Molecular Bio Lecture Terms
Outline how an electron microscope works -- Where is the source of electrons
(Source of electrons is at the top of the microscope) Shoots electrons with wavelengths that become focused by magnets which are then used to image
What is the time of flight proportional to in MALDI-TOF?
(m/z)^1/2 m = mass of particle z = charge T is directly proportional to mass -- The greater the mass the long the time
Outline the Methylation of K9 on H3
- A site specific enzyme will methylated K9 on H3 on all of the nucleosomes - HP1 binds to the specific methylation site, HP1 causes the protein to fold up and be heterochromatic -- Like the sir proteins
What are the results of the processing of mRNA experiment?
- Absorbance showed that most of the RNA is rRNA - Most of the RNA in the nucleus is small - Newly synthesized RNA is big
What were the six things that Watson and Crick's structure determined?
- Bases were facing inwards - the two strands were held together by hydrogen bonds - the phosphate group is attached to the 5' carbon - 2 strands are anti-parallel - 10 base pairs per turn - Double helix is right-handed
Outline how poly-adenylation occurs (DONT HAVE TO MEMORIZE)
- CPSF binds an AAUAAA sequence in the 3' UTR - CStf (stimulatory factor) binds to GU-rich sequence in the 3' UTR downstream of where CPSF binds - A cut occurs 11-30 nucleotides downstream from AAUAAA -- Poly A gets added here after the cut - Poly(A) polymerase (PAP) adds about 200 As - Multiple Poly(A) binding proteins binds to the A-tail (length of the tail determines how many proteins can bind)
Limitations of Northern Blotting
- Can only look at one gene at a time, only use one probe - Not great for detecting genes expressed at very low levels
Outline the Example of Affinity Chromatography with Fusion Proteins Produce an example of control group (negative control) - Interested in finding all of the proteins that bind to protein X
- Clone gene for protein X - Create a fusion protein gene with protein X and GST (binds glutathione) - Put cloned fusion gene into a plasmid, and then into bacteria - Purify fusion protein by binding it to beads coated with glutathione - Prepare mouse cell extract over the column (see which proteins bind to protein X) - You can see which proteins bind to protein X by adding excess glutathione so that the protein complex will bind to the free glutathione Use just a sample of GST to see if any extra proteins stick
How does FISH work?
- Denature DNA (Chromosomes stay together) - Attach biotin molecule to a nucleoside triphosphate (Can make Bio-dCTP) - Use DNA polymerase to synthesize a strand with biotin coated C (Make a probe to hybridize to a specific sequence) - Wash away unhybridized DNA - Probe recognizes DNA in centromeres (repeated DNA sequence) -- Hybridize the probe to the chromosomes and have it reanneal - Avidin will bind the biotin and allow for fluorescent light
Outline the experiment that determines how we know the significance of the 3 cis-acting elements (TATA, CAAT, GC)
- First set the transcription efficiency of a complete promoter to 100% - Perform deletion mapping of elements and remove each region one-by-one to analyze the change in efficiency - Put your promoter of interest, and the luciferase gene into a plasmid, transfect it into cells - Add luciferin and ATP to the transfected plasmid to analyze how much luciferase there is - You are able to quantify the amount of light and can determine how well the promoter worked
Advantages of Northern Blotting
- Gives the exact size of RNA - gives the abundance of RNA too
Sodium dodecyl sulfate
- Ionic detergent and coats the protein with negative charge to linearize the molecule
Outline the processing of mRNA experiment 1
- Label eukaryotic cells with [32P] phosphate for 30 mins - Purify RNA - Centrifuge the RNA - Fractionate RNA - Analyze radioactivity by measuring UV absorbance
Outline replication at the ARS What happens after initiation?
- ORC is phosphorylated - Helicases (MCM proteins) start moving down the strand - ORC remains on one strand - Another ORC binds to the new sequence and the MCM proteins fall off - The ORCs remain but the MCMs are exported out of the nucleus in yeast
What are the functions of the 5' cap
- Protects 5' end of RNA - Helps export from the nucleus - Important in the start of translation
Isoelectric Focusing
- Proteins stop migrating once they get no net charge -- stop migrating once they get to their isoelectric point Proteins aren't separated on size, but on their isoelectric point
Outline a Northern Blot Experiment
- Retrieve the nuclei in a pellet by differential centrifugation - Isolate RNA from the nuclei pellet - Separated the RNA on a gel - Transfer the RNA to a membrane - Probe the membrane with radio-labeled (#2)) single-stranded DNA that corresponds to your gene of interest - Subject the blot to x-ray film
Outline Western Blotting
- Run an SDS page gel without staining - Transfer it to a membrane electrophoretically - Incubate the membrane with an antibody against one protein and detect the bound antibody
How to use MALDI-TOF?
- Separate proteins from a protein sample and find your protein of interest - Digest the protein of interest with trypsin digestion that cuts at lysine and arginines residues to get tryptic peptides - Pulse the tryptic peptides with a laser -- Based on the time, the computer can tell you what the mass is, produces a graph on mass to charge ratio and will tell you what the protein is - Can also be used to detect post-translational modification
How does transcription start after the PIC is established?
- The largest subunit of RNA polymerase II has an extended C-terminal domain - Every fifth serine is phosphorylated by TFIIH - Phosphorylation activates the complex and initiation takes place
How did John Gurdon clone a frog?
- Took an enucleated female frog egg - Took a differentiated cell nucleus - Put the nucleus of the differentiated cell into the enucleated egg and got it to start dividing
Outline the Meselson-Stahl experiment BEFORE THE RESULTS
- Used two stable isotopes of nitrogen N-14 and N-15 - Grew E. coli cells in an N-15 medium -- Incorporated N-15 into their nitrogenous bases -- Allowed for multiple generations - Resuspended the E. coli cells in an N-14 medium, allowing them to replicate once - Kill the cells and collect/purify the DNA - Performed Density-Gradient Equilibrium Centrifugation for days to form the gradient (Mixed the DNA perform beginning to spin) - Lower density DNA floats higher up than the higher density DNA, Hybrid DNA floats in the middle
Outline the detection of Okazaki Fragments Experiment What is the name of the radioactively labeled substance and what does it bind to?
- Used velocity centrifugation with a high pH gradient to disrupt the hydrogen bonds with a swinging bucket rotor - Use radioactive hydrogen [3H] to label newly replicated DNA that only attaches to thymidine of living bacteria cells and do short pulses - Isolated total DNA, only the new DNA will be radioactively labeled - Denature the DNA with NaOH - Sediment the DNA - Collect gradient fractions to determine CPM
What is the octamer structure? What is the stepwise prices?
