Exam 2 Flashcards
Transcription
DNA to RNA, uses RNA polymerase to make RNA polymers. Initiation, elongation, termination.
Why do prokaryotes need txn
Prokaryotes change gene expression often to break down and process their surroundings. Different enzymes need activated based on substance being consumed.
Prokaryotic promoters and regions
TT-35GACAT, TAT-10AAT
RNA polymerase I roles in txn
Core enzyme plus sigma factor is holoenzyme. Alpha, alpha(2 total), beta, B’, omega. Sigma factor binds to the promoter and the core enzyme to initiate txn. Core enzyme is used in elongation phase. Breaks Hydrogen bonds of DNA, not sugar phosphate backbone, and creates new SP backbone with RNA.
During elongation, RNA poly.
Releases sigma factor, core continues to transcribe. Only ~15 base pairs unwound at a time. Transcribed DNA is rewound and reforms bonds.
Termination in prok.
Uses either a Ro protein to stop, or more commonly Ro-independent termination where repeating sequences of AGCCC and UGCCC bond to make a hairpin loop secondary structure. The hairpin forces the release of core RNA poly. from DNA to end the process.
Eukaryotic Nucleus contains
Nuclear membrane (2 phospholipid bilayers), Nuclear Pore Complex, Nuclear Lamina
Outer nuclear membrane is continuous with
ER membrane, interacts with cytoskeleton and ribosomes.
Inner membrane
Interacts with the nuclear lamina and chromatin DNA in the nucleus. Contains lamin B receptors, and emerin. LBRs interact with the lamina, emerin interacts with chromatin.
Nuclear pore structure
Cytoplasmic filaments on outer end, channel in the middle, nuclear ring towards the nucleus on the inside, and nuclear basket on the inside of the nucleus. Made of 150 nucleoporin proteins. Structural polarity present, with differential head and tail.
Nuclear Lamina
Network of proteins on the inside of the nucleus along the inner membrane. Made of inner filaments lamin proteins. Disruption in lamins can disrupt nuclear shape overall.
Nuclear Transport
Histone proteins need to enter nucleus to make chromatin. All proteins for DNA replication and Txn must enter the NPC. Essential for any substance moving in or out of the nucleus.
Steps of nuclear transport
Example Erk trying to enter nucleus. Protein bind to Erk that contains a nuclear localization signal in its DNA sequence. NLS binds to importin and whole structure moves through NPC. Simultaneously, monomeric G protein Ran-GDP enters nucleus through passive diffusion. Ran-GDP uses its GEF inside the nucleus to become Ran-GTP. Ran-GTP then binds to the importin attached to the NLS, forcing it to change conformation and split off of Erk. Erk released in nucleus with NLS.
Export cycle of nuclear transport (Ran-GTP and importin portion)
Ran-GTP diffuses out of nucleus passively while still attached to importin. In cytoplasm, a GAP removes its phosphate group to make Ran-GDP, splitting off the importin. Both substances recycle into the import process.
mRNA leaves the nucleus by
binding to exporter protein exportin, which carries it out of the nucleus
Polymerases used in Eukaryotic txn
RNA polymerases I, II, III.
RNA polymerase I
makes most ribosomal RNA, 45s
RNA polymerase II
makes mRNA
RNA polymerase III
makes tRNA and one rRNA, 5s
Eukaryotic Promoters
Most prominent TAT-25AAA, also BRE sequence, CAT box, GC box.
Eukaryotes contain ___ for txn
Assisting proteins known as txn factor proteins. Examples are the TFII(letter) proteins.
TFIID in txn
uses TATA-binding proteins to bind to TATAAA box at -25 site.
After TFIID bound to promoter
TFIIA, then TFIIB bind to TFIID to create a txn complex. TFIIF and RNA poly II then bind to D,A,B, and DNA
After Txn complex receives TFIIF and RNA poly II
TFIIE and TFIIH bind to DABF, RNA polymerase II, all bound to DNA. TFIIH is a helicase AND a kinase, so it unwinds DNA near the +1 site, and phosphorylates RNA polymerase II.
Once RNA polymerase II is phosphorylated and where
Phosphorylated on the C-terminal end, looks like the wick on a bomb, activates the RNA poly II. This starts the txn process.
