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Biological Sciences
Dan Riggs

Lec 11 Transcription factors have both dna binding and activation. They have binding domain and transcription factors that can turn on genes or repress it. So one allows for it to bind for the right sequence and one affects its function. In the major groove of the helix scructure allows for nucleic acids to be more easily accessible. Within that major groove, the nitrogenous bases are easily accessible. Many different classes of transcription factor: Major ones: zinc finger TF – will form a loop facing the major groove. The helix loop helix transcription factor bind as dimers, usually in the form as heterodimers. Leucine is a hydrophobic side chain. Leucine is found on every 7 amino acid. The leucine motif is to zip up.. the interaction between the molecule is to shield themselves from the water. This motif is used in the bZIP factor. B domain Is the basic domain. It mediates the specificity of DNA binding. For microarray, you get a robot that samples all genes. Another robot uses a printing process to print spots on a solid surface. The grid or chip generated has specific sequences in specific grid positions. The detection and quantitation is based on the fluorescent labeled cDNA that hybridizes to the grid spots. It is a hybridizing technique. If there’s microarray sites where they will complement both red and green cDNA, you’ll get a yellow site. In a red spot, beta galtosidase expressed when lactose is present and if green, it means it’s absent. If yellow, it means beta galactosidase is rexpressed under both conditions. Microarray can be used to find which genes are regulated by transcription factors. In a mutant, no transcription factor and if the transcription factor activates that gene, then no target transcription take place. Up list are genes that are up regulated in the mutant, they are expressed at a higher level in the mutant. In the wildtype, transcription factor negatively regulates them. So when they’re not present, theyre knocked up? Lec 12 - Gene regulation Epigenetics alter gene expression but don’t change te sequence of DNA. The histones that package that DNA are often modified. Often what happens is a methyl group gets added to the histone or dna (or removed). Most of the cell cycle, chromatin is a decondensed state like a bowl of spaghetti. Other times, it is incredibly condensed like a bread stick. Most of the time the chromatin in the decondensed state, is called interphase. Mitosis the time of segregation of the condensed chromosomes go on. They have to go to the condensed, silent state to an active decondensed state. Most of the dna from daughter cell is labeled as euchromatin which means it returns to dispersed reconfiguration state after mitosis. Some of the chromatin remain condensed is known as the heterochromatin. =constitutive (means always) heterochromatin. Some fraction of the chromosome remains condensed no matter what. E.g. repetitive dna of the centromeres. =faculatative heterochromatin remains condensed depending on the cell stage. E.g. x chromasomes in females. Human females have 2 x chromosomes. And males got one x chromsosome from mom and one y from dad. But females really only one active gene. =one of the two x chromosome in every cell will be randomly inactivated. The calico cat has a mosaic of fur type. Coat color controlled by several genes. One controlled by the x chromosome will be for black. The other for orange coat. =random activation during activation, will cause some regions that are black and some are orange. XIST gene is a very large gene that encodes a very large non-coding RNA, meaning it wont transcribe to protein. There are many copies of it and will encode the entire chromosome. Methyl dna will come in and shut down one of the x chromosome. In fig 12-14, the Ac are the groups that added, sometimes lysine, serine, etc. The n terminus tail of H3, you can see large letters of A and R, which is for activating or repressing. 12-17, heterochromatinization packaging of chromatin into a very tight structure. Dicer wil cut the rna into pieces. =the guide strand is coming with a protein complex to a place where it needs to be condensed. =as rna is rolling off, there’s a sequence complex that is being produced. =suv39h1 is a histone methyl transferase. It transfers methyl groups to some of the histone molecules. K=one letter code for LYS. H3 is for histone 3. The boundary element serves as a stop sign for heterochromatin to stop at this point. It is to keep adjacent genes to be condensed, so they don’t become transcriptionally active. Hormones are key regulated of gene expression. Cortisol is escorted into the cytoplasm and interact with soluble receptors. This will translocate to the nucleus. Then you’ll prevent certain gene activations. The steroids induce or repress the expression of genes. E.g. transport processes or growth home, etc. Glucocorticoid receptor binds to the GRE- glucocorticoid response element. So when GR binds to GRE, CPB is a histone acetyltransferase. It transfers acetyl groups to certain histone molecules. Note: tata box seems to be tightly wounded, so it’s not readily accessible. So the goal is to loosen the nucleus from the tata box to be revealed to ttb? Once CBP acetyls histones, it changes. SWI/SNF is a chromatin remodeling complex. General transcription factors will then bind when TATA box is revealed. The steroid binds receptor, finds cis sequence, events happen to open chromatin to allow RNA polymerase assemble and eventually allow transcription(?) fig 12-49. You acertylate to make things active. So HDAC takes off the acetyl group. =to prevent this from reversing, methylation of formerly acetyl histones will be in a tight states, denying polymeraization and transcription is essentially shut off. =so methyl group is the seal to put it into a tight state to deter access. Chromatin regulators will come in and facilitate the loop? Alternative splicing is to make multiple mrna from the same type of transcript. Imagine you have a heterogeneous rna. E3B E3A are exons highlighted in blue. In some cases, in say a tissue of fibroblast, exons need to be present in all mRNA. In other cells, 2 exons must be spliced out so that we get a somewhat different mRna like a liver mrna. In order for splicing to take place. Splicsome