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FCH 532 Lecture 17

FCH 532 Lecture 17. Extra Credit Assignment for Friday Mar. 2 DeLisa seminar, 148 Baker, 3:00PM Study guide 8 posted Exam scheduled for Friday, March 9-will cover up to translation (Chapter 32). Amino acid metabolism will be next exam Chapter 31.

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FCH 532 Lecture 17

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  1. FCH 532 Lecture 17 Extra Credit Assignment for Friday Mar. 2 DeLisa seminar, 148 Baker, 3:00PM Study guide 8 posted Exam scheduled for Friday, March 9-will cover up to translation (Chapter 32). Amino acid metabolism will be next exam Chapter 31

  2. Figure 31-27 The kinetics of lac operon mRNA synthesis following its induction with IPTG, and of its degradation after glucose addition. Page 1240

  3. cAMP is the signal molecule for lack of glucose • cAMP is the signal molecule indicating a lack of glucose. • In the presence of glucose, cAMP levels are diminished. • Addition of cAMP overcomes catabolite repression by glucose. • cAMP binding protein responsible for the action-catabolite activator protein (CAP); cAMP receptor protein (CRP). • CAP is a homodimer of 210 residue subunits that undergoes large conformational change upon binding to cAMP. • CAP-cAMP complex binds to the lac operon and stimulates transcription in the absence of lac repressor.

  4. CAP-cAMP promotes high levels of expression for a weak promoter • CAP-cAMP complex binds to the lac operon and stimulates transcription. • CAP is a positive regulator-turns on transcription • lac repressor is a negative regulator - turns off transcription • lac operon has a weak (low-efficiency) promoter because it differs significantly from the consensus sequence. • CAP interacts directly with RNAP via the C-terminal domain (CTD). • CTD binds to dsDNA nonspecifically but with higher affinity to A-T rich sites (UP elements).

  5. Figure 31-28a X-Ray structures of CAP–cAMP complexes. (a) CAP–cAMP in complex with a palindromic 30-bp duplex DNA. Page 1241

  6. Figure 31-28b X-Ray structures of CAP–cAMP complexes. (b) CAP–cAMP in complex with a 44-bp palindromic DNA and the aCTD oriented similarly to Part a. Page 1241

  7. Figure 31-28cX-Ray structures of CAP-cAMP complexes. (c) CAP dimer’s two helix-turn-helix motifs bind in successive major grooves of the DNA. Page 1241

  8. CAP-dependent promoters • Class I promoters (lac operon) require only CAP-cAMP for transcriptional activation. CAP binding site can be located at various distances on the DNA. • Class II promoters also only require CAP-cAMP for transcriptional activation. CAP binding site only occupies a fixed position that overlaps the RNAP binding site. • Class III promoters require multiple activators to maximally stimulate transcription. May be more than one CAP-cAMP complexes or a CAP-cAMP complex in concert with promoter specific activators.

  9. DNA binding motifs • CAP proteins form a supersecondary structure called a helix-turn-helix (HTH) motif that binds to DNA. • HTF motifs associate with target base pairs mainly via side chains extending from the second helix of the HTH motif (recognition helix). • HTH motifs are observed in the lac repressor, trp repressor, cI repressors, and Cro proteins from bacteriophages. • Another type of structural motif observed in DNA binding proteins are -ribbons or two stranded anti-parallel b-sheets. • -ribbons are found in the met repressor (MetJ).

  10. Figure 31-29 X-Ray structure of the N-terminal domain of 434 phage repressor-target DNA complex. (a) A skeletal model (b) HTH (a2, a3) interaction with target DNA (c) A space-filling model. Page 1243

  11. Figure 31-30X-Ray structure of the 434 Cro protein in complex with DNA. (a) A skeletal model. (b) HTH (a2, a3) interaction with target DNA (c) A space-filling model. Page 1243

  12. Figure 31-31 X-Ray structure of an E. coli trp repressor– operator complex. Page 1244

  13. Figure 31-32a X-Ray structure of the E. coli met repressor- SAM-operator complex. (a) The overall structure of the complex as viewed along its 2-fold axis of symmetry. Page 1245

