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Unformatted text preview: Section Notes Wk 7 GSI: Nikki Kong Transcription regulation in Prokaryotes continued: Trp operon: synthesis (Lac operon regulation is involved in metabolism). This is an example of negative feedback regulation: since there’s already Trp around, you don’t want to make more Trp * Important elements: promoter, operator, leader sequence, attenuator.  ­ ­> 1st negative regulation: Trp repressor that binds to the operator. Only binds major groove when tryptophan is around to change the conformation of the alpha ­ helices in the repressor.  ­ ­> 2nd negative regulation: only achievable in bacteria because translation and transcription are coupled. Three sequence elements are important: A. Leader sequence = Region 1, encodes a full 14 a.a. peptide that has both start AUG codon and a stop codon, as well as 2 UGG codons for tryptophan B. Attenuator = Regions 2, 3, 4, which all have certain level of complementarity, so 2/3 can form a hairpin (antiterminator), and 3/4 can form a hairpin, the former being more preferable. (Side note: 1/2 can also pair and is the strongest; so when ribosome isn’t around or when translation isn’t started, 1/2 pair and 3/4 pair, the end result is still transcription termination. See supplementary fig) C. UUU’s at the end of region 4; transcription terminator Trp present: leader sequence is translated by ribosome which stop at the stop codon, but physically covering region 2 as well, so 2 cannot pair with 3; 3 and 4 form hairpin with the UUU’s at the end, RNAP falls off Trp absent: ribosome will start translating Region 1, but stall at trp codons in the leader sequence (because the tRNA with the complemetary ACC sequence is empty without Trp amino acid, stuck and cannot make peptide bonds), region 2 pairs with 3 with no U’s after, thus RNAP keeps transcribing through the operon to make more tryptophans Two ­component transcription regulation: sensor and response regulator A. PhoR/B pathway: responds to low phosphate levels in bacterial periplasm  ­ ­> PhoR is the sensor, it’s a transmemebrane (i.e. part of it extends to the periplasm of bacteria and part of it stays in the cytoplasm) receptor that normally binds PPi. When there’s low PPi level in the periplasm, PhoR releases the PPi it’s bound, which changes its conformation in the cytoplasmic domain. This conformational change allows PhoR to be autophosphorylated (it grabs an ATP molecule and phosphorylates itself at a histidine residue).  ­ ­> PhoR, once phosphorylated, transfers that phosphate group onto the response regulator in the pathway, PhoB. Phosphorylated PhoB can then turn on transcription of many genes (also cross ­talks with other pathways) that help bacteria cope with the low PPi concentration in their periplasms. B. NtrB/NtcC pathway: responds to low nitrogen level and thus low glutamine (Gln) level. This pathway turns on many genes, one of which encodes Gln synthetase, GlnA.  ­ ­> NtrB is the sensor and also a kinase. It also autophosphorylates (by forming a dimer, and each monomer phosphorylates the other monomer— transphosphorylation), then phosphorylates NtrC, the response regulator.  ­ ­> GlnA gene promoter needs a special sigma54 ­RNAP complex, which only binds DNA to form a closed complex but cannot unwind the promoter, unlike the sigma70 ­ RNAP  ­ ­> Phosphorylated NtrC can bind to  ­108 and  ­140 regions of GlnA gene and interact with sigma54 ­RNAP, thus forming a DNA loop. Then NtrC can use ATP hydrolysis to unwind/separate the DNA duplex in the template strand and help sigma54 ­RNAP form an open complex and initiate transcription of GlnA gene. Eukaryotic transcription Diversified RNAPols in eukaryotes I: pre ­rRNA for 18, 5.8 and 28S, insensitive to alpha ­amanitin II: pre ­mRNA, nearly all mRNA in eukaryotes, and some snRNAs in the spliceosome, very sensitive to alpha ­amanitin: 1ug/mL III: pre ­tRNA, 5S rRNA and some snRNAs, somewhat sensitive to alpha ­amanitin: 10ug/mL Components of Pol II: general shape is similar to bacterial RNAP. 2 alpha ­like—contacting co ­activators 1 beta ­like—RBP2, DNA binding 1 beta’ ­like—RBP1, the largest subunit containing C ­terminal domain (CTD) that has many copies of amino acid sequence “YSPTSPS”. 