1 H3/H4 tetramer 2 H2A/H2B Dimers 1 H3/H4 dimer --> 1 H3/H4 dimer -> 1 H2A/H2B dimer --> 1 H2A/H2B dimer
How many origins of replication do bacteria have? How many replication forks? How many origins of replication does a Eukaryotic chromosome have?
1 origin of replication 2 replication forks MANY
What must the chromatin packaging of DNA accomplish?
1. Effectively condenses the DNA to fit in the nucleus 2. Allows the DNA to function as a genetic material (must be copyable) 3. be adjustable
Outline experiment 2 for the processing of mRNA
1. Label eukaryotic cells with [32P] phosphate for 30 mins 2. Add actinomycin D 3. Chase for 3 hours NO RNA SYNTHESIS
4 levels of gene regulation
1. Transcription 2. RNA processing 3. Translation 4. Protein stability
How many base pairs are present per turn in DNA?
10 base pairs
What is the diameter of a histone octamer + DNA?
10 nm
Angstrom
10^-10 m
How many base pairs are present in a nucleosome? With no linker DNA
146 base pairs
How many bp are okazaki fragments in eukaryotes?
150-200 base pairs
What is the amount of base pairs with two full turns?
166 bp
What is the ratio of mass of DNA to the mass of histones in the nucleus?
1:1 Mass of DNA = Mass of histones
What is the ratio of new DNA to old DNA in the first generation of dispersive replication?
1:1 first
What is the ratio of new DNA to old DNA in the first generation of conservative replication? What about the second generation? What is the key characteristic of conservative replication?
1:1 first generation 1:3 second generation The parental double helix persists
# of hydrogen bonds between adenine and thymine # of hydrogen bonds between guanine and cytosine
2 hydrogen bonds 3 hydrogen bonds
What is the diameter of DNA?
20 angstroms
Wha is the limit of resolution of the light microscope?
200 nm (Tiniest distance you can see)
At what range does DNA absorb light maximally?
260nm
What wavelength do proteins absorb UV light at?
280nm
How many methyl groups can you have per lysine? Is methylation reversible?
3 methyl groups -- Yes by demethylases
How many amino acids are in one turn of an alpha helix?
3.6 amino acids
If there is ten base pairs per turn in DNA, how many angstroms are there per turn? How many angstroms per base pair? -- CAN VARY
34A/turn 3.4/bp
How long is the original transcript for the three rRNA transcripts + transcribed spacers? What is the order?
45S 18, 5.8, 28
Which carbon is not in the ring of DNA or RNA?
5'
What are the introns and exons flanked with in pre-mature RNA?
5' and 3' UTRs
From 5' to 3': State all of the elements of a fully-processed mRNA strand
5' cap 5' UTR Exons 3' UTR Poly-A Tail
What RNA are bacterial ribosomes lacking?
5.8S
What RNA size is not present in bacterial genomes?
5.8S
What are the three types of rRNA?
5.8S, 18S, and 28S
What is the ratio of new DNA to old DNA in the first generation of semi-conservative replication?
50:50 or 1:1 ratio
What are the four RNAs that make rRNA?
5S, 5.8S, 18S, 28S
What is the diameter of the nucleus?
6 micrometers
how many n-terminal tails are there per histone?
8
What is the g value for retrieving a nuclear pellet?
800g/10 min
How, molecularly, is the lariat formed?
A 2' to 5' phosphodiester bond is formed between A and G within the intron because the 3' is already being used
What are the four letters of coded chemical information of the bases in the major groove?
A = Hydrogen bond accepter D = H-bond donor H = Nonpolar hydrogen M = Methyl group (bulky hydrophobic)
Confocal Scanning Microscopy (light)
A fluorescent specimen can be illuminated by a laser with a focused point of light from a pinhole Only seeing stuff from a single plane to create optical sectioning
Outline 5' capping Where are the two methyl groups?
A guanosine with a methyl group on the 7th nitrogen links with the top of the 5' end of the RNA strand Creates a 5'-5 bond A methyl group is present on the 7th nitrogen of the guanosine and on the 2' carbon of the 5' end of the RNA
Shadow Casting
A heavy metal like platinum gets vaporized by wires and piles up on one side of a specimen to shadow one side of it to create contrast -- Produces a 3D image
MyoD protein
A helix-loop-helix protein that homo-dimerizes -- When it binds, it stops the cell cycle and turns it into a muscle cell
TFIIH
A kinase that phosphorylates Serine 5 on the c-terminal domain
What is a co-activator protein?
A protein brought in by an activator protein (Mediator brought in by an activator protein that binds to the enhancer)
Replicon
A region of DNA controlled by a single origin of replication.
What is a labeled probe?
A segment of base pairs that has been labeled for identification purposes (Can be labeled with biotin, fluorescent, or radioactive) Can use a single-stranded piece of DNA to find another piece of DNA
Polycomb + What does it co-localize with?
A silencing protein in fruit flies Co-Localizes with H3methK27
What is a crystal
A solid that has atoms in a repeating structure
Differential Centrifugation
A technique to separate different cellular components based on their size and density. - Homogenize cells and spin them in a centrifuge, collect the pellet, decant the supernatant, and repeat the process several times ANOTHER EXPLANATION Procedure for separating cellular components according to their size and density by spinning a cell homogenate in a series of centrifuge runs. After each run, the supernatant is removed from the deposited material (pellet) and spun again at progressively higher speeds.
Column Chromatography
A way of protein purification -- Things separate based on some sort of principle
Relationship between absorbance and the strand amount of DNA
Absorbance increases when DNA is single-stranded
Where do HATs get their acetyl group from? What do HDACs do with the acetyl group?
Acetyl-CoA HDACs put the acetyl group back on acetyl-CoA
What histone modification codes for histone deposition?
Acetylation of lysines 5 and 12 on H4
What post translational modification can occur on lysine?
Acetylation or methylation
What is the drug that stops RNA synthesis?
Actinomycin D
How do you isolate affinities from each other in affinity chromatography?
Add excess bait! Add excess insulin so that the insulin receptors bind to the free floating insulin instead of the insulin on the bead you can also use salt, detergents, etc
How to engineer a chimeric protein
Add the appropriate promoter, the gene of interest, and the GFP gene -- Put it into a plasmid which drives the transcription of the gene and GFP -- Put it into cells and they will make the chimeric protein
Pulse
Adding radioactivity
What does phosphorylation of S and T do?
