Bio final
Define post-transcriptional control.
(after mRNA production)
mutation in a regulatory sequence
1, change timing of gene expression/cell division/function 2,organism's phsyiology 3, location, which cells can express gene 4, level of gene expression/ amt of protein made
Transcriptional factor/regulators are same thing.
Activator: turn on gene expression. Bring in mediator and general transcription factors and RNA poly to initiate transcription. Bring in Hats and chromatin remodeling factors Repressor: turn off, block activators by binding in same region, HDACs to condense chromatin.
induced pluripotent stem cells
Adult differentiated cells that have been engineered to express transcription factors which reverts them to embryonic-like stem cells. Yamanaka factors
Adult stem cells
Adult stem cells are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues.
Embryonic Stem cells
After egg fertilized by sperm
cortisol
As discussed earlier in this chapter, when this hormone is present, liver cells increase the expression of many genes, including those that allow the liver to produce glucose in response to starvation or prolonged stress. To switch on such cortisol-responsive genes, the cortisol receptor—a transcription regulator—first forms a complex with a molecule of cortisol. This cortisol-receptor complex then binds to a regulatory sequence in the DNA of each cortisol-responsive gene. When the cortisol concentration decreases again, the expression of all of these genes drops to normal levels. In this way, a single tran- scription regulator can coordinate the expression of many different genes
homologous recombination (gene knockout)
At DNA level, we eliminate all or part of DNA required to make protein. Supply the information to repair damage to cell, eliminate coding sequence. we can use PCR and put in a selectable marker. we can also replace sequence with mutation
Housekeeping proteins
DNA poly, RNA polymerase, Metabolic enzymes, stucture elements, found in most cell types
Explain how eukaryotic activator proteins can enhance transcription even when bound to sequences hundreds or thousands of nucleotide pairs away from a gene's promoter.
DNA wraps around you get these loops of these activator proteins contacting mediator and forming pre-initiating complex.
RNA SEQ
Determine which genes are expressed (relative levels), can sequence all of mRNA, no need to know sequences ahead of time, all RNA inside cell. Disadvantage is that it is expensive.
mass spectrometry
Determine which proteins are present in a sample
CRISPR (knockouts but can also put in mutation, altering, repair gene)
Discovered through bacteria. CAS9 cuts. guide RNA that matches target. Cas9 unzips dna, matches RNA, and uses scissors to DNA. CAS9 and guide RNA base pari with DNA that matches and the protein cleaves DNA leaving break. Cause frameshift and premature stop codon, don't make full protein.
RNA interference (i)
Early adaptive immune system, foreign RNA is degraded. use sequences from viral RNA against itself. subtle difference is that it is cleaved by dicer, and bind with RISC, leaving single strand to search for complementary and base pari with forge in RnA.
Mutation within a gene
Errors that arise from mistakes during DNA replication and repair.
Heterochromatin
Eukaryotic chromatin that remains highly compacted during interphase and is generally not transcribed. High levels of methylation. "silencing"
Differentiate between general transcription factors and transcription regulators and compare how they participate in gene expression.
General Transcription factors are required for the binding of RNA polymerase to the core promoter and its progression elongation stage; necessary for transcription to occur general transcript factors are required for transcription to occur and regulatory transcription factors use control elements, regulatory elements and regulatory sequences to regulate specific genes during transcription Transcription factors bind at different cites (will activate or inhibit transcription, it recognizes a very specific sequence) while transcription requires GTF, additional modulate and effect activity of GTF (get polymerase to right place).
Reporter genes
Green fluorescent protein, Lac Z, luciferase enzyme. At the sequence level, at DNA, we insert into organism were interested in studying. We detect this light using equipment in lab. where/when a gene is expressed in a cell
Review how reporter fusion proteins can be used to track the location of proteins within cells.
If we want to know where a protein is localized, we hook up GFP to our protein. Called fusion b/c we contain our gene with gene fpr GFP. Start at DNA level, We put coding sequence of fav gene (5'-3') with GFP, made into MRNA (combined sequence), monstronic, one RNA to one protein, that protein have our gene sequence and GFP sequences. Protein is dragging GFP around the cell. Look at where protein is localized. This can be done in living organisms. We can hook up our fav gene to other sequences "tags." To express specifically in muscles, we put in promoter elements that pull muscle cells (muscle specific transcriptional factor), promoter region will bind transcription factor specifically for muscle cells. Example: express a fusion protein of 35 glutamines followed by GFP, then place the sequence for the fusion protein downstream from a muscle specific promoter. Look at protein and see where its localized. Can be done using living organisms and not limited to GFP.
illumenia sequencing
Illumina sequencing, smiliar to sanger (PCR) on a glass slide we place out DNA that need to be sequenced. We only include fluorescent and reversible NTPs, one base per cycle. We use reversible NTPs we can get rid of things and add another base only doing one
Explain how the accessibility of the AUG start codon can determine whether a message will be translated into protein or not.
