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Chapter 6

Chapter 6. How Cells Read the Genome: From DNA to Protein. Test Your Knowledge. What are two major differences between transcription in prokaryotic and eukaryotic cells?

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Chapter 6

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  1. Chapter 6 • How Cells Read the Genome: From DNA to Protein

  2. Test Your Knowledge • What are two major differences between transcription in prokaryotic and eukaryotic cells? • RNA polymerase and DNA polymerase enzymes catalyze the “same” reaction, but there are some distinct differences in what is required to make them begin catalysis and end catalysis. What are these differences? • Which is more accurate, DNA replication or RNA transcription? • Explain the proteins and mechanisms involved in the initiation of transcription • What determines how many copies of a transcript (mRNA) are made? • How are elongation and termination of the transcript regulated? Video overview of transcription

  3. “The Protein Players” - RNA polymerases, transcription factors, initiation factors, enhancers, repressors

  4. Prokaryotes? Prokaryotic transcription video

  5. Prokaryotic Transcription

  6. DNA Sequences Important to Transcription • Prokaryotes • Promoter – • Pribnow Box (also called the -10 element) – TATAAT • -35 element - TTGACA • Eukaryotes • Promoter –(asymmetrical sequence) • Basic core promoter –TATA box (TATAAA(A)); within 50bp upstream of start site; found in unicellular eukaryotes • Core promoter PLUS • Downstream promoters • Proximal promoter elements

  7. Initiator sequences • Regulatory Elements/Response Element - Response elements are the recognition sites of certain transcription factors Most of them are located within 1 kb from the transcriptional start site.  • Enhancer elements -upon binding with transcription factors (activators), can enhance transcription; located either upstream or downstream of the transcriptional initiation site.  • Upstream enhancer elements • Downstream enhancers • Distal enhancer elements • Silencers - upon binding with transcription factors (repressors), can repress transcription. • Insulators

  8. Gene Regulatory Networks – control the number and type of transcripts made by a cell.

  9. video Simple core promoter UAS = upstream activator sequence RE = regulatory elements INR = initiator sequence DPE = downstream promoter elements

  10. Sequences that can act as promoters (TATA is preferred)

  11. Proteins Involved in Transcription RNA Polymerase General (or Basal) Transcription Factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH Transcription Factors that Bind to Regulatory Elements Holoenzyme or Initiation Complex

  12. Transcription Factors Have Common DNA Binding Motifs • Zinc finger • Helix-turn-helix • Leucine zipper

  13. RNA Polymerase

  14. Recognizes and binds to TATA box; TBP + 10 TBP associated factors; position set

  15. TBP bends DNA ~80o and forces open the minor groove.

  16. Recognizes and binds to TATA box; TBP + 10 TBP associated factors Binds and stabilizes the TFIID complex Recruits RNA pol II + TFIIF to the location Two subunits - RAP38 & RAP74. Rap74 has a helicase activity; RAP38 binds RNAPolII Two subunits - recruits TFIIH to the complex thereby priming the initiation complex for promoter clearance and elongation complex of 9 subunits. One w/ kinase activity; one w/ helicase activity; one is a cyclin (cdk7)

  17. -30 +1 Inr TATA TAFs } or TBP TFIID TBP IIA IIB IIF Eukaryotic RNA polymerase II Pol IIa CTD of large subunit of Pol II IIE helicase IIH IIH IIH protein kinase IIF IIE IIA IIB Pol IIa Inr TATA preinitiation complex ATP hydrolysis IIF IIE IIA Pol IIa IIB Inr TATA initiation complex, DNA melted at Inr Sequential Binding Model for assembly of preinitiation complex = PIC Polymerization of 1st few NTPs and phosphorylation of CTD leads to promoter clearance. TFIIB, TFIIE and TFIIH dissociate, PolII+IIF elongates, and TFIID + TFIIA stays at TATA. Activated PIC

  18. Transcription initiation in the cell often requires the local recruitment of chromatin-modifying enzymes, including chromatin remodeling complexes and histone acetylases - greater accessibility to the DNA present in chromatin

  19. RNA polymerase is also assisted by DNA supercoiling

  20. Phosphorylation of the carboxy terminal domain (CTD) of one of the subunits of RNA PolII; RNA polymerase II dissociates from the transcription factors and other protein complexes that were required for assemblyand elongation begins Phosphorylation also promotes the accumulation of elongation factors – other proteins that arrest transcription long enough to recruiting RNA processing enzymes

