LS3 Exam 1 Study Guide
Griffith experiment
(1920s) Microbiologist Frederick Griffith studied Streptococcus pneumonia • S. pneumonia exists in two types: non-virulent (rough appearance) and virulent (smooth appearance) • The difference lies in a polysaccharide capsule coat present only on the virulent strain Adding heat-killed virulent bacteria to a live nonvirulent strain (each harmless to mice on their own) permanently transformed the live strain into lethal, virulent, encapsulated bacterial.
Chargaff's rules
(1940's) -analyzed the composition of DNA molecules of different organisms - Each organism had different ratios of A/T/C/G but certain ratios were always constant -For all organisms, the nummber of purines (A+G) equaled the number of pyrimidines (T+C) - conclusion: base composition (A:T to G:C) varies between species, but Total purines= Total pyrimidines (A+G=T+C)
Western blot: purpose, procedure, applications, data interpretation (you do not need to know the different detection methods for visualizing secondary antibodies)
(Immunoblot) After you run your protein on a gel... Transfer to a nitrocellulose membrane that binds proteins very tightly. Looks blank once you have transferred the gel, so to visualize we use tagged antibodies! We wash with primary antibody, and then use a secondary antibody in order to detect the first. We use a secondary antibody in order to amplify the signal, we get larger signal output when we use a second antibody A lot more secondary antibodies can bind to the primary antibody making our signal brighter. We can detect by: Autoradiogram: image that is produced by decay emissions from a radioactive body (beta or gamma rays) Fluorescence: image produced by light emission by fluorescent antibody Chemiluminescence: image produced by light emission from an enzymatic reaction. - aka immunoblotting- identify the presence of certain proteins in a sample
Hershey and Chase experiment
- 32P was DNA and 35S was protein; confirmed that DNA was the genetic material of phages - when bacteria are grown in media containing P32 or S35, everything synthesized within the bacteria containing phosphorus or sulfur with become radioactive. If you infect these bacteria with bacteriophages, the progeny bacteriophages they produce have either radioactice DNA (from P32-exposed bacteria) or radioactive protein (from S35-exposed bacteria). Certain amino acids contain sulfur, therefore all proteins will become radioactive when synthesixed in the presence of S35. All nucleic acids contain phosphorus and are hence radioactive when synthesized in the presence of P32 ==centifuge that spins at very high speeds allowing separation of bacteria and phage- phage supernatant and bacterial cells in pellet - S35 detected in supernatant and P32 detected in pellet 1950s Bacteriophage T2 (bacteriophage that infects E. coli) and radioisotopes -variate the amount of time allowed for a nucleotide to be incorporated - how okazaki fragments were discovered -harvested the dna at different time points and measured length of dna strand- separate smaller from larger dnas - pulse: grow cells in radiolabeled nucleotides - chase: density gradient centrifugation of dna after indicated periods of time - the closer to the top of the tube, the smaller the isolated dna fragment most of the radiation from the ³⁵S was recovered in the phage ghosts, wherease the radiation from the ³²P was recovered in the cell, indicating that the genetic material injected into the cell was DNA
-DNA replication: know the complete mechanism
- Catalyzed by DNA POLYMERASES (Diff in PKs and EKs) -- nucleotides are not added spontaneously - need enzyme to catalyze this reaction; rely on pyrophosphate group to make rxn energetically favorable - None of the DNA poly alone can initiate DNA chains, in either direction = they require a PRIMER molecule -- Primer = nucleic acid sequence (DNA or RNA) w a free 3' OH group on the end -- DNA pol REQUIRES A FREE 3' OH to function as nucleophile to attack alpha-phosphate of incoming triphosphate -- RNA IS used as the PRIMER - b/c unlike DNA poly, RNA poly can start adding nucs out of nowhere into growing strand -- Use a type of RNA poly to add short segment of RNA to single strand of DNA once separated so we begin replication -- Allows DNA poly to have free 3' OH group to start adding DNA nucs to it - Direction of synthesis is 5'-3' DNA replication Parental DNA strands have to separate, and are then filled in with complementary nucleotides
-relationship of GC/AT content to DNA melting point 6
- Tm (melting point) is the temperature at which 50% of the DNA is single stranded (denatured) Single-stranded DNA absorbs more UV light than double-stranded DNA. The G-C base pairs contain 3 hydrogen bonds while A-T only contain 2 so DNA with a greater amount of G-C base pairs will take longer to reach melting point. However, the stacking of nitrogenous bases in native DNA interferes with UV absorption, resulting in a lower absorbance. Denaturation disrupts such stacking, allowing for more absorbance by the bases. The phenomenon of UV absorbance increasing as DNA is denatured is known as the hyperchromic shift. The purine and pyrimidine bases in DNA strongly absorb ultraviolet light. Double-stranded DNA absorbs less strongly than denatured DNA due to the stacking interactions between the bases. Single deoxynucleotides absorb more strongly than denatured DNA