Adds a negative charge
Purines
Adenine, Guanine -- Bigger
Names of the five nucleosides
Adenosine Thymidine Cytidine Uridine Guanosine
What chromatography gives the most purity?
Affinity chromatography
How does RNA elongation link to RNA processing?
All of the RNA processing enzymes (spliceosomes, 5' capping enzymes) bind to the phosphorylated CTD polymerase drags these enzymes elongation -- brings processing to the point of transcription
Dichromic Mirror
Allows visible light to pass but not UV light
Ribozyme
An enzymatic RNA molecule
What is the c-terminal domain?
An extended domain of a 7 amino acid repeat that repeats 52 times on the largest subunit of RNA polymerase 2
Explain the relationship between the pK of 4.4 of glutamic acid What about 10.0 pK of lysine
At 4.4, 50% of glutamic acid has a charge. Our pH is closer to seven within our bodies so a higher pH means there is a lower ocncentration of H+ ions so it is going to be more negatively charged At 10.0, 50% of lysing has a charge. Our pH is closer to seven within our bodies so a lower pH means that there is a higher concentration of H+ ions so lysine is going to pick up a charge and be positive
Where do the nucleotides start being numbered?
At the end of the promoter (NO ZEROTH AMINO ACID)
Outline the Cutting Chromatin Experiment -- How did they verify the repeatable structural component
Attack the linker DNA to get individual nucleosomes -- Chops up linker DNA to separate the nucleosomes via Mnase They cut in different integers of nucleosomes -- pieces of 2, 1, 3, 4 -- Determined they were integer multiples of the smallest size by running it on a gel and removing the histones
ARS
Autonomously Replicating Sequence Origin of replication in yeast
What form of DNA is present in humans?
B-DNA
List two substances that break disulfide bonds
BME and DTT (Dithiothreitol)
What do you call RNA Pol II + GTFs?
Basal Transcription Apparatus -- The general components to be able to transcribe a gene
Density-Gradient Equilibrium Centrifugation How is the gradient formed?
Based on density alone Use a very dense gradient (20% to 70%) -- Typically it is a heavy, dense salt (CsCl) -- Chlorine towards the top and Cesium towards the bottom The samples will stop at region of equal density after being spun for days Formed during centriguation
MALDI-TOF Mass Spectrometry
Based on the principle that ions of different molecular weights travel at different speeds if they're accelerated with the same potential A laser beam ionizes a sample which then causes the ions to move towards towards the detector -- Measures the time of flight of the ions
Affinity Chromatography
Beads physically bind the component you want to isolate The beads are coated with a specific bait (receptor, substrate, antigen, etc) to catch the protein of interest) If you want to purify the insulin receptor, coat the beads in insulin and everything else will flow through so you'll be left with only insulin
Why doesn't it work 3' 5'?
Because 5' end already has a phosphate
Why is the DNA not damaged during this experiment?
Because not every thymidine is radioactively labeled and 3H is pretty weak
Why is cysteine sometimes considered nonpolar?
Because the S-H bond is much less polar than an O-H bond
Is RNA polymerase 2 recruited before or after the addition of TFIIH?
Before
What does immersion oil do?
Bends light more
What keeps polymerase on DNA? (Processive) Are RNA polymerases processive?
Beta Sliding Clamp (PCNA IN EUKARYOTES) No
What were the results of the Reiji Okazaki experiment?
Big peak in the beginning showed the lagging strand Big peak in the middle showed the leading strand and ligated okazaki fragments
Heterodimers
Bind two different sequences
Homodimers
Bind two of the same sequences
Avidin -- What color of fluorescence is it with UV light?
Binds biotin -- Turns yellow in UV light
Similarities and differences between HP1 and Polycomb
Both cause hetero-chromatization Different silencer proteins on different residues
Why does methK bind to HP1 and polycomb?
Both proteins have chromodomains only bind methylated lysine The methylated lysine sits in a hydrophobic pocket of the "chromodomains" of polycomb and HP1 -- Can only be in that pocket when it is METHYLATED
Can leucine zippers be repressors or activators?
Both! They could recruit co-repressors to close up a gene
What does proline's structure allow?
Breaks up higher order structures
What does an acetyl group look like?
C2H3O
What enzymes brings in new H3/H4 domains?
CAF1
What brings new H3/H4 to new DNA?
CAF1 -- Brings H3/H4 in properly so that they bind to DNA properly
Fluorescence Microscopy -- How does it work When can you use it?
CAN ONLY USE IT WHEN CELLS ARE DEAD Molecules can accept one wavelength and give off another Use an antibody that can recognize something (tubulin) and have a fluorescent molecule attached to the anitbody -- Will emit visible light when excited by UV light
Why does the tighter structure have advantages?
Can reveal binding sites for specific proteins when loosened
Two Dimensional Electrophoresis
Can run SDS in one dimension and an IEF in the other dimension
Theodor Svedberg
Categorized how things spin in the ultracentrifuge Developed (S) the sedimentation coefficient
HP1 + Where it binds
Causes heterochromatization binds to H3methK9
What is the main method of fractionation?
Centrifugation
Ion-Exchange Chromatography
Charged beads in the matrix (positive or negative) will either attract or repel specific proteins in a mixture Repelled proteins will flow through faster than proteins that are attracted -- Positive beads will cause positive proteins to flow through faster
Microsequencing
Chopping off one amino acid at a time from amino terminus
Who determined what chromatin really looked like?
Christopher Woodcock and Ada and Don Olins
What is the template for nuclear DNA replication?
Chromatin
What is in the nucleus during interphase?
Chromatin (DNA + Protein) Nucleoli Nucleoplasm
How do enhancer sequences work?
Chromatin is flexible, causing it to loop around and bind to the PIC, enhancing its ability to transcribe
Sir John Gurdon
Cloned the first vertebrate (Frog) in the 1950s
What do you call the strand not being transcribed? Why? What is another name for the strand not being transcribed?
Coding Strand It has the same nucleotide sequence (except T for U) as the RNA strand Sense strand
What were the three predictions of the experiment?
