac repressor constitutively. b. block the binding of RNA polymerase to the promoter. c. express -galactosidase constitutively. d. prevent the inducer from binding to the repressor.
c
Characteristics of transformed cells can include all of the following except a. aneuploidy. b. ability to differentiate. c. tight junctions. d. presence of integrated viral genes.
c
During the import of proteins into the nucleus, the importin subunit binds directly to a. FG nucleoporins. b. Ran·GDP c. basic nuclear localization signals in cargo proteins. d. all of the above
c
GPI-anchoring serves a special function, especially in polarized epithelial cells because this modification serves to target proteins to the: a. RER b. Golgi c. Plasma membrane d. Nucleus
c
The glucose uniporter GLUT1 has a Km of 1.5 mM for D-glucose and 30 mM for D-galactose. At a concentration of 5 mM for each, what is the rate of glucose transport relative to galactose transport? The Vmax may be assumed to be the same for both. a. 3.7-fold slower b. equal c. 5.5-fold faster d. 20-fold faster
c
The mediator complex a. can form a molecular bridge between activators of transcription and DNA replication machinery. b. can function to maintain a promoter in a hypoacetylated state. c. has histone acetylase activity. d. none of the above
c
This serves as the promoter for 60-70% of eukaryotic genes and typically serves as a control region for genes that are transcribed at relatively low rates. a. TATA box b. enhancers c. CpG islands d. UAS (upstream activating sequences)
c
Transcriptionally inactive genes a. are always located within euchromatin. b. are not located within nucleosomes. c. often are methylated. d. are not resistant to DNase I.
c
Transepithelial transport requires a cell layer. a. polarized b. sealed c. both a and b d. permeable
c
Ultracentrifuges allow cell biologists to isolate mitochondria from lysosomes based on organelle differences in a. isoelectric point. b. ionic composition. c. equilibrium density. d. size.
c
Unassembled or misfolded proteins in the RER can be damaging to the physiology of a cell and therefore are transported to the cytosol where they are degraded. This transport process is referred to as a. polyubiquitination. b. disulfide isomerization. c. dislocation. d. O-linked glycosylation.
c
Unlike mitochondria and chloroplasts, peroxisomes can arise from precursor membranes, as well as by division of preexisting organelles. a. as condensate b. as dispersions c. de novo d. as vaporware
c
Which of the following is not a step in the run on transcription assay? a. isolation of nuclei b. incubation with 32P-labeled ribonucleoside triphosphate c. exposure of cells to a labeled RNA precursor d. hybridization of labeled RNA to cloned cDNAs
c
a. the C-terminus of the precursor protein. b. amino acid position 173 in most mitochondrial and chloroplast proteins. c. the N-terminus of the precursor protein. d. b or c
c
All the following elements can function as eukaryotic promoters except a. a TATA box. b. an initiator element. c. CpG islands. d. an enhancer.
d
All the following statements about heterochromatin are true except: a. Heterochromatin stains more darkly with DNA dyes than does euchromatin. b. Heterochromatin contains more highly condensed DNA than does euchromatin. c. Heterochromatin is associated with inactive genes. d. Heterochromatin is more susceptible to DNaseI than is euchromatin.
d
Cholesterol and phospholipids are transported between organelles by a. Golgi-dependent mechanisms. b. incompletely characterized vesicle populations. c. direct contact between membranes and, to some extent, small, soluble lipid-transfer proteins. d. b and c
d
If a cellular homogenate were subjected to differential centrifugation, which of the following would be expected to pellet first? a. the endoplasmic reticulum b. mitochondria c. the cytosol d. nuclei
d
Protein import into the mitochondrial matrix is supported by energy input from a. ATP hydrolysis by chaperone proteins in the cytosol. b. ATP hydrolysis by chaperone proteins in the mitochondrial matrix. c. the proton-motive force across the inner mitochondrial membrane. d. all of the above
d
The TATA box a. serves as a promoter sequence for genes transcribed by RNA polymerase III. b. is located approximately 100 base pairs upstream of the start site for mRNAs. c. is present in all eukaryotic genes. d. acts to position RNA polymerase II for transcription initiation.