Basal Txn Machinery
The Completed txn complex with D,A,B,F,E,H, and RNA polymerase II. After transcription starts, most txn factors release aside from TFIID and RNA polymerase
Which txn factor first binds to TATAAA box-25
TFIID
Txn factor that is a helicase and kinase, phosphorylates RNA poly II
TFIIH
Order of txn factor binding to DNA and then each other
TFIID, TFIIA, TFIIB, TFIIF + RNA poly II, then TFIIE + TFIIH
Which DNA strand is the promoter found on?
Non-template. TATAAA and TATAAT are found on the 5’-3’ strand.
Which strand, NT or T, is transcribed?
Template, non-coding strand is transcribed, while non-template, coding strand is a mirror image of the RNA
Which strand is nontemplate? Which is template?
Non Template is top, 5’-3’. Template is 3’-5’, on the bottom.
Polymerase Chain Reaction PCR
Experiment that amplifies or makes additional copies of ~1000basepairs of DNA. First, collect DNA from sample, then Heat to denature it, ~90-100 C, breaking it up into single strands. Cool to anneal primers, (~55C) allowing complimentary pairing of primers to DNA, ~20nucleotide sequences. Reheat to ~75C to elongate the DNA, allowing DNA polymerase to synthesize more DNA to make more copies. Can be repeated to make more and more DNA copies, usually ~30 cycles. Improves observation of specimen DNA, able to detect changes in sequences. Can be used with bacterial plasmid to make observable proteins, and detect viral DNA by finding a viral sequence. Can be used to clone RNA as well.
Primers in PCR are
Complimentary, antiparallel, SINGLE-STRANDED. Both on opposite ends, looking at each other.
First, Second, Third Cycle of PCR produces
2 double stranded DNA molecules, then 4, then 8.
Southern Blot
detects specific DNA sequence using electrophoresis, blotting, and nucleic acid probe. Collect DNA, run through elec., transfer to nitrocellulose membrane, then add single stranded DNA probe. Probe is visualized with fluorescent, radioactive, or chemiluminescent enzyme to label. Probe illuminates where it binds to the target sequence. The target sequences will illuminate, and the lit up fragment bands’ intensity and distance traveled are observed. The more copies of the sequences in the fragment, the brighter the bands will be.
DNA footprinting methods and purpose
Studies if and where proteins binds to DNA sequences. Starts with PCR to amplify a select region of DNA, then label 1 end of each piece with a radioactive label. Split into one group with the protein to be observed, Ex. RNA poly. Holoenzyme, and a control group with just DNA. Then, add DNAse to both groups to digest the DNA. If the protein in the experimental group is bound to a certain sequence, it will inhibit digestion of the DNA in that area. Therefore after electrophoresis, the protein group will be missing fragments where the protein bound, and the control will have all fragments present. The protein group will have larger fragments in areas that were not cut due to protein activity. If both groups have same fragments, then no enzyme bound to the DNA. Determines if a protein is present that prevents digestion in the desired area.
How do Prokaryotes regulate transcription?
Txn is regulated using operon sequences near the promoter sequence. Activators or repressor proteins bind based on the type of operon. Can be negative or positive, and inducible or repressible. Operons control txn based on their type.
Negative Inducible
Repressor protein that is active and bound by default, txn is off. Txn can be INDUCED to turn on. Substrate can bind to the repressor, removing it from the operon and inducing Txn until there is not enough substrate to bind to the repressor.
Negative Repressible
Repressor protein that is inactive and unbound by default, txn is on. Txn can be REPRESSED to turn off. Once enough product is produced, it binds to the repressor to activate it, binding it to the operon and turning Txn off until product decreases.
Positive Inducible
Activator protein that is inactive and unbound by default, txn is off. Txn can be INDUCED to turn on. Once substrate binds, activator is activated, binding to operon to start txn until enough substrate is product.
Positive Repressible
Activator protein that is active and bound by default, txn is on. Txn can be REPRESSED to turn off. When product increases, it deactivates the activator, turning txn off until product levels lower.
TFIID and TATA binding proteins play the same role as ________ in prok. txn?
Sigma factor on holoenzyme, TFIID and TBPs bind to the promoter to initiate assembly of BTM.
If TFIIH’s helicase or kinase functions were disrupted, ………………..