In Prokaryotes, if the ribosome binding site is accessible we can make protein, but if repressor blocks shine dalgarno, no protein will be made. Euk blocks on other side For bacteria, Ribosome can't access start codon as it is in hairpin but ribosome is exposed when it melts as it gets inside human cell
combinatorial control
In eukaryotes, such regulatory inputs have been amplified, so that a typical gene is controlled by dozens of transcription regulators that bind to regulatory sequences that may be spread over tens of thousands of nucleotide pairs. Together, these regulators direct the assembly of the Mediator, chromatin-remodeling complexes, histone- modifying enzymes, general transcripton factors, and, ultimately, RNA polymerase
Asymmetric cell division
In this case, the cell direct fate determining factors to one side of cell. Cell fate determinants are Proteins (transcription factors), they can be small regulatory RNAs, mRNA (localized within cell, one has this one one does not, drive differences in proteins. Seggregation of cell fate factors
Describe how interaction with a small effector molecule and/or phosphorylation can alter the function of a transcription factor.
Phosphorylation can affect gene expression in different ways and it depends on the circumstance whether gene expression is turned on or off. One instance is that a transcription factor can be phosphorylated, which prevents the transcription factor from entering the nucleus to turn on genes. (The example given for this in the lecture was Yki.) Another instance is that there can be phosphorylation of proteins that bind to transcription factors. (The example given for this in the lecture was Rb for retinoblastoma patients.) Retinoblastoma patients can have mutations that prevent Rb from inhibiting the transcription regulator that it binds to, thus resulting in cell division. Additonally, It can do both. For example, in the Rb protein, the phosphorylation of it can cause it to not suppress the activator of the gene which causes expression. While another example would be the phosphorylation of another TF that when it is phosphorylated, it cannot enter the nucleus and thus the phos is repressing. In short it depends on the environment and the specific gene
Relate how positive feedback, DNA methylation, and histone modification allow differentiated cells to maintain their identity and to pass this information to daughter cells during cell division.
Positive feedback loop: where a master transcription regulator activates transcription of its own gene, in addition to that of other cell-type- specific genes. Each time a cell divides, the regulator is distributed to both daughter cells, where it continues to stimulate the positive feedback loop (Figure 8−22). The continued stimulation ensures that the regulator will continue to be produced in subsequent cell generations. The Ey protein and the transcription regulators involved in the generation of ES cells and iPS cells take part in such positive feedback loops (see Figure 8-18B). Positive feedback is crucial for establishing the "self-sustaining" circuits of gene expression that allow a cell to commit to a particular fate—and then to transmit that decision to its progeny.. Epigenetics: Change how genes are turned on and off, acgt sequence order not changed. Marks to histones in terms of acetylation, methylation, phosphorylation. DNA methylation found in CG pairs, cytosine gets methylated; maintain methylation so cell can remember what state its in. Enzymes maintain methylation--turn genes off in this area. CPG islands are in which CG pairs are methylated lots of them. Also enforced by histone modifications: When a cell replicates its DNA, each daughter double helix receives half of its parent's histone proteins, which contain the covalent modifications that were present on the parent chromosome. Enzymes responsible for these modifications may bind to the parental histones and confer the same modifications to the new histones nearby.
Transcription factors
Proteins that will bind DNA and can bind/inhibit transcription. Activators/repressors. Bind to enhancer regions. They recognize a very specific sequence/specific sequence. One can turn on many genes. Not like general transcription factors at promoter region that get RNA poly to bind to activate transcription.
Summarize how eukaryotic repressor proteins decrease transcription.
Repressors bind to the DNA and physically block activator form binding, or bind directly to activator. Goal is to prevent complex from forming and preventing initiation of transcription. by inhibiting assembly of the transcriptional initiation complex, or they can recruit histone-modifying complexes like deacetylases, which remove the activating acetyl groups from nearby histones.