mRNA
- carrier of information from DNA -template for protein synthesis -generally has short life span -makes up small percentage of cellular RNA (5%) -translated into proteins, typically a simple structure that is mostly single stranded, short half life, very diverse (many types of mRNAs are present in a cell, but they do not account for most of the RNA in a cell)
how proteins 'read' the major/minor groove of DNA (the pattern of chemical signatures for each type of base pairing will be provided)
- nucleotide bases are paired in the middle of the double helix but different portions of the base are accessible for interaction with proteins through major and minor grooves -minor groove (12A; 1.2nm) and major groove (22A; 2.2nm) -helical formation has major and minor grooves- there is a narrow angle (120) and a wider angle (240)- the angle at which bases are oriented towards each other causes the phosphodiester backbones to be closer to each other in the minor groove than in the major groove - edges of each base pair are exposed in the major and minor grooves -patterns of available hydrogens varies by base pair therefore the types and numbers of H bonds available in the major/minor grooves differ- this allows proteins to recognize specific DNA sequences without altering the DNA helix - minor groove is not as useful for distinguishing base pairs The major and minor grooves are opposite each other, and each runs continuously along the entire length of the DNA molecule. They arise from the antiparallel arrangement of the two backbone strands. Note that the grooves are actual structural features of the molecule, not consequences of the way it is drawn. The grooves are important in the attachment of DNA Binding Proteins involved in replication and trascription. To explain in words, if the glycosidic bonds (which attach the nucleic base to the sugar in the backbone) stuck straight out at 90 angles on both sides, then the grooves in double-stranded DNA would be symmetrical. Because the glycosidic bonds are at an angle (relative to the interface between the AT or GC pairs), one of the "faces" of the base pair is larger than the other. you can distinguish base pairing from the major groove How do specific binding proteins usually search for specific DNA sequences through H bonding with the major groove
end replication problem
- the end replication problem in linear (eukaryotic) chromosomes =RNA primer will be removed but DNA pol I can't synthesize new DNA as there is no further 5' prime as a substrate - result: chromosomes get shorter with each cycle of replication In linear (EK) chromosomes, not PK - the inability of DNA polymerases to replicate the final segment of DNA at the 3' end of lagging strand where there's no primer to provide a 3' OH group PROBLEM = RMA primer will be removed but DNA pol I can't synthesize new DNA as there is no further 5' primer as a substrate - If RNA primers occurs on EDGE of chromosome = primer removed and left w small 3' overhang - two 3' overhangs = single stranded DNA -- NOT stable, subject to degradation by exonuclease that chews away at end - if happens every time we replicate -> chromosomes become shorter and lose critical info - SOLUTION = TELOMERASE! - Fixed by adding telomeres via telomerase enzyme
tRNA
-adaptor molecule that "decodes" mRNA -each tRNA is specific for a single amino acid -classic cloverleaf shape -accounts for 10% of total cellular RNA - not translated, but have a function in protein translation by delivering amino acids to the ribosome, complex 2D/3D structure containing many double stranded regions
role of beta clamp
1. Beta clamp = A component of the E. Coli DNA polymerase III holoenzyme - A Ring-shaped homodimer that encircles and slides along the duplex DNA ahead of the Pol III core to which it's attached - Greatly enhances processivity of DNA synthesis - beta clamo keeps polIII associated with the DNA- pol III has low affinity for DNA
role of pol III
2. Pol III holoenzyme = the REPLICATIVE polymerase in E.Coli (does bulk of job) - 17-subunit E. Coli DNA pol III complex - responsible for chromosomal replication - Includes three Pol III (enzymatic cores), three sliding clamps, and a clamp-loading complex - the replicative polymerase in E. coli (it does the bulk of the job) - DNA pol III "falls of" the DNA when it encounters RNA primers - 17-subunit E. Coli DNA pol III complex - responsible for chromosomal replication - Includes: -- 3 Pol III enzymatic cores = catalytic units -- 3 Beta sliding clamps = each associated w one pol III core; wrapped around DNA to help keep DNA pol core enzymes closely associated w DNA -- A clamp-loading complex = large structure that everything's attached to
Meselson and Stahl experiments: how was the model of DNA replication proved from the three different models?