Conservative: 100% Heavy DNA is always present, no hybrid DNA Semi-Conservative: 100% hybrid in the first generation; Both 100% light and hybrid in the second generation Dispersive: 100% hybrid in first generation; DNA progressively gets lighter in following generations
Conserved "A" at branch point near 3' end of intron
Conserved in the middle of an intron -- Portion of a primary transcript Helps with the formation of the lariat
Helix-Loop-Helix Proteins What are the properties of the DNA-binding alpha-helices
Constructed similarly to zippers -- Basic, positively charged alpha helices that recognize different sequences, can be hetero or homodimers
Leucine Zipper
Contains two regions, one region binds DNA, and the other region dimerizes to create a coiled-coil
Density-Gradient Velocity Centrifugation
Create a 5% to 20% sucrose gradient Uses sucrose because it can be highly concentrated and doesn't interfere with the components Cell components separate based on molecular weight, size, and shape
Trypsin
Cuts proteins after lysines and arginines
Level of chromatin organization
DNA --> Nucleosome -->DNA + Histones string (10nm fiber) --> 30nm fiber --> Chromosome
How do we know that eukaryotic chromosomes have many origins of replication? What is the technique called? What are the results?
DNA Fiber autoradiography - Use radioactive thymidine that only goes into new DNA - Hot pulse cells with 3H thymidine - Isolate DNA and let radioactive DNA expose and X-ray film Diagram shows origins of replication because it radioactively binds to the areas that have new thymidine
What is chromatin?
DNA and Protein (Histones and nonhistones)
Satellite DNA
DNA in centromeres
Why does replication need a primer?
DNA polymerase needs a pre-existing nucleotide chain to figure out where to start
DNA polymerase 2
DNA repair
What is rDNA?
DNA sequence that codes for ribosomes
Cis-acting elements + Example
DNA sequences on the same gene or chromosome TATA Box CAAT Box GC Box
What is the paradigm controversy in science? What was the paradigm of chromatin?
Data that contradicts the paradigm of reality will be discounted That it was stringy
What causes chromatin to coil into the 30nm fiber? What causes chromatin to uncoil?
Deacetylation Acetylation
How does a peptide bond form? Where does this happen?
Dehydration synthesis reaction between amino terminal of one amino acids and the carboxyl terminal of the other amino acid In a ribosome
What is PCNA a subunit of?
Delta and Epsilon DNA Polymerase
What is PCNA a subunit of?
Delta and epsilon polymerase
What is an alternate way to determine the difference of dispersive and semi-conservative replication based solely on looking at the first generation?
Denature the DNA Semi-Conservative would produce a light band a heavy band Dispersive would produce a hybrid band
DNA Fiber Radiography
Determination of multiple origins of replication through the use of 3H radioactivity
James Watson and Francis Crick
Determined the structure of DNA through X-ray diffraction and Chargaff's Rule
Protein Motifs + Examples
Different chains of proteins come together to create structural elements Coiled coils and beta barrels
Charles Francois de Cisternay du Fay
Discovered the two charges of electricity (vitreous (+) and resinous (-))
What do you call DNA that has not been transcribed? What do you call DNA that has been transcribed?
Downstream Upstream
Summary of Polyadenylation What is the purpose?
Downstream of PAP, you get a cut in the 3' UTR where the poly-A tail is added Regulates translation and protects the 3' end of the RNA
When does processing begin?
During transcription
What are the three properties of DNA polymerase?
Elongation 5' to 3' exonuclease (removing previous primers) ONLY IN BACTERIA 3' to 5' exonuclease (proofreading)
Difference between the things binding to promoters and enhancers
Enhancers become bound by specific transcription factors found in a certain tissue Promoters become bound by transcription factors found in all cells
The four main affinities
Enzyme - Substrate Receptor - Ligand Antibody - Antigen Protein - Antibody
What modifies the tails on the histones?
Enzymes
What are nonhistones involved with?
Enzymes and transcription factors
What is the stain used for gel electrophoresis with DNA and RNA?
Ethidium bromide
Two types of chromatin higher order structure
Euchromatin -- Transcriptionally active (light) Heterochromatin -- Transcriptionally silent (Dark)
Is euchromatin more acetylated or deacetylated? How about heterochromatin?
Euchromatin is more acetylated Heterochromatin is more deacetylated
DNA polymerase alpha
Extends RNA primer for 20 nucleotides
Chargaff's Rule -- What was the name of the rule
First Parity Rule A%=T% G%=C%
How do you analyze dead cells with microscopy?
Fix them, make the membrane permeable, and stain the cell
Three types of rotors
Fixed Angel rotor (Think chem lab) Swinging-bucket rotor (horizontal), vertical rotor (Up and down)
What does the condenser do on a microscope?
Focuses light on the specimen
Why can base pairs sometimes flip out of the DNA?
For enzymes access, repair, and recombination
What does histone acetylation usually mean?
Gene activation
What does histone methylation usually mean?
Gene repression
What does an unmodified tail typically mean?
Gene silencing
What does methylation mean on histone H3? What amino acid is being methylated?
Gene silencing in heterochromatin Lysine 9
GTFs
General transcription factors
What are samples fixed with in transmission microscopy?
Glutaraldehyde and Osmium tetroxide
What amino acid typically forms the beta sheets?
Glycine
What nucleotide is the 5' end of the intron?
Guanine
What is the name of a guanine base, a sugar, and two phosphate groups?
Guanosine Di-Phosphate
What does H1 do?
H1 helps to stabilize the 30nm fiber
What is the smallest subunit of the nucleosome? What is the biggest?
H1 is the biggest H4 is the smallest
What are the histones that the nucleosome core particle contains?
H2A, H2B, H3, and H4 Two of each
What subunit has four alpha helices?
H3
What enzymes add acetyl groups? What enzymes remove acetyl groups?
HATs -- Histone acetyltransferases HDACs -- Histone deacetylases
Two ways to melt DNA
Heat or High pH
What are transmission electron microscopy samples stained with?
Heavy metals like uranium that absorb electrons
MCM Proteins
Helicases that unwind the DNA, are involved in permission to replicate
Tao Factors
Help assemble the complex
Example of a quaternary structure
Hemoglobin, Antibody
hnRNA
Heterogeneous nuclear RNA; the primary transcript made in eukaryotes before splicing.
What does the acetylation of lysines 5 and 12 on H4 do?
Histone deposition
What controls the coiling and uncoiling of chromatin? What was the experiment done to accomplish this?
Histone tails are required to form 30nm fiber and histone H1 - Shaved off the positive tails, using trypsin digestion - Showed that the tails did not supercoil without the tails
MyoD:Id
Homodimerizes with MyoD -- It is a truncated protein and does not have a DNA binding domain -- It prevents MyoD from binding to DNA and stopping the cell cycle and differentiation
What does absorbance correlate to?
How much RNA there is
Synonym for reannealing
Hybridizing
What holds the alpha helix together?
Hydrogen bonding between the various layers
Are nonpolar amino acids hydrophobic or hydrophilic?
Hydrophobic
What holds the higher order folding in the tertiary structure together?