must make specific cuts and then put the introns back together. Snrps, etc, have to be attracted to splice sites. They are attracted to the splice sites due to regions of splicing enhancers (as well as supressors). If spliceosome is not recruited, exon 2 will be removed with the adjacent exons. When fertilization takes place, it is associated with CPEB phosphorrylation. Maskin is a molecule that binds to CPEB and binds to eukaryotic eIF4E. CPEB holds it so that ribosomes cant transcribe it. When CPEB is phosphorylated, the cap structure will now be able to the ribosome? Poly a polymerase will elongate the tail. The tail is what provides stability. Ferritin is to control iron homeostasis. But it can be toxic. IRE – iron response element that is a structure within 5’ untranslated region of the ferritin rna. It creates a binding site of the regulated protein. When you have low iron concentration you don’t really need ferritin, so the Iron regulated protein prevents it from binding to mRNA? If there’s a lot of iron, which is toxic, iron is the brown square will bind to the IRP (fig 12-55), the binding of small molecule changes the structure and lets go of the iron response unit. The poly a tail is at the end of eukaryotic molecules. 3/5/13 Lec 10? The ends of Eukaryotic chromosomes are linear chromosomes. Telomere repetes exists at the ends. They are useful for capping the end of the chromosomes. Because the ends are linear, this causes a problem. Since dna polymerase cannot initiate change and can only operate 5’ to 3’ direction. This leaves a 5’ at the newly synthesized strand. Fig 12-20a, at the top you have double black stranded dna. After replication takes place, at the 5’ end, The processing of the 5’ ends occurs will generate a little 3’ strand. Since the telomeres is repeats of the strnads. This will allow the strand to fold on itself and sine it is homologus, it can base pair just fine. Capping proteins will come and protect the ends. There is essentially no free ends for anything to come in and attach to. Telomerase replicates telomeric regions. It’s not just an enzyme but an rna complex – it carries an rna molecule that helps replication of the chromosome. 12-20c note there’s base pairing and note the telomeric telomerase acts as a reverse transcriptase. Using rna template, it can make a copy. It makes a dna copy based on the rna telomerase. After replication you have a sequence. Telomerase will come in and elongate the 3’ template. This will give the system enough room for primase to come in. Dna polymerase and ligase will come extend the chain and the primer will remove. What will happen if telomerase does not exist? If captain proteins are no longer present – fusion of ends may happen. If we put two chromosomes together, we have a dicentric chromosome. Di meaning 2, centric from centromeres. So it has 2 centromeres. So in normal cell division, microtubules will pull one direction, the other one will be pulled to the other direction. The chromosomes wont segregate properly and will be essentially torn in half. As we age, telomerase activity will become lower. Cancer often results from telomerase activity enhanced. As we age, telomerase are designed to essentially have cell death in order to prevent them to becoming cancer cells. Dna repair – replisome operates at 1000nucleotides/sec. so imagine an assembly line, doing 1000/sec. so you would expect there to possibly be errors. In addition to their 5’ to 3’ synthesis function, they might contain nuclease activities. Nuclease does essentially the opposite of synthesis. It destroys it. Fig 13-16, at the top is 5’ to 3’ exonuclease activity. Exo means outside. 5’ to 3’ exonuclease means you nibble from 5’ to 3’. You’re acting on the free 5’ end to the 3’ end. Indo nuclease (e.g. restriction dna) Bottom one, 3’ to 5’ activity and is needed for proofreading. Proofreading like when you’re erasing your mistake and rewrite it. So, if you mistakenly put in a T, so at that point, the hydroxyl end is going down making the geometry wrong. The exonuclease finds where the pairs are correct and go there and thus will restart the base pairing to get it right this time. Dna polymerase will realize what is in front of it is not dna? So it replaces the rna with deoxynucleotides. Fig 13-17 , the distance is about 11 nanometres. The pitch angle is the nitrogenous base to the figure the sugar is attached. If an incorrect base pair occurs, this will give us a distorted geometry. The strand is essentially kinked. When that happens, repair processes is activated. It will reverse engine, go back to where it is correct and proceed from there again. Pyrimidine dimer, nitrogenous bases will be linked. So note how there’s nothing for it to pair with on the other side. Uv light can promote this. Suppose a lesion occurs, how will you repair it. Damae anerpaired on the NERP – nucleotide excision repair pathway. It gets rid of the part with mistake. The transcription coupled pathway where rna polymerase goes along. Global pathway is a slower mechanism. BERP is a base excision repair pathway. Both of these pathways use enzymes to remove the area containing the region an dreplace it by using rna polymerase. Transcription coupled pathway, as rna polymerase is going along and finds a distortion it will stall at this point and this will attract some factors. Once helix distortion sees it, it will stop there and this will attract other things that will target transcription factors. Components of transcription factor 2h. on both sides of the region, note how it is unwound. Some other activites like endonucleases will cut both 3’ to 5’ of the region. After the single strand is released, along comes dna polymerase and will correctly fill in the nucleotide. Dna ligase comes in and axe and gets rid of the damaged region. Base excision repair pathway. Note in 13-27, u doesn’t base pair with g. Glycosylases will come in (it is base specific). Glycosylase will cleave a glycosytic bond. Note the sugar phosphate backbone is still attached. First there’s an endonuclease at the site. Next is a phosphodiester activity will trim it. But note we will now have a perfect area for dna polymerase to replicate. Dna ligase comes in, seals it, and repair has now been completed. Suppose dna polymerase has made
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