3 proposed models: 1. Conservative 2. Semi-conservative 3. Dispersive Grow bacteria in 15N -> All DNA is heavy 15N Transfer to 14N -> DNA incorporates light 14N - Isolate DNA from cells - Place in solution of CsCl - Centrifuge solution 140,000 x g for 48hr - Examine location of DNA - Expect DNA to get lighter the longer it is in 14N solution Expected experimental results for each model after one round of DNA replication: 1. C - one double helix is all parental DNA (heavy) and the second is synthesized with all light nitrogen = two different bands; light and heavy -- Trend = a HEAVY band and LIGHT band gets darker w each round of replication 2. SC - two double helices that have one light daughter strand (light) and one parental strand (heavy) = one band that's in b/t light and heavy b/c mixed -- Trend = HYBRID band and a LIGHT band that gets darker w each round of replication 3. D - All mixed; would not see two diff bands b/c both bands contain 14 and 15N = one band in b/t light and heavy -- one band that migrates from Heavy to light with each round as it gets darker Before N14 transfer (0), only H-H DNA (heavy) ~ 1 generation (0.7-1.5), only H-L DNA (hybrid) ~ 2 generations (1.9-3.0), both H-L (hybrid) and L-L (light) DNA ~ 4 generations after, only L-L DNA (light) What happened after ONE round of DNA replication? Before transfer to 14N (all DNA heavy) = heavy band (further down in gel) After one round = band is in between the 15 and 14N mark Eliminate conservative as potential hypothesis for DNA rep because C would have been one light and one heavy band What happened after TWO rounds of DNA replication? If Dispersive -> all mixed trending to the lighter because only light DNA can be incorporated If Semi -> two mixed double helices and two light double helices -- One molecule completely made up of light DNA; one molecule mixed w one light strand and one heavy strand RESULTS = one HYBRID and one LIGHT band - Rules out dispersive Meselson and Stahl experiment summary Before N14 transfer (0), only H-H DNA (heavy) ~ 1 generation (0.7-1.5), only H-L DNA (hybrid) ~ 2 generations (1.9-3.0), both H-L (hybrid) and L-L (light) DNA ~ 4 generations after, only L-L DNA (light) Conclusion: Semi-conservative hypothesis is correct: original parental strands are conserved = little light-heavy band, but majority of DNA contains light over time
mechanism of synthesis
3' hydroxyl group of existing chain is the nucleophile and the alpha phosphate (closest the carbon) of the incoming nucleotide is the electrophile - nucleophilic attack--> condensation reaction-> a covalent bond formed called a phosphodiester linkage and water is released
phosphatase/ kinase
= Abl kinase is involved in checkpoint regulation during cell cycle - kinase adds a phosphate group (covalent attachment to the -OH group of Ser, Thr, or Tyr) - phosphatase removes a phosphate group - adding or removing a phosphate can activate or inactivate a protein - phosphorylation can change: conformation, ligand binding site, binding with another protein
you will not be asked to recognize specific nucleotides, but you do need to know their structural components and how RNA/DNA nucleotides differ
A nucleotide is composed of a pentose sugar, a base, and a phosphate (nucleoside means no phosphate). Phosphate attached to the 5' carbon and base attached to the 1' carbon of the sugar. RNA differs from DNA in that it has ribose instead of deoxyribose in the backbone (which makes it more reactive bc of the OH and thus less stable) (less stable in alkali than DNA), uracil instead of thymine, single stranded, and the secondary/3D structure that will allow it to catalyze other reactions and act as an enzyme/ribozyme. Also, RNA can form non-canonical base pairs due to lower structural restraints as a single stranded molecule.
free energy
A thermodynamic quantity equal to the enthalpy (of a system or process) minus the product of the entropy and the absolute temperature. How much energy is available in the system. Y-axis on a energy hill graph.
predict consequences of AA substitutions for active site/transmembrane/cytoplasmic regions of a protein
Amino acid substitutions will change in the function of the protein, especially if it causes a change in the net charge (non polar/polar) or depending on its size (proline/glycine). Amino acids in active sites are very specific, so the active site would no longer work in receiving the substrate to start any process. In the transmembrane proteins should be hydrophobic and non polar. Cytoplasmic regions are generally hydrophilic and polar.
N terminus
Amino terminal, N-terminus (extracellular). Polarity of protein is an amino acid on one end and carboxyl group on the other. N-terminus to C-terminus.
domain
An independently folding region of a protein that usually confers a specific structure or function to the protein it is part of. Domains are made up of motifs. It is a region that specifies binding/kinase. It could be considered a tertiary structure if the protein consist of only one domain. You could cut it out of the sequence, it can fold on itself. You could swap domains to make new proteins https://www.youtube.com/watch?v=aZCZCbanfe0 difference between domain and motif: when separated from other protein structures, domain would keep its shape and therefore function but a motif would lose its shape and therefore function since its shape was dependent on the other protein structure as well proline kinks amino acid chains- proline would ruin the regular structure of a helix proline-glycine turn- kink in amino acid followed by a compact amino acid that can handle the sharp turn- creates sharp turns in polypeptide tert structure
quaternary protein structure and the chemical bonds that govern these levels of organization
Arrangement of multiple subunits into a larger complex, arrangement of multiple independently folded peptide units into one mule-subunit complex. Has all types of bonds, example of structures are cofactors, coenzymes, dimers, and hemoglobin again because it has an independent function.