Sanger sequencing
Sanger sequencing based on PCR, (we only use one primer), ddNTPs (prevent chain from extending, chain terminating). Our goal here is to read DNA bases. If we terminate the chain, we can see what base was added last. Gel exlextropheorsis allows separation of DNA fragments by size. Top of gel is larger fragments. We use labeled bases now. Chain terminating ddntps are labelled fluorescently, and then the machine reads these different colors. Normal DNTPs added in excess. By chance, we get the incorporation of labeled ddntp. Sequencing human genome powered with disease analysis
exon shuffling (MIX AND MATCH NEW PROTEINS)
Scientists have taken advantage of these relatively modular protein domains to control gene regulation. They can put pieces together. Protein domain is a region of a polypeptide chain that can fold independently and usually has its own cellular fate. Proteins can be created from a parts list, mix and matches by knowing what these domains do. In evolution, introns are important because we can pop in new exons and create new proteins. Exons can jump in intronic regions, because splicing can occur, we splice together different exons. As long as the splicing event doesnt alter the reading frame the new exon will become part of the protein. This allows a mix and match of proteins that create new proteins
Point Mutations
Silent: no change in Amino acid, Nonsense-change to a premature stop codon, Missense-change in amino acid (conservative, change to an amino acid with sim. Properties to original), and missense (diff properties), Read Through-change in stop codon to something that would be read as a amino acid, adds extra. -Amino acids change alters secondary and tertiary structure, it is not always good ro bad. -Insertions and deletionsL causes frameshifts will add or remove amino acids. -Mutations to splice sites can alter exon inclusion and amino acids in the protein (only EUK)
Euchromatin
The less condensed form of eukaryotic chromatin that is available for transcription. high levels of acetylation
Master Regulators
They can turn on many different genes. They can drive their own expression but can also drive each other's expression. They have overlapped genes they can turn on, they also turn each other on. Single transcription factors can bind many different regions in genome. Can drive formation of entire structure.
Waddington's landscape
Totipotent (zygote), Pluripotent (embryonic stem and germ cells egg n sperm), Multipotent (adult stem cells), Unipotent (specialized cell types). Changes in gene expression dictate changes in these cells.
Yamanaka factors
Turning on certain transcription factor pushes cell up hill, adult cell ---> embryonic cell state. Three factors Oct4, sox2, KLF4.
miRNAs (micro) Reduce Amt of protein RISC can be reused!!
Use complementary base pairing to seek out target. Can repress translation and target for destruction. Protein production control. Interact with RISC and unzip double stranded RNA. Search for great match and then recruit in proteins that degrade the RNA. with less extensive match it represses translation.
RT-PCR
Used to determine mRNA concentration, fast and cheap. disadvantage is need to know mRNA sequence, limited to looking at a few mRNAs at a time. Allows you to detect specific mRNAs
The Formation of an Entire Organ Can Be Triggered by a Single Transcription Regulator
a single transcription regulator called Ey triggers the differentiation of all of the specialized cell types that come together to form the eye. How the Ey protein coordinates the specification of each type of cell found in the eye—and directs their proper organization in three-dimensional space—is an actively studied topic in developmental biology. In essence, however, Ey functions like the transcription regulators we have already discussed, controlling the expression of multiple genes by binding to DNA sequences in their regulatory regions. Some of the genes controlled by Ey encode additional transcription regulators that, in turn, control the expression of other genes. In this way, the action of this master transcrip- tion regulator, which sits at the apex of a regulatory network like the one shown in Figure 8−18, produces a cascade of regulators that, working in combination, lead to the formation of an organized group of many differ- ent types of cells. One can begin to imagine how, by repeated applications of this principle, an organism as complex as a fly—or a human—progres- sively self-assembles, cell by cell, tissue by tissue, and organ by organ.
Compare how eukaryotic activator and repressor proteins exploit the mechanisms that regulate chromatin packaging to enhance or suppress transcription. (HATs and HDACs)
histone acetyltransferases promotes the attachment of acetyl groups to selected lysines in the tail of histone proteins; these acetyl groups themselves attract proteins that promote transcription, including some of the general transcription factors, And the recruitment of chromatin-remodeling complexes makes nearby DNA more accessible. These actions enhance the efficiency of transcription initiation. HATS neutralizes charge and loosens. gene repressor proteins can modify chromatin in ways that reduce the efficiency of transcription initiation. For example, many repressors attract histone deacetylases—enzymes that remove the acetyl groups from histone tails, thereby reversing the positive effects that acetylation has on transcription initiation.
CAN HANDLE mutation
intergenic regions, introns
RNAi Knockout
knocking down using our own RNA, post transcriptional study effects of knocking it down. Cheap and easy, but is transient, and not complete knockout. We make double stranded RNA from bacterial plasmid, we put 2 promoters.
Ribosome profiling
look at mRNAs, trap ribosome with mRNAs, sequence mRNAs
in situ hybridization (find where RNA is inside cell)
looking at RNA (DNA) fixed in a tissue or a cell. To determine in a cell where messenger RNA is and how many. Can look at newly-forming RNA. Disadvantage is need to know sequence of mRNA and need fixed sample, can only look at a couple samples at a time. use fluorescence and probe that hybridize with mRNA, DNA where gene is located on a chromosome.
If we want to think about where a gene is expressed
reporter or fusion protein. Reporter genes mimic which
stem cells
unspecialized cells that have potency
