BSCI222 Lecture 7 (9/24/13)- HW Chapter 17 (to review for the test): 2, 3, 9, 13, 15, 25. Turn in next Wednesday with additional HW (will be assigned Thursday). Test will be on material through today.- Attenuation requires translation, so what book calls 5’ untranslated, should be called leader peptide.- Control of Gene Expression in Eukaryotes (Chapter 17)- All the different places where there could be molecular interventions that regulate gene expression, cost versus benefit.- 7 potential points of regulation (EXAM: Essay question, name 4 and give example of each)o Modification of chromatin structure: DNA is packaged up, wrapped around a coreof 8 different histone proteins (nucleosomes are the spools, full of histone proteins; look like wire wrapped around spools). While it’s wrapped tightly around, relatively inaccessible to other proteins. Have to loosen the histones’ grip to get to the DNA, for RNA polymerase to go through. Requires methylation or acetylation of the histones. About 150 base pairs per nucleosome, and another 50 in between nucleosomes. The nucleosomes are a form of protection; not there when the gene is being actively transcribed. Some regions though, just because of their DNA sequence, tend not to have nucleosomes (called Nucleosome Deficient Regions). There are also proteins that can get in there and move the nucleosome along the sequence, after which transcription factors can get in and bind to the region and stabilize it. Now we can form a Pre-Initiation Complex (PIC): all the proteins that bind at the promoter in order to initiate transcription, doing things that allow the RNA polymerase to come in and begin to transcribe through that region.  Number of proteins capable of moving the nucleosomes from where transcription wants to happen. Specialized on the job of remodeling the chromosome. They can modify the histone or they can modify the DNA; scientists have mostly been studying modifications of the histone. Methylation and acetylation. Methylation of histone: amino acid, Hydrogens get replaced with methyl groups. This helps to attract other remodeling factors; they recognize it as a signal that this region needs to be modified. Acetylation: amino acid has a charge (Lysine, pretty highly positive, makes it good for interacting with DNA which is negative, this iswhat makes the DNA and the histones bind so tightly), acetylation puts big negatively charged Oxygen instead of a Hydrogen, weakens the bond because now the histone is less positive, less complementary charges. Arabidopsis, FLC protein represses flowering when expressed, by inhibiting FT gene (which would otherwise promote LFY, getting flowers). (Diagram: arrows mean promotes, line that ends in a / means inhibits) When FLD is expressed, it deacetylates the chromatin aroundFLC, blocking its expression (tightens the histone, transcriptor proteins can’t get in). Thus you get reduced FLC, and the plant flowers. Can also methylize the DNA itself. In humans, close to 90% of CpG sequences are methylated, and clusters of them are found near genes. Methylate the C, tends to repress transcription; have a whole cluster of them, will completely block transcription. Directly blocks transcription factors, binds other proteins attracted to it (methyl-CpG-binding domain proteins -- MBDs), and the MBDs recruit deacetylases. Would have to reverse the methylation in order to allow transcription. (Demethylated or acetylated is looser binding, methylated or deacetylated is tighter binding).Called epigenetic modifications, outside of the classical genetic ACTG process. Starting to see evidence that these kinds of mechanisms can also be inherited, epigenetic inheritance.  siRNA induced transcriptional silencing: Small interfering RNA, binds to a protein and becomes its recognition part, and attracts a methylation enzyme. o Initiation of Transcription: The Pre-Initiation Complex, lots of proteins that have to assemble, TATA-binding protein, TF2D, etc. The PIC looks different for different genes, never remember all the subunits, but always have a core promoter (TATA-binding and etc.) and an upstream regulatory promoter (binds other proteins, assist) and even more, farther upstream. Requires direct contact between the proteins in order for the activation of transcription to occur. The GAL4 system: part of the galactose operon in yeast. GAL4 is a protein that binds to the upstream activation sequence as an enhancer. GAL80 binds to that dimer of GAL4, and prevents its interaction with the transcription apparatus. If there is galactose in the environment, it will bind to GAL3, which brings about a change in GAL80 and removes it, therefore allowing GAL4 to interact with the basal transcription complex and allow transcription. When it’s there, GAL80 wedges itself between GAL4 and the basal transcription complex. GAL4 expression construct: start expressing green fluorescent protein wherever GAL4 is being expressed, can be done with any gene. Can now follow the development of a particular organ. Can put in another construct,again with the upstream activation sequence, again will be activated by GAL4, but this time with a different gene for a different fluorescent protein, will only be expressed in that tissue that you want.  Enhancers and Insulators: insulators (also called boundary element) prevent the action of an enhancer, from acting on any other gene beside the one it’s supposed to. Boundary elements hold to the nuclear lamina, hold the enhancers back.  Polymerase pausing: Most understanding of transcriptional complexes is from yeast, where transcription proceeds without stopping to the end ofthe gene. In metazoans, there is evidence for regulated pausing of Polymerase III during transcription. To know where the polymerase is spending its time on a gene, we cross-link all of the proteins that are attached to the DNA, attach them tightly with covalent cross-links by treating with a form of formaldehyde. Then chop up the DNA and use an antibody to recover the particular protein that we’re interested in, in this case the bit of DNA to which RNA polymerase is bound. Find where the polymerase spends most of its time. Different for different genes. On paused genes, it binds and transcribes a little but then pauses right at the beginning, waits for a long time for an additional signal, then continues through the entire thing without stopping again. Why would this evolve? A hundred or more