Avery experiment
Avery wanted to identify the component from the dead virulent S. pneumonia that transformed the rough nonvirulent strain into smooth virulent strain. He heat killed the smooth strain, divided this amongst different tubes and treated each with a different enzyme that breaks down a specific macromolecule before introducing the mixture to live rough strain. By systematically eliminating DNA, RNA, proteins, carbohydrates, and lipids from the heat killed smooth strain, AVery was able to demonstrate that treating heat killed smooth strain with DNAse prevented it from transforming rough strain into smooth strain - DNases- degrade DNA into individual subunits -RNases- degrade RNA into individual subunits - Proteases- degrade protein into individual subunits Conclusions: DNA destroyed ability of heated extract to transform R strain into lethal smooth (S) strain; DNA must contain the transforming agent, DNA must contain the genetic info
BME
Beta- mercaptoethanol is a reducing agent removes disulfide bonds.
cell lysate
Break open the cell, usually done by disrupting the plasma membrane. The rupturing of a cell by sonicatation, detergents, physical disruption. Break open the cell using sonication: application high energy sound waves. Submerging cells in a detergent (solution that makes non-soluble things more soluble) , this will make holes in the membrane. Feeding the cells through a homogenizer: uses high pressure in order to force cells through a small hole. After we apply these methods a homogenate: thick soup made up of the insides of the cell like the cytosol, enzymes, ribosomes of the cell will be left over.
C terminus
Carboxyl terminus, C-terminus (cytoplasm). Polarity of protein is an amino acid on one end and carboxyl group on the other. N-terminus to C-terminus.
Southern blots: purpose, procedure, applications, data interpretation
Carried out for the purpose of detecting particular DNA sequences in DNA samples. Combines both electrophesis and probe hybridization. Probes are pieces of DNA, with a sequence of gene of interest will only bind with gene of interest on chromosome. We can make single stranded DNA due to the separation of H bonds but they can anneal to another piece of DNA so that is why we are able to utilize these. DNA is cut up by restrisction endonucleases and placed on Agarose Gel to be electrophoresed and separated by size. DNA is denatured in alkaline solutions and so the single strands are prepped for later hybridization by probes After transferring the gel unto the membrane, it is treated and the membrane is exposed to the labeled probe and then visualize on X ray film or autoradiogram. - require gel electrophoresis to separate macromolecules by size all require transferring macromolecules from the gel to the membrane -all require incubating the membrane (blot) with a type of labeled probe -all require "developing" an image of the probe signal - use DNA probes
CsCl gradient
CsCl = cesium chloride - create CsCl Equilibrium centrifugation - DNA and CsCI mixed evenly - same density at t = 0 - Centrifugation for 24 hours: This is so sensitive, it can distinguish b/t DNA labelled w diff isotopes - At t = 24hr: CsCl forms a concentration gradient (separation); DNA accumulates at a point of equal density - DNA migrates in the tube to match its density to density of CsCl - less dense at the top of tube - more dense at the bottom of tube - After centrifugation = find region of DNA in midst of CsCl - special b/c able to detect v slight differences
product
Differs from the substrate by one or more covalent bonds, has been affected by the enzyme.
-FISH: purpose and applications
Fluorescent In Situ("in place"- since its on a slide) Hybridization is used to detect the physical location of a gene on a chromosome. The DNA chromosomes are adhered on to a slide. Cells that have been arrested in metaphase, are treated to make them swell The slide is treated so that chromosomal DNA is denatured on the slides. Probes are flooded on to the slide Binding via Heterologous Hybridization and excess are washed away. Probes are either fluorescent or they are chemically treated so that they're visualized. Application of the FISH technique: the use of 2 probes can allow us to detect whether Philadelphia chromosome is present (chromosome 9 is fused with chromosome 22). - used to detect specific sequences in the context of the cell or tissue
tertiary protein structure and the chemical bonds that govern these levels of organization
Folded structure of the entire polypeptide chain. Overall 3D geometric formation/shape of a protein. Occurs when certain attraction are present between substructures like alpha and beta sheets. Bonds involved are non covalent ionic, hydrogen, and van der waals bonds, with di-sulfide covalent bonds. Examples would include global molecules that make up hemoglobin, which is made up of 2 alpha and 2 beta
principles of DNA/RNA gel electrophoresis
Gel Electrophoresis: Uses electricity to separate macromolecules, like DNA and RNA, based on their size. Gel acts as a form of resistance for the macromolecules moving through it, its main purpose is to separate the macromolecules based on size & conduction of electric current. Example of gels are: Agarose gels (DNA & RNA). Smaller molecules will pass through the gel faster than the larger ones, they separate by size. Conformation plays a huge role, a supercoiled plasmid will travel differently than linearized molecule. In order to denature DNA/RNA formamide and urea can be used. Hook electrodes up to the gel apparatus, and induce electric field from one side of gel to the other.
