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SectionNotesweek6 - Section Notes, wk 6 GSI: Nikki...

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Unformatted text preview: Section Notes, wk 6 GSI: Nikki Kong * This is going to be the last detailed section notes posted on bSpace. From now on, only supplementary figures + comments and an outline of discussion during section will be posted DNA  ­> RNA  ­> Protein Multiple levels of regulation: micro RNA  ­> RNA; Protein  ­> post ­translational modifications, etc Transcription in prokaryotes versus eukaryotes Bacteria 1 compartment for transcription and translation, which can be coupled Eukaryotes Nucleus separates transcription and translation, the latter occurs in the cytoplasm 1 mRNA ­no introns, can be polycistronic mRNA are monocistronic, and needs to be spliced because of introns No 5’ cap 5’ cap for export out of the nucleus Poly (A) tail is mainly for degradation Poly (A) tail is mainly for export, translation initiation; shortened tail signals for destruction One RNAPol makes mRNA, tRNA, and mRNA is processed more; have rRNA specialized polymerase (RNA Pol II) Comparing RNAP in E. coli to DNA polymerase:  ­> RNAP also uses DNA template, but only the non ­coding strand serves as template  ­> 5’  ­> 3’ polarity, Mg+2 requirement are the same  ­> no primer needed for making RNA—de novo synthesis  ­> Like DNA pol, RNAP also generates positive supercoils in front of the transcription bubble, which needs to be resolved by topoisomerases  ­> rNTPs drive chain elongation instead of dNTPs  ­> Also very processive like DNA pol, because once it falls off, needs to find the promoter sequences again  ­> 10^5 less accurate than DNA pol, because it doesn’t have to be very accurate: mRNAs are made in high copy numbers, degraded faster, mistakes in the wobble base or introns don’t matter, etc RNAP in prokaryotes: mRNA, rRNA, tRNA Core enzyme: alpha ­contacting activators; two in the enzyme beta – catalytic site beta’ ­ binds DNA omega ­ holds complex together Holoenzyme: all of the above + sigma: sequence specific recognition of promoters Most sigma factors recognize consensus sequences at  ­10 and  ­35 upstream from the transcription start site (TSS); strong promoters are those that look like the consensus sequence. There are specialized sigma factors to form different holoenzymes Core binds non specific DNA sequences tightly; holoenzyme binds specific seuqneces not as tightly, for promoter clearance later DNA footprinting: in vitro experiment to test where a sequence specific protein (such as RNAP or another transcription factor) binds a piece of DNA Steps: 1. Radiolabel at one end, usually 5’, of the DNA of interest with 32P, usually with a kinase reaction. This is for visualization 2. Two tubes, one is your negative control which is just the labeled DNA, the other is your DNA + the purified protein of interest. 3. Add DNaseI, which cuts DNA nonspecifically, at a really low concentration to ensure that on average, each strand is only cut once 4. Separate DNA from protein (protease treatment e.g.) 5. Denature the strands by heat (just like Southernblotting) 6. Separate the digested fragments by size via gel electrophoresis, which will reveal pieces of DNA extending from the radiolabeled end to the site of DNaseI cleavage. If your DNA is bound by the protein of interest, the protein will block DNaseI from cutting the DNA at the position where it binds, and thus tube 2 will contain a pattern that shows some missing fragments comparing to tube 1 – the protein’s footprint To obtain the sequence of the binding site, you will need to sequence the DNA fragment used in the above experiments: Maxam ­Gilbert sequencing Transcription in E. coli 1. Initiation: Scanning holoenzyme looks for  ­35 and  ­10 consensus sequences in the promoter with the help of sigma once found promoter sequence, Holo enz becomes the closed complex and binds to DNA tightly (the step captured in DNA footprinting) When rNTPs are around, hydrolysis of these high energy phosphate bonds could occur, a few nt of the mRNA are synthesized, transcription bubble opens and sigma dissociates the remaining core enzyme can escape the promoter and elongate 2. Elongation: RNAP generates positive supercoils in front of the bubble, and negative supercoils behind; the mRNA synthesized always maintain ~8bp RNA ­DNA hybrid at the bubble. Requirements for elongation: non ­coding DNA template, rNTP, Mg+2, no primers are needed 3. Termination A: Rho ­independent—two sequence requirements: i. hairpin that can be formed by mirror sequences on either side of some non complementary sequences (rotational symmetry) ii. Three U’s right after the hairpin. Because the A(DNA) ­U(mRNA) bond is weak, after pausing at the hairpin, RNAP would encounter these weak bonds and fall off B. Rho ­dependent: 5’  ­> 3’ hexameric helicase. It loads onto a C ­rich rho utilization site (rut) on the mRNA and translocates along the mRNA to get it off of the DNA template. Rho translocates by contacting each RNA nt with one subunit in the hexameric ring, which goes through 4 catalytic states: ATP ­ binding (coordinated with RNA binding), ATP ­hydrolysis, exchange (translocation), and product (exit). Therefore, it uses 1 ATP molecule per nt of translocation Regulation of transcription: simplified Positive regulation: an activator is involved Negative regulation: a repressor is involved Both could either require an inducer to bind DNA, or fall of DNA because of an inducer binding changing the conformation Lac operon in E. coli: polycistronic, advantageous since the protein products made from this mRNA participate in the same pathway, similar functions  ­ ­> negative regulation: Lac repressor, which isn’t part of the Lac operon, is expressed when glucose is the main nutrient. It’s made from the LacI gene, and binds to operator sites in the Lac operon  ­> the homotetrameric Lac repressor binds the primary operator O1 and one of the two secondary operator sequences, O2 and O3. Binding of two sites increases the local concentration of the repressor  ­> Once lactose is present (or lactose mimetic IPTG), it binds to Lac repressor, inducing a conformation change and the repressor dissociates from DNA. Lac operon is activated to metabolize lactose  ­ ­> positive regulation: catabolite activator protein, which normally cannot bind DNA  ­> low glucose in nutrient means there’s no breakdown product from glucose, so adenylate cyclase is not inhibited.  ­> so under low glucose condition, adenylate cyclase can convert ATP to cAMP  ­> cAMP binds to CAP, inducing a conformational change, so CAP can bind to an enhancer site in the Lac operon. Lac ZYA genes can then be transcribed and polypeptides can be made to metabolize lactose.  ­ ­> When both glucose and lactose are present, CAP doesn’t bind DNA, but Lac repressor doesn’t either, so some Lac mRNAs are synthesized. But when no glucose is around but lactose is, positive regulation is on and negative regulation is off. You will get a lot of Lac mRNA transcribed. Each gene in this polycistronic mRNA will have its own translation initiation signal sequence, and can recruit ribosomes to start synthesizing polypeptides! ...
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