d
The major ATP-powered pump responsible for maintaining ion gradients across the plasma membrane of mammalian cells is a. the calmodulin-activated plasma membrane Ca2+ ATPase. b. the sarcoplasmic reticulum Ca2+ ATPase. c. the vacuolar F-class proton pump. d. the plasma-membrane Na+/K+ ATPase.
d
The oxidation of fatty acids occurs in the a. mitochondria. b. rough endoplasmic reticulum. c. peroxisome. d. a and c
d
The pH of lysosomes is lower than that of the cytosol because of the action of a. Na+ and OH− transport proteins in the lysosomal membrane. b. H+ and Cl− transport proteins in the plasma membrane. c. acid-producing enzymes in the lysosomal lumen. d. H+ and Cl− transport proteins in the lysosomal membrane.
d
Which of the following could be used to visualize subcellular structure in living cells? a. transmission electron microscopy b. scanning electron microscopy c. brightfield microscopy d. differential interference light microscopy
d
Which of the following is not a structural motif found in a DNA-binding domain? a. homeodomain b. zinc-finger c. helix-loop-helix d. random-coil acidic domain
d
Which of the following proteins involved in cotranslational translocation of proteins into the ER membrane is not a GTP-hydrolyzing protein? a. ( subunit of the SRP receptor b. elongation factors in ribosome mediated mRNA translation c. P54 subunit of SRP d. Sec61 translocon
d
Which of the following statement(s) is (are) true of V-class proton pumps? a. They are ATPases. b. They are present in membranes of plant vacuoles. c They serve to decrease the pH inside a lysosome. d. all of the above
d
Which of the following statement(s) regarding the transcription initiation and RNA Pol III is (are) true? a. ATP hydrolysis is not required for initiation. b. Pol III is responsible for synthesizing tRNAs and 5S-rRNA. c. The promoter elements of tRNA genes lie entirely within the transcribed sequence. d. all of the above
d
Which of the following synthesizes the phospholipids that form the plasma membrane? a. the rough endoplasmic reticulum b. the plasma membrane c. the Golgi apparatus d. the smooth endoplasmic reticulum
d
Which of the four classes of ATP-powered pumps share overall similarity: several subunits, the same general organization, and a similar function in being H+ transporters? a. ABC superfamily and P-class pumps b. ABC superfamily and P-class pumps c. F-class pumps and P-class pumps d. F-class pumps and V-class pumps
d
transepithelial transport
epitheial cell is polarized. apical = lumen of intestine. basolateral = inside organism between two cells are cell junctions. need tight junctions. transepithelial transport of glucose = glucose taken into the cell from the intestine through the glucose/2 Na+ symporter glucose diffuses from cell to the blood via GLUT2 passiv transport. BUT! the Na+ has to go out. so it does so via an Na+/K+ ATPase. (K+ might leave through a resting channel) basically two transport proteins--a symporter and a pump help glucose get into the blood.
Distinguish between homologous and heterologous desensitization
feedback repression. when GPCRS are continuously stimulated, several serine/threonine residues on cytosolic domain get phosphorylated by a B protein kinase. this phosphorylated receptor can bind its ligand, but not sufficiently activate the G proteins. = homo then there's PKA that can get activated by any ligand that stimulates a receptor that stimulates Galpha-s, prolonged exposed to any hormone that does this can desensitize the one receptor. = heterologous
fatty acids synthesized how?
formed in cytosol from acetyl CoA and elongated and desaturated in the ER. get transported by fatty acid binding proteins with a hydrophobic pocket.
phospholipid biosynthesized how?
glycerol plus phosphate plus two FAS. goes to ER. phosphate head removed. choline added on cytoplasmic face. then flipped to ER ;luminal side by flippase.