15N/14N
Grow bacteria in 15N -> All DNA is heavy 15N Transfer to 14N -> DNA incorporates light 14N - Isolate DNA from cells - Place in solution of CsCl - Centrifuge solution 140,000 x g for 48hr - Examine location of DNA - Expect DNA to get lighter the longer it is in 14N solution - used in meselson and stahl experiment Grow E. Coli in presence of heavy nitrogen so DNA is labeled w heavy nitrogen - N isn't radioactive, just a different density - transfer bacteria to media w only 14N -> trace how much the light label gets incorporated into DNA - Expect mixture to get lighter the longer it is in 14N solution -A
rough strain
Lacked the capsule
immunofluorescence: purpose and applications
Proteins can be detected by antibody that is treated by fluorescent tags. Can also use the Indirect method of tagging. Sometimes proteins can be bound to small fluorescent proteins (in living cells only). An example is GFP. - localize specific proteins in within cells
RNA polymerase
RNA polymerase is an enzyme that is responsible for copying a DNA sequence into an RNA sequence, duyring the process of transcription. As complex molecule composed of protein subunits, RNA polymerase controls the process of transcription, during which the information stored in a molecule of DNA is copied into a new molecule of messenger RNA.
motif
Referred to as a super-secondary structure. A common, stable combination of alpha helices and/or beta sheets, like zooming in on a portion of a protein. It is not considered a tertiary structure. - example: various helix motifs: 1) helix turn helix motif 2) four helix bundle motif - example: beta sheet motifs 1) antiparallel- beta hairpin, antiparallel beta sheet, beta barrel 2)parallel
smooth strain
Secretes a polysaccharide capsule that protects bacteria from immune system of animals
affinity chromatography (how column chromatography separates proteins)
Separates proteins based on specific interactions, by affinity to a specific antibody. Beads are covalently linked to antibody. Antibodies are specific for the protein of interest you want to study. Then proteins are recognized by the antibody and bond, the other proteins not specific to those antibodies elute out. Continue to was in order to wash out what isn't attached to antibodies. At the end use agents to disrupt matrix and interactions to get protein of interest. - isolate of specific proteins + binding partners
ion-exchange (how column chromatography separates proteins)
Separates proteins by charge. Same charge as beads = elute faster, oppositely charged proteins will bind to the beads. Come out in different fractions. First most negative proteins, then slightly negative, then neutral. In order to get the positive proteins you add high salt to disrupt matrix at end and elute them.
gel exclusion (size- exclusion) (how column chromatography separates proteins)
Separates proteins by size. Larger = elute faster, smaller = elute slower. Smaller proteins enter the pores in the beads, they flow through the column slower. Larger proteins flow through the column faster and elute first, them medium, then small.
GFP fusion proteins: purpose, applications
Sometimes proteins can be bound to small fluorescent proteins (in living cells only). For example, if GFP is added to the DNA of living cells it is expressed throughout the organism. Glowing mice and fish.
peptide bond formation (know the nucleophile and electrophile)
The 3' hydroxyl on the ribose sugar from the most recently incorporated nucleotide causes a nucleophillic attack on the alpha phosphate of the incoming nucleotide triphosphate, breaking off the other two phosphates. A new phosphodiester cold (covalent bond) is formed between the 3' ribose and the alpha phosphate. They are linked in the 5' to 3' direction bc it doesn't have the energy to go the other way, with the 5' always being the phosphate and the 3' always being the hydroxyl (OH). It is a hydrolysis reaction because a water molecule is released (condensation rxn as well).
5'/3' polarity
The 5' end refers to the phosphate connected to the 5' carbon of the deoxyribose sugar on the "top-most" nucleotide in a single stranded DNA chain. The 3' end refers to the OH group connected to the 3' carbon of the deoxyribose sugar on the "bottom-most" nucleotide at the end of a single stranded DNA chain
immunoprecipitation: purpose, applications
The act of isolating and extracting a protein from a mixture using an antibody specific to the protein that you want. Isolate specific proteins + binding factors. The antibody is attached to a magnetic field (apply magnetic filed to precipitate) or heavy bead (centrifuge to precipitate). You would express a tagged protein in a cell, then precipitate that tagged protein with a specific antibody (make it go down to the pellet or to the side with magnet), then make cell extract after centrifuging. Example: Antibody linked with bead is added to protein mixture. It binds to a specific protein/protein complex. Mixture is centrifuged. The heavy beads are pulled into a pellet at the bottom, along with the protein of interest. Then use more antibodies to verify, one antibody to isolate protein from solution with beads (magnetic/heavy) and another antibody to verify purified form of protein, to confirm identity (radioactive/fluorescent/enzyme).
primary structure
The amino acid sequence (blueprint) that determines all other structures. Bonds involved are peptide bonds (covalent bonds). No important substructures.