1 omega ­like subunit and many others. Core promoter elements: more complex compared to bacterial  ­10 and  ­35 regions a, Simple eukaryotic transcriptional unit. A simple core promoter (TATA), upstream activator sequence (UAS) and silencer element spaced within 100–200 bp of the TATA box that is typically found in unicellular eukaryotes. b, Complex metazoan transcriptional control modules. A complex arrangement of multiple clustered enhancer modules interspersed with silencer and insulator elements which can be located 10–50 kb either upstream or downstream of a composite core promoter containing TATA box (TATA), Initiator sequences (INR), and downstream promoter elements (DPE). Note RE = regulatory element such as the BRE (TFIIB RE). Figure (a) illustrates promoters of simple unicellular eukaryotes such as yeast; Figure (b) illustrates metazoan transcriptional control. Proximal promoter elements often contain GCboxes that are recognized by Sp1 transcription factor; enhancer = distal sites that are bound by specific activators that often loop to interact with the promoter; distal/upstream enhancers + proximal promoter elements are collectively called cisacting control elements. Levine and Tjian, Nature 2003 --> These elements are not usually all represented in the promoter of a specific gene, some promoters might contain TATA but not INR, or vice versa, or neither. Often, a promoter that is TATA-less contains a compensatory DPE to facilitate pre-initiation complex (PIC) recognition. --> Housekeeping genes (those expressed at steady levels in the cells) often don’t have TATA or INR but CpG islands (stretches of CGs) that facilitate global activation or global repression (through DNA methylation, e.g.) Formation of PIC on gene promoters: many factors/complexes since Pol II doesn’t recognize promoters on its own The order of assembly is still debated; many think that some components of PIC pre ­ assemble before loading onto the promoter of a gene to be transcribed. Euk RNAP still follows the closed  ­> open  ­> elongation  ­> termination steps Formation of the CLOSED complex on promoter TFIID: TATA ­binding protein (TBP) and its associated factors (TAFs, can be tissue ­specific) TFIIA and TFIIB load onto either side of TBP. Recognizes TATA box and other regulatory regions by TAFs TFIIB binds BRE, TFIIA holds B and D together in a complex called DAB; A is dispensable in an in vitro transcription assay, but could help in vivo when promoter and Tbp bindings are weak Hypo(low)phosphorylated Pol II + TFIIF TFIIF helps targeting Pol II to specific DNA sequences, and not non ­promoter regions TFIIE Loads and recruits TFIIH TFIIH Helicase that separates DNA duplex at the promoter (analogous to the function of phosphorylated NtrC on GlnA promoter) Also a kinase complex that phosphorylates all Serine 5’s (so 52 residues in humans) on CTD tail of the RBP1 subunit of Pol II using the Cdk7 subunit, thus changing the conformation of the CTD and signaling for promoter clearance After TFIIH opened up the promoter and phosphorylated CTD Ser5 to signal promoter clearance, the PIC becomes an OPEN complex After this switch to the OPEN complex, Pol II synthesizes a few nt but stalls at the promoter until P ­TEFb complex (another kinase complex containing Cdk9) can phosphorylate all Serine 2’s in the CTD of Pol II. This signals for release of Pol II from pausing and productive elongation can begin. Only Pol II and TFIIF participate in elongation, where as DAB complex remains at the promoter to poise for the next round of mRNA synthesis, and TFIIE/H dissociate after 70nts of mRNA are made. After transcription is completed, a phosphatase (FCP1) gets rid of the Ser2/5 phosphorylation on the CTD, and Pol II is recycled to the promoter for another round of mRNA synthesis, as long as the DAB complex is bound to the promoter and directs Pol II there. Techniques In vitro: DNaseI footprinting and gel shift (EMSA)—both test the ability of a protein (often recombinantly purified) to bind a piece of DNA (often radiolabeled in these assays). Footprinting has a black ground (many DNA fragments) so it’s not as sensitive since it requires >80% of probes being bound to detect a footprint (white on black); gel shift is more sensitive since the background is white, and all you are visualizing is the difference in mobility between naked DNA and protein ­bound DNA (only require <1% of the DNA being bound by the protein to visualize). However, gel shift cannot tell you the sequence and it requires very stable protein ­DNA interactions since running in a native gel takes hours. DNA affinity chromatography—purifies a sequence specific DNA ­binding protein such as Sp1. Must know the sequence of the DNA your protein prefers to bind. It’s just a way to enrich for a protein In vitro transcription—an experiment to test whether a protein of interest can activate transcription and whether it’s dependent on a particular promoter sequence. You add DNA template containing the promoter or other regulatory sequences, individual components of PIC (sometimes nuclear extracts from cells) and radiolabeled rNTPs. Then you either include or exclude the protein of interest and compare the amount of synthesized mRNA (now radiolabeled) under either condition. ...
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