Helicase disruption would cause the DNA to not be unwound. Kinase disruption would inhibit the phosphorylation of RNA polymerase. In both cases, transcription would not occur.
If any transcription factor TFII doesn’t bind,
Every subsequent factor would also not bind. Transcription will not occur.
How could one tell if RNA polymerase bound to the promoters, or if the promoter was present at all?
DNA footprinting could check interaction between RNA polymerase and the DNA. Southern Blot could probe for the complimentary sequence of the promoter.
Elongation in Eukaryotic transcription
Once ~10nucleotides are transcribed to RNA, txn factors detach other than RNA polymerase and TFIID. RNA polymerase continues to unwind, transcribe, and rewind DNA as it moves down the sequence.
Termination in Eukaryotic Txn
RNA polymerase will eventually transcribe a poly-adenylation RNA sequence, AAUAAA. The poly-A signal sequence signals termination and the addition of 3’ poly-A tail. ~10-30 nucleotides down are indicated by poly-A signal sequence to cleave the desired RNA at the 3’ end, creating a new 5’ end on the strand behind it. RNA polymerase continues to transcribe until Rat 1 endonuclease detects the new 5’ end, attaching to the remaining RNA and digesting it, moving towards the polymerase. Once it catches up to the RNA polymerase, it digests the RNA until it pushes both substances off of the DNA, ending transcription.
What DNA sequence is required at the end of a gene to cut off desired portion?
TTATTT
Rat 1 endonuclease
Protein that digests nucleic acids, rests on C-terminal domain of RNA polymerase until it detects a new 5’ end.
Why does termination need to be efficient?
Energy is wasted making RNA that is digested again.
Cap and tail modifiers
5’ cap is added as RNA is being made to prevent Rat 1 from attaching and digesting. Poly A tail
Format of an Experiment, including hypothesis, 2 variables, control, data, methods
Include a control group with a placebo. Independent variable changes the dependent. Data should designate span of time for study if necessary, AND how it is going to be obtained. Hypothesis form - If “indep.” changes, then “dependent” will “change” because “reasoning”
How do eukaryotes regulate transcription?
Eukaryotes use cis-acting elements and chromatin regulation to control initiation of txn, as it is the most effective way to decide whether txn starts at all, or not.
Cis-acting elements and how they act with DNA
CSEs are DNA sequences near the gene that they regulate, usually upstream before +1 site. Examples are CCAAT (cat box), GCGCGC… (GC box), usually within 100basepairs from +1 site. BUT some can be found thousands of BPs away, such as enhancers and silencers, which bind to trans-acting factors(activator, repressor).
Enhancer sequences
Allows activators to bind, which causes a loop in DNA, allowing for interaction with the activator and BTM as a result. Transcription occurs in activated areas.
Silencer sequences
Allows repressors to bind to disable it. If silencers are repressed transcription occur, but if unbound, txn is silenced.
Multiple Specific Activators and Enhancers, Gene expression
Only specific activators can bind to specific enhancing sequences. The same enhancers can be used for different genes, so in the case of multiple corresponding activators, more than one enhancer can be active at once. Allows for multiple genes to be expressed at once, or one specific gene in the case of unique enhancers. Specialized gene expression
Insulator sequences
found in between genes and enhancer to block it from activating txn on the gene. Prevents the enhancer from looping with the gene on the other side of the insulator, so only the designated genes are activated.
Activator proteins
Trans acting factor that binds to enhancers to stimulate txn, above the basal level. Impacts the BTM. Has 2 domains, DNA-binding domain for enhancer binding, and activation domain that binds to the BTM’s txn factors to assemble them. Activation domain also modifies chromatin structure to allow for transcription.
Repressor proteins
Trans acting factor that binds to silencers to repress txn. 2 domains, including DNA binding domain, and repressor domain. 6 mechanisms for repressor function.
Competitive DNA binding (repressor mechanism 1)
enhancer and silencer region overlaps. When repressor concentration is high, they bind to the silencer regions more than the activators can bind to the enhancers. Prevents activation of the enhancers, and therefore txn initiation.
Repressor masking (repressor mechanism 2)
Repressor directly binds to activation domain in activator, occupying it and preventing binding to enhancers.