fraction
The different groups of proteins that you are extracting at different times from the chromatography. Because they aren't the protein of interest.
substrate
The ligand that binds to the active site of an enzyme. A molecule that undergoes an enzyme-catalyzed reaction.
supernatant
The liquid on top of material deposited by settling or centrifugation. Clear liquid remaining after sediment settles Lighter organelles that remain suspended after centrifugation.
transition state
The maximum energy in a system, where everything is most unstable. Catalyst lower the activation energy by interaction with the transition state.
origin of replication
The nucleotide sequence or site in DNA where DNA replication is initiated (ORI) = where double strand is unwound - Rep bubble = Separated DNA into two strands (pulling on circle) - BI-DIRECTIONAL rep fork; each rep bubble has TWO REPLICATON FORKS - At each fork, DNA is replicated both "forwards" and "backwards" in PKs = one ORI -- Circulation genome (pro) -- replicate in both directions -- Rep forks -> replication -> separate the two complete replicated rings from e/o into daughter duplexes in EKs = multiple ORIs -- Linear genome (euk) -- wouldn't be efficient if we had to start from one origin to replicate DNA -> rep all DNA in much smaller fraction of time -- these bubbles replicate in BOTH directions and eventually collide/meet - DNA replication is initiated at 'origins of replication' (ORI) where the double strand is unwound - prokaryotes typically one ORI - eukaryotes typically multiple ORIs DNA replication is Bi directional: two forks at every origin - terminus region about 180 degrees opposite the origin
column
The tube-like thing where you have you beads and proteins, where the chromatography happens in trying to extract the protein of interest. They travel down the column to be eluted.
elute
To extract certain unwanted proteins from the bottom of the column in order to isolate your protein of interest. You elute out your "other" proteins. At the end when you only have your protein of interest you can elute it out as well by destroying the matrix or you can extract them. remove (an adsorbed substance) by washing with a solvent, especially in chromatography.
homodimer
Two of the same structures that bind and work together, 2 identical subunits bind.
nitrogenous bases
Uracil- 2 H bonds Thymine-2 H bonds Adenine- 2 H bonds Guanine- three H bonds Cytosine- three H bonds
gel
Used in gel electrophoresis, it is what proteins must travel through to reach the positive end of the gel, acts as the resistance. it is also very fragile so we often transfer proteins over to a membrane so it si easier to work with and add antibodies. - DNA loaded at top of gel - agarose or acrylamide gels are very fragile and will fall apart when handled - you can detect total protein on a gel...
primary antibody
Used to detect the protein of interest, when put into the column or onto the membrane and it is specific for the protein of interest, it will bind only that particular protein and help isolate it from the rest. target and directly bind to antigen
principles of protein gel electrophoresis
Uses electricity to separate macromolecules, like DNA and RNA, based on their size. Gel acts as a form of resistance for the macromolecules moving through it, its main purpose is to separate the macromolecules based on size & conduction of electric current. Smaller molecules will pass through the gel faster than the larger ones, they separate by size. Conformation plays a huge role, a supercoiled plasmid will travel differently than linearized molecule. Hook electrodes up to the gel apparatus, and induce electric field from one side of gel to the other. "pulling" energy by the positive electrode at end of gel (end side), and "pushing" force created by negative electrode (loading side). - SDS- polyacrylamide gel electrophoresis (SDS-PAGE) separates proteins by size - use porous gel - SDS-PAGE= sodium dodecyl sulfate polyacrylamide gel electrophoresis - SDS: strong ionic detergent; denatures proteins and coats proteins with a uniform negative charge BME= beta mercaptoethanol reducing agent- removes disulfide bonds
secondary antibody
We wash with primary antibody, and then use a secondary antibody in order to detect the first. We use a secondary antibody in order to amplify the signal, we get larger signal output when we use a second antibody. A lot more secondary antibodies can bind to the primary antibody making our signal brighter.
active site
Where the substrate/ligand binds too, most of them are highly specific to their particular pathway. Can be inhibited or conformational changed by enzyme regulators.
disulfide bond
While free cysteine residues do occur in proteins, most are covalently bonded to other cysteine residues to form disulfide bonds. Disulfide bonds play an important role in the folding and stability of some proteins, usually proteins secreted to the extracellular medium. Since most cellular compartments are reducing environments, disulfide bonds are generally unstable in the cytosol with some exceptions. Inside the cell, disulfide bridges between cysteine residues within a polypeptide support the protein's tertiary structure. Insulin is an example of a protein with cystine crosslinking, wherein two separate peptide chains are connected by a pair of disulfide bonds. - rarely see disulfide bridges in the cell because the cytosol is a reducing environment ( yes there are exceptions) - a disulfide bond greatly increases stability of a folded protein because of the strength of covalent bonds, and crosslinks formed between different segments/regions/proteins - alpha helix breakers- proline and glycine -cysteine- in reduced form cystine- oxidized form - extracellular environment favors formation of disulfide bridges since it is an oxidizing environment- intracellular environment is reducing and therefore no bridges
conservative dna model
a double stranded copy is generated and the parental DNA is conserved as a double strand Parental -1st generation = 2 old strands in one + 2 new strands in another -2nd generation = 2 old strands + 2 new strands + 2 new strands + 2 new strands
endergonic reaction
absorbs free energy from its surroundings and is nonspontaneous Requires energy to start the pathway, has a positive delta G, non-spontaneous.