  14. Figure 31-32bX-Ray structure of the E. coli met repressor-SAM-operator complex. (b) The antiparallel b ribbon (yellow) in the DNA’s major groove. Page 1245

  15. trp operon regulation. • Encodes 5 polypeptides that make 3 enzymes mediating the synthesis of tryptophan from chorismate. • Under control of trp repressor (homodimer, 107 residues)-binds L-tryptophan to form a complex that binds to the trp operator to reduce the rate of transcription. • Trp forms a hydrogen bond to DNA phosphate group increasing the repressor-operator association (corepressor-acts in conjunction with trp repressor). • Controls 2 other operons: trpR and aroH involved in chorismate synthesis.

  16. trp operon attenuation • Transcriptional control through which bacteria regulate the expression of certain operons involved in amino acid biosynthesis. • Discovered with E. coli trp operon-before they thought it was just the trp repressor responsible for regulating operon. • trp deletion mutants downstream of trpO increased trp operon expression 6-fold-additional transcriptional control elements. • Sequence analysis revealed trpE is preceeded by a 162 nt leader sequence (trpL). • The new control element is located in trpL ~30-60 nt upstream of trpE.

  17. trp operon attenuation • When W is scarce, the entire 6720-nt polycistronic trp mRNA, including trpL is synthesized. • As W increases, rate of trp transcription decreases as a result of the trp-repressor-corepressor complex. • Of the trp mRNA that is transcribed, an increasing amount consists of only a 140-nt segment corresponding to the 5’ end of trpL. • The availability of tryptophan results in the premature temination of the trp operon transcription. • The control element responsible is an attenuator.

  18. Figure 31-39 A genetic map of the E. coli trp operon indicating the enzymes it specifies and the reactions they catalyze. Page 1251

  19. Figure 31-40 The base sequence of the trp operator. The nearly palindromic sequence is boxed and its –10 region is overscored. Page 1251

  20. trp operon attenuation: mechanism • The attenuator transcript has 4 complementary segments that form one of two sets of mutually exclusive base paired hairpins. • Segments 3 and 4 together with the succeding residues make a normal rho-independent transcription terminator: G-C rich sequence that forms a hairpin with several sequential U residues. • Transcription rarely proceeds beyond this termination site when W is scarce. • A section of the leader sequence (segment 1) is translated to form a 14-residue polypeptide with 2 consecutive Trp residues. • This provides a clue to the mechanism.

  21. trp operon attenuation: mechanism • When an RNAP that has escaped repression initiates the trp operon transcription, a ribosome attaches the ribosomal initiation site of trpL mRNA and begins translation of the leader peptide. • When W is abundant, lots of tryptophanyl-tRNATrp, the ribosome follows closely behind the transcribing RNA polymerase to sterically block the formation of the 2-3 hairpin. • The prevention of the 2-3 hairpin allows the formation of the 3-4 hairpin which results in the termination of transcription. • If there are low levels of Trp, the ribosome stalls on the 1 position and the 2-3 antiterminator forms allowing transcription of the trp operon.

  22. Figure 31-41 The alternative secondary structures of trpL mRNA. Page 1252

  23. Figure 31-42a Attenuation in the trp operon. (a) When tryptophanyl–tRNATrp is abundant, the ribosome translates trpL mRNA. Page 1253

  24. Figure 31-42b Attenuation in the trp operon. (b) When tryptophanyl–tRNATrp is scarce, the ribosome stalls on the tandem Trp codons of segment 1. Page 1253

  25. Table 31-3 Amino Acid Sequences of Some Leader Peptides in Operons Subject to Attentuation. Page 1253

  26. Regulation of rRNA synthesis: Stringent Response • Under optimal conditions, E. coli divides every 20 min. • These cells contain up to 70,000 ribosomes so 35,000 ribosomes must be made per cell division. • RNAP can initiate transcription at 1 gene per sec. • In order to meet the needs of the cell for ribosomes, there are multiple copies (7) or the rRNA operon in the E. coli genome. • Rapidly growing cells contain multiple copies of their replicating chromosomes. • The rate of rRNA synthesis is proportional to the rate of protein synthesis. • Stringent response: a shortage of any species of amino acid charged tRNA that limits the rate of protein synthesis triggers a metabolic adjustment.