Hydrophobic interactions Hydrogen bonds Ionic Interactions Van der Waals forces
Quaternary structure distinction
If you take away one subunit, the polypeptides will not function -- It is one protein
Antonie Van Leeuwenhoek -- What did he use as the light source?
Invented the microscope, discovered bacteria, capillaries, and protozoa -- Sun
Three types of column chromatography
Ion-Exchange Chromatography Gel-Filtration Chromatography Affinity Chromatography
What type of bond is present between histones and DNA?
Ionic bonds
Transformed Cell Culture + Examples
Isolated cells from tumor HeLa cells (isolated from cervical cancer)
What happens when light slows down? When does light slow down?
It bends When it goes into different media
What is the benefit to using a fixed-angle rotor?
It can be spun much faster
What can magnets do to light?
It can bend light
What would explain why there is an increase in efficiency when you remove a section of DNA?
It could have a repressor protein bound to it
What does luciferase do in the presence of ATP and luciferin?
It glows
How is DNA able to fit in the nucleus?
It is very thin and associates with proteins to form chromatin
What does the lagging strand actually do when replicating?
It loops backwards so the polymerases can act as a complex
What does the cell need to do after adding new H3/H4 tetramers?
It needs to remove the acetyl groups so it can be folded up again This is done by adding the H2A/H2B dimers
How does CAF-1 recognize new DNA?
It recognizes the clamp at the replication fork
What are the enzymes that add phosphate groups? What are the enzymes that remove phosphate groups?
Kinases add, phosphatases remove
What type of helices do proteins have?
Left and right
What direction is the nucleosome coiled?
Left-handed
Chase
Letting things exist
Two source of cells
Living organs or cell culture
Polypyrimidine tract
Located right before the 3' splice site on the introns
What are the three steps to removing introns and connecting exons?
Looping Lariat Formation Cut and Splice
Two examples of diseases caused by mis-folded proteins
Mad Cows Disease and Creutzfeldt-Jakob Disease
How do you accomplish deletion mapping?
Make a synthetic gene to manipulate the promoter
What do enhancers do? Do they have to be near the start site? Where can they be?
Make transcription more efficient They can be no where near the start site, both downstream and upstream of the promoter
What does methylation of lysine and arginine do?
Makes them more hydrophobic -- doesn't change the charge
Fluorescence Energy Resonance Transfer (FRET)
Measures protein-protein interaction Example: If protein's X and Y interact with each other, you could use the combination of BFP (Excited by violet and emits blue) and GFP (excited by blue and emits green) to see if it'll emit green by uses violet light You tag proteins with specific fret dyes to analyze their interactions Donor Excitation Acceptor Emission
If acid phosphatase removes phosphate, what is a good wait to measure its activity?
Measuring the amount of phosphate release -- operationalizing it
What were the names of the scientists that determined the semi-conservative modeling of DNA?
Meselson and Stahl
Fluorescene in situ hybridization (FISH)
Method to locate positions of genes in chromosomes
What post translational modification can occur on arginine?
Methylation
What is the DNA-cutting enzyme? What special property does it have?
Micrococcal Nuclease (MNase) It attacks the Linker DNA before the DNA in the nuclease
What are the two ways to determine the identity of an unknown protein from a Western Blot?
Microsequencing MALDI-TOF Mass Spectrometry
What is the marker for mitochondria, lysosomes, the cytoskeleton, and the nuclei?
Mitochondria -- Cytochrome C Lysosomes -- Acid phosphatase Cytoskeleton -- Actin/Tubulin Nuclei -- Histones, nuclear genes
Trans-acting factors + Example
Mobile and act at a distance (transcription factors like proteins and nucleic acid molecules)
Difference between a multi-protein complex and quaternary structure Give example of multi-protein complex
Multi-protein complexes have subunits that have an independent function Quaternary structure proteins cannot survive without all of the subunits together Pyruvate dehydrogenase
Outline how MyoD and MyoD:Id operate with each other
MyoD, when it binds, has the cell cycle stop and differentiates the cell into a myoblast When MyoD:Id is present, MyoD:Id inhibits the binding of MyoD because it does not have a DNA-binding domain and cannot bind to DNA to stop the cell cycle
Does N-15 or N-14 cause an atom to be more dense? Does N-14 or N-15 represent new DNA?
N-15 N-14 represents new DNA
Resinous
Negative
Two components of a nucleoside
Nitrogenous base and 5-carbon sugar
Three components of a nucleotide
Nitrogenous base, 5-carbon sugar, phosphate group
Do bacteria do alternative splicing?
No
Does cDNA contain introns?
No
Do bacteria have nucleosomes? What about archaea? What can we conclude?
No Archaea have primitive histones -- They protect about 60 bp and have no tails Our histones evolved from archaea histones
Can DNA be lost from development? What causes different cell?
No Cells have differential gene regulation to differentiate into different cells
Does the clamp loader stay on while polymerase is replicating? What happens when the beta clamp and DNA polymerase hits another RNA primer?
No It stays behind briefly and then recruits another DNA polymerase It stays behind so that CAF1 can put the histones on the DNA
Difference between DNA and RNA
No "O" on carbon-2 for DNA (Deoxy)
Are all RNAs transcribed from the same DNA strand? What is a commonality amongst all RNA strands?
No -- Both Watson and Crick strands can be used for RNA synthesis They are all made 5' to 3'
Does the nucleosome core particle wrap around twice?
No because a little bit of the linker DNA was chopped off
Do atoms on the bases have prime symbols?
No, only on the sugar
Is the 30nm fiber transcribable?
Nope
Are coactivators always present?
Nope, sometimes activator proteins can bind directly to the PIC
How do you measure RNA abundance?
Northern Blot
What is typically found at the first layer of differential centrifugation?
Nuclei, unbroken cell, large components
Is RNA typically bigger in the nucleus or the cytoplasm?
Nucleus because it hasn't been processed yet
Relationship between ORC and ARS
ORC binds to ARS
ORC + How many proteins
Origin Recognition Complex -- 6 proteins
Where does reverse transcriptase occur?
Out in the cytoplasm
How are disulfide bonds created?
Oxidation creates the bond (+2H+ + 2e-), reduction breaks the bond
What is the beta sliding clamp called in eukaryotes?
PCNA
What are histones involved with?
Packaging and Gene Regulation
List all of the techniques that can be visualized with a light micrscopy
Phase-Contrast Microscopy in living cells Fluoresence Microscopy in living and dead cells Sectioning embedded tissue with dyes
What techniques do you use for viewing living cells?
