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Purdue University. Biology 438 Exam 2 Study Guide.

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Transcription Initiation Regulation via: • Constitutive genes: expressed continuously • Regulated genes: code for inducible enzymes need in certain environments • Negative Control: stops/repr... esses transcription of an operon in presence/absence of a signal (ex. Lac or trp operons of E. coli) • Positive Control: turns on/activates transcription of an operon in the presence/absence of a signal (ex. Ara operon of E. coli) o Consensus Promoter= most efficient promoter o What is the function of -35 to -10 sequences?  They constitute the recognition site for sigma-70 form of RNA poly o LacZYA promoter has sequence: TTTACA (N18) TATGTT. Why the sequence suggests that it needs a transcriptional activator, such as CRP-cAMP complex, for transcription  Both -35 and -10 sequences differ from the consensus sequences, making this sequence a poor promoter and requiring a transcriptional activator Operons: DNA sequence that encodes one or more genes that are transcribed as a single mRNA • Operons regulated by Positive Control: o have poor promoters o need transcriptional activators to assist binding of RNA poly. to poor promoters o has operator (before the promoter) that is the binding site for a transcriptional activator so transcription occurs o has transcriptional activator proteins have two conformations: 1) binds to operator and assists promoter recognition by RNA poly. 2) doesn’t interact with DNA and/or RNA poly. so no transcription occurs o EXAMPLE:  AraC controls expression of araBAD operon; when L-arabinose is present, AraC assists in RNA poly. binding to pBAD & araBAD operon induced = transcription; when L-arabinose is absent, AraC blocks binding of RNA poly. to pBAD & araBAD operon repressed = no transcription • Operons regulated by Negative Control (if repressor present, no transcription occurs): o Operator is close to the promoter o Has a transcriptional repressor proteins have 2 conformations: 1) binds to operator and shuts down transcription 2) doesn’t bind to operator and allows transcription to occur o EXAMPLES:  Lactose controls the expression of lac operon: if lactose absent, lac operon repressed = no transcription (b/c lacI binds to operator); if lactose present, lac operon induced = transcription (b/c allolactose binds to repressor)  Tryptophan controls expression of trp operon; if tryptophan absent, trp operon is induced = transcription (b/c TrpR protein can’t bind to operator without tryptophan); if tryptophan present, trp operon repressed = no transcription (b/c tryptophan binds to TrpR proteins and then they bind to operator) o Transcription of lac operon produces a polycistronic mRNA o Allolactose= intracellular inducer of the E. coli lac operon o E. coli lacY mutants that are defective for lactose uptake are not inducible for beta-galactosidase(lacZ)., and lacZ mutants are not inducible for lacY. Why?  Allolactose is formed from lactose via lacZ. Mutants defective in lacY can’t take up lactose, so mutants defective in lacZ can’t make lactose o Ex: Explain mechanism of regulation of trp operon by trpR  Tryptophan enzymes encoded by trp operon are expressed at max level when tryptophan is not available. TrpR is an aporepressor that can’t bind to trp operator as long as tryptophan levels are low. For repression to occur, tryptophan must be present at high concentration to bind the aporepressor to convert it to a repressor that will bind to operator to stop transcription of trp operon • PhoR/PhoB Signal System o If there is enough phosphate, PhoR is inactive; when phosphate low, protein kinase activated in region of PhoR o PhoB becomes activate from phosphorylation; in return, activates transcription of genes that will increase phosphate level again o Positive control Regulation of Transcription Elongation—controls transcription termination via attenuation • Intrinsic Transcription Terminators o Length of self-complementary stem and loop structure; followed by 6+ U’s in the mRNA strand o When RNA poly. runs into this, the RNA poly. falls off the DNA and transcription stops • Attenuation Control o Trp Operon: independent of TrpR-mediated negative control  Level of transcription of trp operon modified to amount of charged tRNAtrp in the cell  Mediated via a short, translated leader sequence at the 5’ end of trp mRNA  Takes advantage of alternate mRNA conformations to regulate early termination  Insufficient amount of tRNAtrp: ribosome stalls in region 1, creates a hair pin at regions 2&3, and transcription continues  Sufficient amount of tRNAtrp: ribosome stalls in region 2, creates hair pin at regions 3&4, and termination occurs o His operon:  If adequate amounts of histidine: termination occurs  If insufficient amount of histidine: transcription still occurs o Ex: (a) What would phenotype of E. coli strain be if terminator of his attenuator was deleted? (b) What would phenotype of E. coli strain be if the 7 histidine codons in his attenuator were replaced by 7 serine codons?  (a) his operon would be expressed constitutively. The reason is because the terminator, which would function to stop transcription before first his structural gene in presence of histidine, will not be there to terminate transcription  (b) his operon would be transcribed at high level in absence of serine and would be down-regulated in presence of serine. The reason is because serine would act as histidine to regulate expression of the operon o Ex: How can you establish that the trpR-mediated control of trp operon is an example of negative or positive transcriptional control of gene expression?  Make mutation in trpR gene and test effect on expression of trp operon. If operon is expressed at high level in trpR mutant, the operon is regulated by negative control (trpR is repressor). If trpR operon not expressed, the operon is regulated by positive control (trpR is transcriptional activator) • Riboswitches: RNA species that bind metabolites/ions, can regulate mRNA expression ::::::::::::::::::::::::::::::::::::::::CONTENT CONTINUED IN THE ATTACHMENT::::::::::::::::::::::::::::::::::::::::::::::::::: [Show More]

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