Direct interaction with BTM (repressor mechanism 3)
Repressor binds to a component of the BTM, preventing its assembly or inhibiting its function
Recruiting Chromatin Remodeling Complexes (repressor mechanism 4)
Repressors use Chromatin Remodeling Complexes to alter chromatin to tighten it into heterochromatin, inhibiting the binding of the BTM to the DNA.
Recruiting Histone Deacetylases (repressor mechanism 5)
Repressor uses Histone Deacetylase (HDAC) to remove acetyl groups from N terminals(branch) of histone proteins, tightening the negative DNA wrapped around the resulting more positive histones.
Recruiting methyl transferase (repressor mechanism 6)
Repressor uses Histone Methyl Transferase to methylate histones, adding a CH3 group to histones and causing inhibitors to bind. Prevents txn as a result.
Histone proteins structure and function
Histones Hold DNA together with their positive charge. Consist of H2A, H2B, H3, H4 (2 of each present). 4 sets of 2 histones with DNA wrapped around it ~1.67 times, with ~150 base pairs. All 8 proteins have N and C terminal ends, which are positive by default with amino acids. N terminal is the amino terminal tail, branches out. Connected to a coiled up C terminal, known as the histone fold domain. The 8 proteins and DNA coiled around it are known as the NUCLEOSOME. Another protein, H1, clamps the DNA down to the histones, making the structure a chromatosome.
Formation steps of Chromosomes
Double stranded DNA coils around 8 histones to form nucleosome, then H1 clamps it down to form chromatosome, making 11nm chromatosome beads on a DNA chain structure. This structure is wrapped more to make a 30nm chromatin fiber. Further wrapped to make a monad chromosome, which then interacts with another monad to become dyad. Coiled DNA and histones throughout. Arms known as telomeres, center is centromere.
Heterochromatin
Highly condensed and packed chromatin found at the ends of telomeres and in the center, centromeres. Also found in prophase, prometaphase throughout the whole chromosome. Found in other areas of chromosome THAT ARE NOT BEING FUCKING TRANSCRIBED
Euchromatin
Relatively decondensed and looser chromatin that is expressed, used DNA sequences being transcribed. Used in DNA replication and most of the cell cycle, decondensed chromatin during telophase. Found between telomeres and centromeres in chromosome
Repressors and activators affect chromatin how
Repressors induce heterochromatin, activators induce euchromatin. (by binding to respective sequences)
How can chromatin regulate txn? (activators increasing euchromatin mainly)
Chromatin can interact with High mobility group HMG proteins such as HMGA and HMGB to bind to histones, bending the DNA and allowing txn. HMGN is similar to H1 in structure and can remove H1 to induce euchromatin from loosening. Can modify histone proteins with Histone Acetyl Transferase HAT by adding a negative acetyl group to their N terminal, loosening the negative DNA around the histones with a decreased charge difference. Histone phosphorylation is similar, but instead adds phosphate groups to increase negative charge using a kinase(and phosphatase for inverse). Chromatin remodeling factors can also be used by activators and repressors to remove, remodel, or replace histones to induce or inhibit txn, respectively to the trans-acting factor used.
DNA Affinity Chromatography method and uses
Used to collect proteins that bind to a specific DNA sequence. Starts with PCR to amplify specific DNA region for study(operator, promoter, enhancer, etc.). Then, adhere DNA to agarose beads, insert the DNA and beads into a column(container like tube). After DNA and beads are in column, add nuclear lysate. The protein of interest will be bound to the DNA sequence, and after rinsing with a low-salt wash, all excess and unbound proteins will be removed. Can then be rinsed with medium-salt wash, denaturing the protein and making it unbind from DNA, allowing it to be collected.
Southern Blot uses
Can be used to determine common sequences in DNA between different organisms’ genomes. Can also determine if there is viral DNA present in a sample.
Northern Blot method and uses
Similar to Southern Blot, BUT uses RNA instead of DNA. Determines whether or not specific RNA sequences are present, and therefore if a gene is expressed or not. Searches for specific sequence of RNA, which determines if transcription occurred for the target gene (expression as a result). Run RNA through Elec., transfer to nitrocellulose membrane, probe with DNA nucleic acid probe(DNA probe is more stable than RNA probe), and examine if target sequences are present based off of illumination distance and intensity in each fragment. Can detect if txn occurred in the area that a sample was taken from. Only observes 1 gene, ~20nucleotides probed. Can detect the presence of RNA viruses.