Phase-contrast microscopy and a different type of fluorescence microscopy (fusion proteins)
Objective Lense
Picks up the light to focus on the specimen
Vitreous
Positive
What is the charge of histones? What amino acids are highly present?
Positively charged (Contain a lot of lysines and arginines)
What do you call the combination of GTFs + the recruited RNA polymerase 2?
Pre-initiation Complex (PIC)
What is a primary culture?
Primary Culture: Isolate an organ, disperse the cells with a protease, grow them on dishes on a complex medium to see how they grow
What stabilizes the different structures of a protein?
Primary: Peptide bonds Secondary: Hydrogen bonding Tertiary: Van der waals, hydrophobic interactions, hydrogen bonding, ionic interactions Quaternary: Same as tertiary
Difference between things that bind promoters and enhancers
Promoters are bound by things in all cells, enhancers are cell-specfici
Pros and Cons of Transformed Cell Culture
Pros: - Immortal cells, need a complex medium but can divide indefinitely - Great for looking at replication and transcription Cons: - Do not contact inhibit - they don't regulate the cell cycle properly, can grow many liters -- not a proper representation of the organism
Pros and Cons to Primary Culture
Pros: Right from the organism, contact inhibit so they don't become crowded Cons: Can not be grown in the lab indefinitely, after ~50 generations they will stop dividing -- Have to keep isolating them
Chromodomain
Protein structural motif that recognizes and binds certain methylated Lys residues in proteins.
Two types of interactions occurring within chromatin
Protein-DNA Protein-Protein
What reads the chemical info of the base pairs? What does this mean?
Proteins -- Allows for them to bind with sequence specificity
Sir Proteins When do they bind? Where do they specifically bind?
Proteins that silence genes and bind to histone tails The bind only when lysines 8 and 16 are deacetylated on H4 They bind at telomeres
Gradient Fractions
Puncture a hole in the bottom of the test tube to collect various different types of tubes to separate cellular components based on how fast they sedimented
Histone Deposition
Putting histones on DNA during DNA replication
What is the name of 2 phosphate removal?
Pyrophosphate removal
What is the symbol for purines? What is the symbol for pyrimidines?
R Y
Outline the experiment for processing RNA What are the results What are the three main characteristics of the radioactive RNA?
RADIOACTIVITY REFERS TO THE SIZE OF RNA ABSORBANCE REFERS TO THE QUANTITY OF RNA SHOWS HOW THERE IS A LOT OF RNA THAT HAS MAINLY BEEN PROCESSED -- THERE IS ONLY A VERY LITTLE BIT OF UNPROCESSED LARGE RNA - Label eukaryotic cells with radioactive uridine for 1-30 mins Radioactive RNA will be - Have large size - Have diverse sequences - Are mainly in nucleus
Is DNA or RNA older?
RNA
How did RNA precede DNA?
RNA can self-replicate without enzymes and is catalytic
What are the three major DNA-dependent eukaryotic RNA polymerases?
RNA polymerase 1,2,3
Where does 5S RNA come from?
RNA polymerase 3
Who took photo 51?
Raymond Gosling -- Under Franklin's direction
What do Tao factors do in the replisome?
Recruit ATPase
Who discovered Okazaki fragments?
Reiji Okazaki
What does acetylation do to positively charged lysine?
Removes positive charge
DNA polymerase delta
Replicates lagging strand
DNA polymerase epsilon
Replicates leading strand
What is the enzymes that copies RNA to DNA?
Reverse Transcriptase
What is an example of a protein that folds spontaneously?
Ribonuclease A
What is typically found in the bottom layer of differential centrifugation?
Ribosomes, viruses, and large macromolecules
What is always the direction of the alpha helix?
Right-handed
What type of symmetry does the nucleosome core particle have?
Rotational Symmetry
List four protein confirmation disrupters
SDS Nonpolar solvents (benzene) Heat BME & DTT
What is an experiment that you can do as a follow up to the previous experiment?
SDS-Page to examine the proteins gathered
Semi-Conservative Replication
Save one strand and keep one old one
What were the results of the second generation of the experiment if replication was semi-conservative? How about conservative? How about dispersive?
Semi-conservative would produce a light band that gradually gets bigger as well as a hybrid band Conservative would produce a light band that gradually gets thicker and a heavy band Dispersive would produce a thickening band between light and hybrid
Fractionation
Separating cell components
What happens to serine 2 during elongation? What can you infer about this?
Serine 2 gets phosphorylated during elongation You are able to analyze whether or not the RNA polymerase 2 has started initiation and has been continuing with elongation based on the phosphorylation pattern of the c-terminal domain
What three amino acids can be phosphorylated, why?
Serine, threonine, and tyrosine because of their OH groups -- loses hydrogen picks up phosphate
What is an alternate way to look at a sample in TEM besides staining?
Shadowing
Scanning Electron Microscopy
Shoots electrons at sample and they bounce off the surface -- Looking at the surfaces of something, not good resolution because of the scattered electrons
What were the results of the first generation of the experiment? Did this prove semi-conservative replication?
Showed a 100% hybrid generation No, because it could also be dispersive
Is RNA single or double stranded?
Single stranded MOSTLY but can be double stranded
What happens if there is acetylation at lysines 8 and 16 on the histone tails?
Sir proteins can't bind
Nucleolus
Site of rRNA synthesis
What does radioactivity correlate to?
Size of the RNA
What do you usually start with to isolate an enzyme?
Size-exclusion chromatography followed by ion-exchange chromatography
How can gene regulation be controlled by the synthesis of RNA?
Some RNAs could be stored or degraded You could produce several proteins that degrade quickly, or one protein that has a lot of function
Immunofluorescence Microscopy with secondary antibodies
Sometimes it's hard to make antibodies that hook up to fluorescent molecules Can use a secondary antibody to bind to primary antibody and the secondary antibody has the fluroescent molecule
When do licensing factors bind?
Soon after mitosis
What are transcribed spacers? What happens to the spacers during processing?
Spacers between the RNA that will end up in the ribosome -- NOT INTRONS because they don't interrupt the functional RNA The spacers get destroyed to make mature rRNAs
Enzymes that removes introns
Spliceosome
How does recoding work in mRNA?
Stop codons can be recoded to be other amino acids UGA can be recoded to a selenocysteine UAG can be recoded to pyrrolysine
Homogenization -- Why do you do it on ice?
Suspend cells in a medium and smash them up in down -- Breaks up the cell You don't want proteases attacking the cell components
Expansion Microscopy Why do we want this?
Swell up sample, make it bigger to take a 3d image Allows for imaging of really small things with fast light microscopes
How is the pellet positioned in swinging-bucket rotor and fixed angle rotor?