P32
all nucleic acids contain phosphorus and are hence radioactive when synthesized in the presence of P32
antigen
antigen (Ag): Foreign substance that elicits production of an antibody - e.g., snake venom, flagella, etc. Antibodies bind antigens very specifically. In some cases, antibody can distinguish between proteins that differ by a single amino acid - pruify antibodies with affinity chromatography using antigen attached to beads - "epitope"= unqiue part of antigen recognized by antibody
DNA polymerase
are enzymes that synthesize DNA molecules from deoxyribonucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two identical DNA strands from a single original DNA molecule. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones.[1][2][3][4][5][6] These enzymes catalyze the following chemical reaction deoxynucleoside triphosphate + DNAn ⇌ diphosphate + DNAn+1 Catalyses DNA-template-directed extension of the 3'- end of a DNA strand by one nucleotide at a time.
film
can also be what the gel is transferred onto in order to more easily work with proteins, can also be used to detect the print of coomassie blue print in light
centrifuge
centifuge that spins at very high speeds allowing separation of bacteria and phage - separates based on density
S35
certain amino acids contain sulfur, therefore all proteins will become radioactive when synthesized in the presence of S35
delta G
change in free energy Only determine the direction of a reaction and not the rate of the reaction. Reactions occur spontaneously when delta G is negative (exergonic) and reactions are unfavorable when they are non spontaneous with a positive delta G (endergonic).
parental DNA/daughter DNA
daughter strands are the complement of the parent strand
role of pol I
degrades the RNA primer and synthesizes DNA in a 5' to 3' direction to fill the gap
how to denature/renature DNA
dna can denature and anneal reversibly and repeatedly - hydrogen bonds hold the strands together under physiologic conditions - at high temps or extreme pH or ion (=salt) concentrations, hydrogen bonds are broken and strands separate Single stranded DNA can re-anneal. Base pair composition determines the energy required to denature DNA strands. In order to denature DNA/RNA formamide and urea can also be used.
semiconservative dna model
each strand is serving as a template and the new double strand contains one parental and one new strand The parental strand is separated into segments and each new strand consists in parts of both parental and new DNA -Parental -1st generation = Each strand 1/2 old, 1/2 new + each strand 1/2 new, 1/2 old -2nd generation = 4 DNA molecules where each strand is 1/2 old 3/4 new
coupled reactions
enzymes couple endergonic reactions with exergonic ones the energy released by an exergonic reaction is used to drive an endergonic reaction
protein probes
labeled antibodies that recognize/bind to a specific protein you want to study
nucleic acid probes
labeled short, single stranded pieces of DNA that are complementary to/bind a specific nucleic acid sequence you want to study
pellet
larger and denser components bottom of tube, contains large components of cell, organelles like nucleus
DNA probes
most nucleic acid and protein techniques rely on PROBES to detect specific nucleic acid sequences or proteins - you can use annealing of labeled probes to visualize parts of chromosomes or particular DNA sequences - probes are labeled small single stranded pieces of DNA - complementary to the region of DNA you want to analyze - labeling the probe lets us detect it: fluorescent labels detected by microscopy; multiple colors - DNA probes in S/N blots and Protein (antibody) probes for western blots sed in Southern Blotting in order to be the complementary strand to the gene of interest, its labelled in order to be recognized once it binds to the particular gene. It is also used in Northern blotting to bind to the complementary strain of RNA in ht particular gene because a DNA probe is more stable than an RNA probe. (RNA has a reactive hydroxyl group).
secondary protein structure and the chemical bonds that govern these levels of organization
ocal structure, 3D formations, occurs when the sequence of amino acids is linked by H bonds. Does not describe the overall atomic position in 3D space. Bonds involved are hydrogen bonds between the oxygen of the carboxyl C=O and the hydrogen of the amino H-N. Important substructures are the alpha helix which is in vertical orientation and within the polypeptide backbone. And the beta-pleated sheets, in horizontal orientation.