  27. Stringent Response • Stringent response: a shortage of any species of amino acid charged tRNA that limits the rate of protein synthesis triggers a metabolic adjustment. • Can cause a 10 to 20-fold reduction in the rate of rRNA and tRNA synthesis. • Stringent controldepresses numerous metabolic processes (DNA replication, biosynthesis of carbohydrates, lipids, nucleotides, proteoglycans, glycolytic intermediates) while stimulating other pathways (amino acid biosynthesis).

  28. Stringent Response • 2 nucleotides regulate the stringent response ppGpp and pppGpp. Together known as (p)ppGpp. • The accumulation and decay of these regulates the stringent response. • Relaxed control mutants designated relA-, do not exhibit the stringent response-lack (p)ppGpp. • (p)ppGpp inhibits the transcription of rRNA genes but stimulates transcription of the trp and lac operons. • Stringent factor (RelA) catalyzes the reaction: ATP + GTP AMP + pppGpp ATP + GDP AMP + ppGpp

  29. Stringent Response • Several ribosomal proteins convert pppGpp to ppGpp. • Stringent factor is only active in association with a ribosome that is actively engaged in translation. • (p)ppGpp synthesis occurs when ribosome binds its mRNA specified but uncharged tRNA. • The binding of a specified and charged tRNA greatly reduces the rate of (p)ppGpp synthesis. • (p)ppGpp degradation is catalyzed by the spoT gene product.

  30. Eukaryotic RNA polymerases RNA polymerase I (RNAP I, Pol I, RNAP A)-located in nucleoli, synthesizes rRNA precursors. RNA polymerase II (RNAP II, Pol II, RNAP B)- in the nucleoplasm, synthesizes mRNA precursors. RNA polymerase III (RNAP III, Pol III, RNAP C)- alos in nucleoplasm, synthesizes precursors of 5S rRNA, tRNAs, and other small nuclear and cytosolic RNAs. Have greater subunit complexity than prokaryotic RNAP. Molecular masses up to 600 kD

  31. Table 31-2 RNA Polymerase Subunitsa. Page 1232

  32. RNAP II • RNAP II is regulated by it’s C-terminal domain (CTD). • Contains 52 highly conserved repeats of the heptad PTSPSYS in mammals (26 in yeasts). • Subject to phosphyorylation/dephosphorylation by CTD kinases and CTD phosphatases. • RNAP II initiates transcription when CTD is unphosphorylated. • RNAP II commences elongation only after the CTD is phosphorylated. • Crystal structures shows it is similar to the Taq RNA polymerase. • RNAP II binds 2 Mg2+ ions in the active site. • Several subunits not observed in Taq RNA polymerase.

  33. Figure 31-20a The X-Ray structure of yeast RNAP II that lacks its Rpb4 and Rpb7 subunits. Page 1233

  34. Figure 31-20b The X-Ray structure of yeast RNAP II that lacks its Rpb4 and Rpb7 subunits. (b) View of the enzyme from the right in Part a showing its DNA binding cleft. Page 1233

  35. RNAP II • Eukaryotic RNAPs cannot independently bind to their target DNA. • They must be recruited to target promoters through complexes of transcription factors. • RNAP II can initiate transcription on a dsDNA with a 3’ single-stranded tail at one end.

  36. Figure 31-21aSecondary structure of an RNAP II elongation complex. Template DNA cyan, nontemplate DNA green, and newly synthesized RNA red. Page 1234

  37. Figure 31-21cCutaway schematic diagram of the transcribing RNAP II elongation complex. Page 1234

  38. Amatoxins • Amanita phalloides(death cap) mushroom produces bicyclic octapeptides known as amatoxins. -amanitin is shown: • Forms a tight 1:1 complex with RNAP II (K = 10-8 M) and RNAP III (K = 10-6 M). • Binding slows RNAP synthesis from 1000s to a few nt per min. • RNAP I, mitochondrial, chloroplast and prokaryotic RNAPs are insensitive.