Swinging-bucket places it evenly at the bottom of the tube Fixed angle orients it on one particular side of the tube
What are the three promoter elements? Are they cis or trans factors? Do all genes always have all three of these elements?
TATA box CAAT box GC box All cis factors No, not all genes have these three elements
What protein does the TATA box bind?
TBP
What happens when TFIID (with TBP) binds?
TBP + TAFs bind together and bind to the TATA box
What are TAFs?
TBP Association Factors -- They are the other proteins within TFIID that are not TBP
Relationship between TFIID and TBP What does TBP do?
TBP is a subunit of TFIID TBP bends and unwinds DNA
Difference between TEM and SEM
TEM = Block electrons with higher resolution SEM = Scatters electrons with lower resolution (surfaces only)
Nomenclature for Transcription Factors
TF = Transcription Factors Roman Numeral = # of RNA polymerase Capital Letter = Identifies Factor Example TFIIA -- Complex of proteins, one factor
What part of the PIC is left behind when initiation takes place?
TFIID -- TAFs and TBP
Where is the promoter in relation to the enhancers?
THE PROMOTER IS ALWAYS GOING TO BE RIGHT WHERE TRANSCRIPTION STARTS -- ENHANCERS CAN BE UPSTREAM OR DOWNSTREAM
What is the negative control in the luciferase experiment?
Take out a large portion of insignificant DNA to determine that the transcription efficiency does not drop by a lot
What do you call the strand that is being transcribed? What is another name for the strand being transcribed?
Template Strand Anti-sense strand
What is the proper model of the 30nm fiber?
The "Two-start" helix -- Wrap around each other in zig-zag -- The nucleosome its attached to is on the other side of the fiber, not next to it
What is the next level of organization after the 10nm fiber?
The 30nm fiber
Resolution
The ability to clearly distinguish two separate points
What does the intensity of a band refer to on a gel?
The amount of DNA/RNA
How many bands will show up on SDS-PAGE?
The amount of subunits in the protein is the number of bands that will show up (Unless it's two of the same sizes)
What does the angle alpha mean in microscopy?
The angle of light -- 1/2 of the angle of light going to the objective lense
What portion of the DNA does the absorbing?
The bases -- They're more free when denatured
What is the crick strand?
The bottom strand that is read 5' to 3' to the left
Relationship between the closeness of an object at the diffraction of the beams
The closer the object, the more diffracted the waves get
Correlation between the amount of protein and the stain
The darker the stain/the bigger the band, the more protein you have
Explain how phase-contrast microscopy works
The different refractive index of a cell causes the wavelengths of light to be out of phase with a normal wavelength of life, leading to the a different wavelength -- converts phase difference to phase contrast
Does the slower or faster component have a higher S value?
The faster component
Relationship between the CPM and the amount of radioactive DNA
The higher the amount of radioactive DNA, the higher counts per minute
Relationship between the bending of light and the refractive index?
The higher the refractive index the more bent the light is
Semi-discontinuous replication
The leading strand is continuous, lagging is discontinuous
What happens to the peaks when you increase the amount of time between each pulse?
The leading strands combine with the ligated okazaki fragments to show a huge peak
What happens to light when two waves are out-of-phase?
The light becomes dimmer
Relationship between the how fast the centrifuging is and the density of a cellular component
The lower the speed, the denser/bigger components will be in the pellet and the tinier components will be in the supernatant
How do yeast control the cell cycle?
The make it so that the MCM proteins can't go back in -- The cell has to go through mitosis for them to get back in
Why does Velocity Centrifugation work? Doesn't it go against Galileo?
The medium used in the test tube allows for the separation of cellular components
Explain how no change in amino acid sequences can cause an infectious disease?
The misfolded insoluble version of a protein can bump into the correct soluble version and flip their solubility causing cells to burst becasue the proteins aren't soluble
Relationship between the density of something and its location in the test tube for density-gradient equilibrium centrifugation
The more dense something is, the lower it'll be in the gradient (Closer towards 70%)
Relationship between the component separation and the increase in U
The more separate the component is the higher the U will be -- Less and less things will be there and you'll be able to see the activity of the enzyme
What does the primary antibody recognize?
The non-fluorescent antigen
Photo 51
The picture of the structure of DNA taken by Rosalind Franklin & Raymond Gosling
Why does polycomb not bind to H3methK9 and why doesn't HP1 bind H3methK27?
The portions of the H3 tail bind at different sites on the two different proteins
Relationship between the length of the wavelength and how bent light is What color is bent the most?
The shorter the wavelength, the more bent the light Blue is bent more than red
Relationship between the size of an object and the significance of its wave
The smaller the object, the more significant its wave will be and will have a wavelength (like an electron)
What is the relationship between how fast something moves through the sucrose gradient and its molecular weight, shape, and size?
The smaller the size, shape, and molecular weight, the slower it'll move through the gradient
What is the Watson strand?
The top strand that is read 5' to 3' to the right
What is the primary structure of a protein consist of?
The type of amino acids, the order of the amino acids, and the number of amino acids
How does the sheet form?
The upwards and downwards orientations of the R groups hydrogen bond with each other to make the sheet
What happens to old H3/H4 tetramers in replication?
They are saved and deposited on new DNA Only replaces half of the DNA
How do transcription factor motifs work?
They bind to sequence-specific sites on the DNA based on the coded chemical information of the nitrogenous bases the amino acids read the coded chemical information
What does the dimerization of leucine zippers allow for?
They can be homodimers or heterodimers Allows for three different transcription factors for two genes R-R B-B R-B
Why is lysine 9 of H3 interesting? Why is lysine 27 of H3 interesting?
They can both be methylated or acetylated
What did Christopher Woodcock & Ada and Don Olins do? What was their experiment?
They did a transmission electron microscopy -Placed cells in water to lyse them and then did electron microscopy of chromatin - They found a string of round structures, that are now called nucleosomes -- Deemed the 10nm fiber
What is interesting about the N-terminal tails?
They do not have a strict high-order structure and are accessible to enzymes
How do the base pairs orient in DNA?
They lie flat
What happens to polar side chains in an aqueous environment?
They stick out toward the aqueous environment
What has the bigger diameter, G-C or A-T?
They're equal
What is the general structure of eukaryotic histones?
Three alpha helices (this histone fold)(sometimes four) and an extended amino-terminal tail H3 has four alpha helices
Difference between uracil and thymine
Thymine has a methyl group on 5 carbon
Pyrimidines
Thymine, Uracil, Cytosine -- Smaller
Two types of spacers
Transcribed and non-transcribed
What does RNA polymerase 1 do?