reducing agent
one that gives its electrons to another element, therefore oxidizing itself the electron donor in a redox reaction
topoisomerase
prevents DNA supercoiling in front of the replication fork An enzyme that catalyzes alteration in DNA topology - Introduces and/or removes positive or negative supercoils in closed, circular duplex DNA
single stranded binding proteins (SSB)
protect and organize single stranded DNA (ssDNA) A bacterial protein that binds single-stranded DNA in a sequence independent fashion
heterodimer
protein composed of two polypeptide chains differing in composition Two different structures that bind and work together.
enzymes
proteins that act as biological catalysts Protein catalyst that speeds up the rate of specific biological reactions- lowering activation energy (neg delta G)- stabilize transition state - the level of activity of a certain enzyme can be affected by both the rate of enzyme synthesis and the rate of enzyme breakdown- transcription and translation can be very important in determining the regulation of enzymes- the breakdown of proteins is also important for determining the concentration of many enzymes
pulse chase experiment
proved the existence of Okazaki fragments and lagging strand DNA synthesis - short 'pulse' treatment with a (radio)labelled compound--> chase time--> measure the labelled compound concentration in molecule of interest at different time intervals (chase) Trace where a label is getting incorporated and how much of it is being incorporated into the macromolecule - two-phased technique used to examine cell processes taking place over a period of time - Short 'PULSE' = treatment with a (radio) labeled compound = expose cell to labeled compound at t = 0 -- Expose DNA of bacteria (E.Coli) to radio-labeled thymine for amt time it takes for one round of replication (in the example) - CHASE = unlabeled form replaces labeled compound; reaction is monitored to see how long it takes labeled form of compound to be replaced by unlabeled form (wikipedia) - Measure the labelled compound concentration in molecule of interest at different time intervals (CHASE) - NOTE: you cannot have multiple time points for one sample -> need a sample for EACH time point; 5 time intervals = 5 different samples of E. Coli On the graph- Pulse w 32P-ATP is the sharp change in the curve from straight line to logarithmic growth x-axis = time; y-axis = radioactivity in total DNA - the longer exposed to radio-labeled thymine, the more DNA becomes labeled w DNA - pulse = expose subject to radioactive material - chase = how much label is incorporated into particular molecule at particular time
exergonic reaction
releases energy during the process of the reaction, has a negative delta G, spontaneous.
rRNA
ribosomal RNA - critical part of the ribosome -contains catalytic activity. is a ribozyme - accounts for up to 85% of cellular RNA - not translated, but are part of the ribosome structure and have a role in protein translation - a rRNA catalyzes peptide bond formation between amino acids, can have highly complex structures
ligase
seals nicks Enzyme that creates a Phosphodiester bond b/t the 3' end of one DNA segment and the 5' end of another
SDS
sodium dodecyl sulfate strong ionic detergent denatures proteins and coats proteins with a uniform negative charge. Destroys non-covalent bonds
coomassie stain:what it does and doesn't tell you
stains all proteins so you can visualize them. Separates proteins by size, lets you know how many proteins there are, detect total protein on a gel but does not tell us any information about the identification of proteins.
nucleotides
strands of DNA and RNA are formed by a linear sequence of paired subunits called nucleotides (monomer) consists of a phosphate, nitrogenous base, and sugar
primase
synthesizes RNA primers An enzyme that catalyzes the formation of RNA oligonucleotides used as primers by DNA polymerases
activation energy
the minimum amount of energy required to start a chemical reaction The input of energy (activation energy) is required to initiate reactions. The activation energy determines th speed of the reaction.
dispersive dna model
the parental strand is separated into segments and each new strand consists in parts of both parental and new DNA Parental -1st generation = Each strand 1/2 old, 1/2 new + each strand 1/2 new, 1/2 old -2nd generation = 4 DNA molecules where each strand is 1/2 old 3/4 new
phage ghost
the protein component of a bacteriophage after it has injected its genome into a bacterium
Northern blots: purpose, procedure, applications, data interpretation
this is identical to the Southern Blott except this time you are analyzing RNA instead of DNA!! However, we keep using a DNA probe here instead of RNA due to its stability. - the probe that will be used is typically made of DNA (more stable) - a method to measure and analyze specific RNA molecules - require gel electrophoresis to separate macromolecules by size - all require transferring macromolecules from the gel to the membrane - all require incubating the membrane (blot) with type of labeled probe - all require "developing" an image of the probe signal
role of helicase
unwinds DNA
bacteriophage
viruses that infect bacteria - embodies all the central principles of molecular biology: replication (DNA) Transcription (RNA) Translation (protein) weak interactions
membrane aka blot
what the gel is transferred onto on order to be easier to work with since the gel is fragile, works better when adding charge and antibodies to isolate proteins. - transfer to a membrane is critical since the agarose or acrylamide gels are very fragile and will fall apart when handled
epitope tags: purpose, applications
you can use and epitope tag to "tag" a protein with an epitope recognized by a strong antibody to aid protein purification. Applied in affinity chromatography/ immunoprecipitation. - epitope= unique part of antigen recognized by antibody
