BBMB 405 1st Edition Lecture 35 Outline of Last Lecture XVI. Chapter 30: Protein SynthesisC. Eukaryotic protein synthesis differs from prokaryotic protein synthesis primarily in translation initiationD. A variety of antibiotics and toxins can inhibit protein synthesisE. Ribosomes bound to endoplasmic reticulum manufacture secretory and membrane proteinsXVII. Chapter 31: The control of gene expression in prokaryotesA. IntroductionOutline of Current Lecture XVII. Chapter 31: The control of gene expression in prokaryotesA. IntroductionB. Many DNA-binding proteins recognize specific DNA sequencesC. Prokaryotic DNA-binding proteins bind specifically to regulatory sites in operonsCurrent LectureXVII. Chapter 31: The control of gene expression in prokaryotesB. Many DNA binding proteins recognize specific DNA sequencesC. Prokaryotic DNA binding proteins bind specifically to regulatory sites in operons1. The lac operon: classic example of transcriptional regulationThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.a.b. Bacteria need to control gene expression because their transcription and translation are combinedc. Activators or inhibitors bind to control sight and turn genes off or on2. Observation: Lac operon production increases as presence of lactose increasesa. Lactose metabolism proteins are only expressed in the presence of lactoseb. There has to be a mechanism for sensing lactose and turning on gene: when regulatory gene and operon are removed the lactose structural genes are expressed all the time3. Reminder: sugar nomenclature (Review chapter 11): Beta-galatosides have an organic R group on the C1 oxygen, lactose (galactosyl glucose) is an example 4. The lac operon controls lactose transport and metabolism in E. colia. Lac Y: lactose permeaseb.c. Lac Z: beta-galactosidased.e. Beta-galactosidase can also be used to cleave X-gal which produces a blue color, this is used to assay activity of beta-galactosidasef. Lac A: thiogalactoside transacetylase- Acetyl-CoA + Beta-D-galactoside CoA + 6-acetyl-Beta-D-galactoside- Not essential for lactose metabolism- May have role in detoxification of compounds transported by permeaseg. Lac I: the lac repressor is an inverted repeat-- The lac repressor binds the lac operator- Binds to the major groove using Arg- Operator DNA is a symmetrical dimer of lac repressor5. Repressors block transcription of genesa.b. Lac repressor DNA binding- Dissociation constant (Kd) of 0.1 pM (10^-13) for operator sequence- Diffuses along DNA to search for operator (1D not 3D search)c. Repressor-inducer complex does not bind DNA- Inducer binding causes conformational change in repressor- Several Beta-galactosides are inducers of Lac operon6. No inducer present (A, figure above): Lac repressor binds the operator tightly7. Inducer present (B, figure above): lac repressor releases operator; note: DNA binding domains are disordered, illustration is organized for show; 8. Lac repressor binds two sites as a tetramera. Two sites within 500 bp of primary operator have similar sequenceb. One dimer binds operator, the other binds at one of the other two sitesc. Intervening sequence loops outd. No other closely matching sites in E. coli genome9. Lactose is not direct inducer of the lac operon; The lac operon is induced in the presence of 1,6-Allolactose10. The lac operon paradoxa. Beta-galactosidase can catalyze two reactions using lactose as substrate: either hydrolysis to create glaactose and glucose or transgalactosylsis to create 1,6-Allolactoseb. A small amount of beta-galactosidase is necessary to create an inducer from lactose11. When would E. coli need to metabolize lactose?a. What is preferred energy source? Glucoseb. When glucose is abundant, other carbon sources are not necessary; high glucose concentrations inhibit lactose permease and adenylate cyclasec. When glucose is depleted, lactose metabolism (or other carbon source) will be upregulated by catabolite-activator protein (CAP)12. Helix-turn-helix DNA-binding motifsa. Second helix of N-helix-turn-helix-C contacts major groove of DNAb. Amino acid side chains make contact with edges of base pairs13. Multiple operons can be controlled by same (or similar) operator sequencesa. Pur repressor binds DNA when bound to guanine hypoxanthine: co-repressors rather than inducers, negative feedback regulatorb. Pur operator sequence is short 5’-ANGCAANCGNTTNCNT-3’: evolved independently at sites around genome, regulatory regions of genes participating in nucleotide
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