Transcribes genes for 5.8S, 18S, and 28S rRNA genes
Outline the 5-step stepwise process of RNA processing
Transcription Capping Polyadenylation Cutting Splicing (Ligating)
What is the other way to culture cells?
Transformed cells (from tumors)
What does TEM show?
Transmitting the light through the specimen to create internal visualizations
What are the two inhibitors of deacetylation? What then happens?
Trichostatin A (TSA) and Sodium Butyrate You get highly acetylated chromatin creating a lot of euchromatin
What do new nucleotides come in as? What happens during replication?
Triphosphates two phosphates are removed and the third one is linked to the growing DNA strand
How many times does DNA wrap around an octamer of histone proteins?
Twice
Coiled Coil
Two alpha helices wrapped around each other Has a hydrophobic residue every 7th amino acid -- Two full turns gives a hydrophobic amino acid and they face each other to create a 7-mer repeat
Zinc Finger (Cys-His)
Two cysteines and two histidines bind to a zinc molecule creating a finger shaped out of a beta sheet and an alpha helix
What is the codon for selenocysteine and pyrrolysine?
UGA and UAG
Svedberg (S)
Unit for sedimentation rate Measure the rate at which particles of a given shape and size travel to the bottom of a centrifuge tube Have to be consistent with shape and size (5S RNA to 10S RNA, can't do 5S RNA to 5S protein)
What is the difference between uranium staining and platinum-shadowing?
Uranium staining produces internal structure and platinum shadowing shows a 3-D image
What substance breaks hydrogen bonds?
Urea
How do you detect one protein out of many after page?
Use Western Blotting!
How do you collect proteins that are attracted to the beads?
Use a salt (0.5 NaCl) which will rip the proteins off and the negative proteins will fall off
gel filtration chromatography (size exclusion chromatography)
Use porous beads Larger proteins come out first because they can't fit in the porous beads
How do you assay each different density component to make sure it's pure?
Use protein or enzyme markers
Visualizing cells by fluorescence microscopy
Use the cell appropriate promote in conjunction with the GFP gene and the gene for protein of interest and insert it into a plasmid This will produce X-GFP
Tryptic Peptide Analysis
Use tryptin to cut proteins into fragments, use chromatography and electrophoresis to separate fragments Shows a difference in amino acids (Think sickle cell anemia)
X-Ray Diffraction
Used x-ray beams at a crystal which causes the beams to diffract -- Bending them at various angles that determine the structure
SDS-Page
Uses SDS to denature proteins and give them a negative charge (smaller proteins run faster)
What is the minimum number of amino acids to be a protein?
Usually greater than 40
Refractive Index Formula
Velocity of light in a vacuum/velocity of light in a medium
Outline how the lariat is formed What does the A end up becoming
When splicing occurs, a lariat is created between the 5' G and conserved A of the intron The ends up becoming the connecting point of the lariat
Where does H1 sit? Why is there no H1 one in the core particle?
Where the DNA enters and exits Because the binding site for H1 is cleaved with the removing of the ten base pairs one each side
Resolution Equation + What do the variables mean? What constitutes the best resolution
YOU WANT A SHORT WAVELENGTH AND A LARGE REFRACTIVE INDEX The minimal distance two points must be separated to be viewed as distinct -- The lower the value of d the better d=0.61λ/(nsin(a)) d = Resolution lamda = Wavelength of light n = refractive index of the medium alpha = 1/2 angle of light from specimen to objective lens Shortest wavelength, and the biggest alpha to capture the most light, bigger refractive index is better (bends light more)
Does RNA polymerase II require other factors to initiate transcription?
Yes
Do the UTRs remain in the mature mRNA? What information do they contain?
Yes Contain information for the regulation of mRNA and translation
Can histone modification occur in the histone domains?
Yes, both there and in the tails
Is acetylation reversible? What about phosphorylation?
Yes, they can be put on or off Yes phosphorylation is reversible
Alternative Splicing
You can make different proteins from the same RNA through different combinations of exons
What is a way to study this specific methylation? What are the three specificities of antibodies?
You can use an antibody because they are histone specific, modification specific, AND site specific - You can merge Anti-H3methK9 and Anti-Hp1 to make yellow fluorescence showing that HP1 modifications always occur with H3methK9
What is the next step after you have collected the collection of proteins at a specific fractionation number from ion-exchange chromatography?
You have to assay proteins for purity based on enzymatic activity
What is the necessity that you must have when using a native gel?
You have to know the charge
How do you get a crystal of DNA?
You have to use a saturated solution, you can dehydrate it in ethanol
What is left-handed DNA called?
Z-DNA
Three main transcription factor motifs
Zinc Finger H-L-H Leucine Zipper
Contact Inhibition
a process that stops additional cell growth when cells become crowded and touch each other
DNA polymerase 3
adds base pairs
Beta Barrel
created when beta sheets are extensive enough to fold back on themselves
Protein Domains
discrete structural units in a protein, often associated with a particular function(s)
DNA polymerase 1
enzyme that replaces RNA primers with DNA nucleotides
What do acetylation of amino acids 8 and 18 mean on histone h4?
gene expression
What does an unmodified histone H4 mean?
gene silencing
Mediator Complex
increases the efficiency of the assembly of the pre-initiation complex
Prions
infectious protein particles that do not have a genome
snRNAs
involved in mRNA splicing
snoRNAs
involved in rRNA modification
Outline how the making of cDNA works
mRNA is processed and sent into the cytoplasm, reverse transcriptase synthesizes cDNA in the cytoplasm
Empty Magnification
magnification without resolution
native electrophoresis vs SDS-PAGE electrophoresis
native: used to separate proteins based on net charge of protein SDS-PAGE: used to separate protein based on approximate size when protein denatures in the presence of an anionic detergent
Native Gel
one without SDS that doesn't change the charge of the protein Can separate on the basis of charge, size, shape, etc
pK
pH at which 50% of an amino acid has picked up a charge
Chimeric Protein (Fusion protein)
proteins created through the joining of two or more genes that originally coded for separate proteins
RNA polymerase 2
transcribes mRNA
What does RNA polymerase 2 do?
transcribes mRNA and miRNA
RNA polymerase 1
transcribes rRNA
RNA polymerase 3
transcribes tRNA
What does RNA polymerase 3 do?
transcribes tRNA genes, 5S rRNA genes
Specific Activity
units of enzyme/total protein mg
What does the clamp loader do?
uses the energy of ATP hydrolysis to lock the sliding clamp onto DNA