  39. Figure 31-22 The proposed transcription cycle and translocation mechanism of RNAP. (a) Nucleotide addition cycle. (b) RNA · DNA complex in RNAP II. Page 1235

  40. Eukaryotic promoters • Mammalian RNA polymerase I has a bipartite promoter consisting of a core promoter element (-31 to +6) and upstream promoter element (-187 to -107) ; GC-rich, recruits transcription factors. • RNA polymerase II promoters are longer than prokaryotic promoeters. • Constitutively expressed genes have 1 or more copies of GGGCGG (GC box) located upstream of transcription start site.

  41. Eukaryotic promoters • Most structural genes have a conserved AT-rich sequence 25-30 bp upstream from transcription start site. • TATA box (sometimes called Hogness box)-resembles -10 region of prokaryotic promoters. • Deletion of TATA box does not eliminate transcription; instead generates differences in transcription start site. • -50 to -110 also contains promoter elements, example: globin genes have a conserved CCAAT box -70 to -90. • Globins also have the CACCC box upstream from the CCAAT box.

  42. Figure 31-23 The promoter sequences of selected eukaryotic structural genes. Page 1236

  43. Enhancers in eukaryotes • Enhancers are transcriptional control regions that can be located several thousand base pairs upstream or downstream from the transcription start site. • Enhancers must be associated with promoters to trigger site-specific and strand -specific transcription initiation. • Required for full activities from promoters. • Enhancers are recognized by specific transcription factors that stimulate RNA polymerase II to bind to the corresponding but distant promoters. • Mediate selective gene expression in eukaryotes.

  44. RNA processing • Most (all in eukaryotes) primary transcripts (the RNA molecule as encoded in the DNA) function after being altered covalently by one or more of the following processing steps: removal of 5’ and/or 3’ nucleotides, addition of nucleotides at the 5’ and/or 3’ ends, covalent modification of the bases, or “editing” of the nucleotide sequence (changing the information content of the RNA). • Require one or more of the following activities: capping enzyme, polyA polymerase, specific ribonuclease, RNA ligase, spliceosomes-specialized RNA processing complexes consisting RNA and protein, and catalytic RNA-ribozymes

  45. Messenger RNA processing in eukaryotes • 1. Capping • 2. Polyadenylation • 3. Splicing • 4. Introns early or introns late?

  46. A gene is not necessarily co-linear with its encoded protein (for eukaryotic genes only) UAG AUG … 3’ UTR 5’ UTR Green=ORF(open reading frame) The linear order is never violated; it is simply interrupted

  47. Eukaryotic mRNA is capped • 5’ cap is a reversed guanosine residue so there is a 5’-5’ linkage between the cap and the first sugar in the mRNA. • Guanosine cap is methylated. (cap-0) • First (cap-1)and second nucleosides (cap-2) in mRNA may be methylated

  48. Capping mRNA-cont. • Capping involves several enzymatic reactions • Removal of the leading phsophate group from the 5’ terminal triphosphate group by RNA triphosphatase • Guanylation of the mRNA by capping enzyme; requires GTP and yields the 5’-5’ triphosphate bridge and PPi • Methylation of guanine by guanine-7-methyltransferase (methyl from SAM). • The O2’ methylation of mRNAs first and maybe second nucleotide by a SAM requiring 2’-O-methyltransferase. Capping enzyme and guanine-7-methyltransferase bind to phosphorylated CTD of RNAPII.

  49. Poly (A) tails • Eukaryotic mRNAs are monocistronic. • Sequences signaling transcriptional termination not identified; not precise. • Mature mRNAs have well defined 3’ ends of poly A tails (~250 in mammals and ~80 in yeast). • Added in two reactions by a complex of at least 6 proteins.

  50. Polyadenylation of eukaryotic mRNA • Transcript is cleaved to yield a free 3’-OH group at a specific site 15-25 nt past an AAUAAA site and within 50 nt before U-rich or G-U rich sequence • Endonuclease that cleaves RNA uncertain but requires cleavage factors (CFI and CFII). • Poly(A) tail is made from ATP by poly(A) polymerase (PAP) which is recruited by the cleavage and polyadneylation specificity factor (CPSF).

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