-35 and -10 TATA box
helps σ70-containing RNA holoenzyme bind with th correct directionality. needed for euks.
explain the cardiac cotransporter
in cardiac muscle, the export of Ca2+ is coupled to (and powered by) the import of Na+. 3 Na in for 1 Ca out.
template in transcription and replication
in replication == both strands are template in transcription == template is the strand copied. the other one is the coding strand.
make a cDNA library
isolate all mRNA. all mRNA has a poly A tail, so your primer = poly T extend via reverse transcriptase. use that cDNA. make ds cDNA, and on the ends, ligate the ends to linker sequences that have target recog seq for restriction enzymes. then you can cut for sticky ends. and ligate them into a plasmid and put them into a cell. multiply the cell.
GPCR rhodopsin
light-sensitive GPCR called rhodopsin. Rhodopsin contains a protein called opsin, which possesses seven transmembrane alpha helices further bound to a light-absorbing pigment, retinal. Rhodopsin is not a typical GPCR because it does not rely on a ligand. It requires photon-induced isomerization of its light absorbing pigment (from 11-cis-retinal to fully trans--also termed meta-rhodopsin II, or "activated" rhodopsin, denoted as "R*"). This causes a conformational change in the the GPCR membrane helices, permitting the subsequent binding of the Gat subunit (the "t" stands for transducin, which is the name of the G-protein coupled to the rhodopsin receptor) and allows for the exchange of GDP for GTP. Then, Gat-GTP may break free and interact with its effector protein, cGMP phosphodiesterase (PDE), which hydrolyzes cGMP to 5'-GMP. When this happens--that is, when the concentration of cGMP drops due to the enzymatic action of PDE--the nonselective Na+ and Ca2+ channels close, preventing further entry of these ions into the cell and thereby promoting hyperpolarization.
other cloning vectors
plasmids = 15000 bp bacteriophages = 23,000 bp bacterial artificial chromosomes = for huge dna yeast artificial chromosomes = even huger DNA. contain yeast ORI, two selectable markers, telomeres and centromeres for proper segregation
transient vs stable transfection
protein is expressed from cDNA in plasmid DNA in transient. loss of DNA over time. unfaithful segregation protein is expressed from cDNA integrated into the host genome, faithful.
misfolded proteins fate =
recognition > dislocation by ERAD and AAA ATPase family > polyubiquination and deredation by proteosome
nuclear import?
requires a nuclear localization signal, A nuclear transport protein called importin binds to this sequence, creating a cargo complex. It can diffuse through the nuclear pore by transiently interacting with FG repeats within the nuclear pore. Once the cargo has reached the nucleoplasm, RanGTP binds to importin, which causes a conformational change, decreasing importin's affinity for the nuclear localization signal of the cargo protein. The importin-RanGTP complex then diffuses back through the nuclear pore and into the cytoplasm. When the complex encounters the cytoplasmic filaments of the nuclear pore complex, the filaments stimulate Ran to hydrolyze its bound GTP to GDP. This forces a dissociation between Ran and importin, allowing importin to find a new cargo protein nuclear localization signal. Meanwhile, RanGDP may diffuse down its concentration gradient through the nuclear pore, into the nucleoplasm, where it encounters guanine nucleotide exchange factor--GEF--that will cause it to exchange GDP for GTP. The import cycle may continue.
screening by functional complementation
shutter vector - can replicate in yeast and e coli has an ORI in e coli has AMPr for e coli has polylinker has ARS (ORI in yeast) centromere in yease URA3 sequence for selection in yeast can select for two features.
stop-transfer anchor sequence signal anchor sequence
type I in ER - hydrophobic membrane spanning AA sequence which allows for proper insertion and positioning of protein in membrane. the c term is outside cell type II in ER that anchors chain in the membrane and forces the N-terminus to flip extracellularly, and the C to go into the cytosol type III in ER. N feeds in first, signal anchor results in the C on the outside.
unfolded protein resposne
unfolded proteins bind Bip (two bips live in IRE1) empty IRE1 can dimerize. >> it promotes formation of Hac1 transcription factor>> these transcrption factors promote transcription of mRNA for agents that help proteins folds.