  21. Elongation is Coupled to RNA Processing • Capping • Splicing • Polyadenylation

  22. RNA Capping enzymes: • Phosphatase • Guanyl transferase – GMP in 5’ to5’ linkage • methyltransferase Video of transcription and capping

  23. CBC – cap binding complex proteins also associate and protect the cap; Later they will direct transcript in its exit from the nucleus

  24. RNA Splicing

  25. How Introns Are Identified: • Consensus sequences at (5’ to 3’ direction) • 5’ splice site • Lariate loop closure site of the intron sequence • 3’ splice site R=A or G,Y=C or U

  26. The Spliceosome Forms • snRNAs (U1, U2, U4, U5 and U6) and associated proteins = snRNPs • U1 binds to the GU sequence at the 5' splice site, along with accessory proteins/enzymes, • U2 binds to the branch site, and ATP is hydrolyzed; • U5/U4/U6 trimer binds, and the U5 binds exons at the 5' site, with U6 binding to U2; • U1 is released, U5 shifts from exon to intron and the U6 binds at the 5' splice site; • U4 is released, U6/U2 catalyzes transesterification, U5 binds exon at 3' splice site, and the 5' site is cleaved, resulting in the formation of the lariat; • U2/U5/U6 remain bound to the lariat, and the 3' site is cleaved and exons are ligated using ATP hydrolysis. The spliced RNA is released and the lariat debranches.

  27. Rearrangements that occur during splicing • U1 replaced by U6 • BBP (branch binding protein) and U2 • U5 complex branch forming enzymes in U6 and U2 • Allows for “check and recheck” at each splice site.

  28. Why is splicing so accurate? Introns are small-large; Exons are about 150bp long Exons might be easily identified, while introns probably couldn’t be.

  29. Accurate………. As the RNA is being transcribed, SR proteins (rich in serine (S) and arginine (R)) sit down on the exons. Along with the U proteins, demarcates the start and end of the exon. Capping proteins or polyA binding proteins act as markers at either end of the transcript. Other hnRNPs (heterogeneous nuclear RNPs) bind along the introns, helping to distinguish these sequences from exons.

  30. ……….But Flexible Changes in splicing patterns caused by random mutations have been an important pathway in the evolution of genes.

  31. 3’ end splicing sequence • AAUAAA • Cleavage site CA – 10-30 nucleotides downstream • Polyadenylation site – GU- or U-rich region about 30 nucleotides downstream from the cleavage site

  32. CPSF = Cleavage and Polyadenylation Specificity Factor CstF = Cleavage Stimulation Factor

  33. Poly A polymerase – no template strand required All of the A nucleotides are derived from ATP Poly A binding proteins remain until mRNA undergoes translation

  34. Only very “select” RNAs can be transported out of the nucleus

  35. Guided diffusion along the FG-repeats displayed by nucleoporins Proteins bound to mature mRNA molecules and that signal completed splicing have nuclear export signals as a part of their sequence

  36. hNRPs “straighten out” the mature mRNA so that nuclear export signals can be “read” • 5’ cap enters the pore first • Many of the RNA binding proteins fall off as mRNA exits the nucleus • Initiation factors (elF-4G and elF-4E) immediately bind to the 5’ capping complex (which falls off) and to the polyA tail, forming a loop

  37. Test Your Knowledge – Translation • Transcription requires only changing a DNA code of nucleotides into a similar RNA code of nucleotides, while translation involves changing the RNA code into what? • What are codons and what “reads” codons? • What is “wobble” and how is it related to translation? • What attaches amino acids to t-RNA? • What are the “parts” of the ribosome? What function does each part perform? • What are the A, P, and E sites of a ribosome? What binds at each of these sites? • Does anything beside the ribosome participate in elongation of the amino acid chain? If so, what is it and what does it do? • What signals where translation starts and stops? • What happens to improperly translated or proteins that don’t fold properly after being translated?

  38. Transfer RNA • anticodon- 3’ to 5’ sequence that matches the complementary 5’ to 3’sequence (codon) on the mRNA • Acceptor arm - Amino acid code on 3’ end • T and D loops – provide structure for interface with aminoacyl-tRNA synthetase ?

  39. A different aminoacyl-tRNA synthetase enzyme for each amino acid

  40. Large subunit does???? Small subunit does ?????

  41. Translation Initiation This is the only tRNA that can bind to the small ribosomal subunit by itself

  42. Protein made in 5’ to 3’ direction, with N-terminal end made first General Mechanism • A site is where new codon is translated • P site is where the growing peptide chain is kept and new aa are attached • E site is where “naked” t RNA exit the ribosome

  43. More Detailed View New tRNA carrying amino acids are accompanied by elongation factor called EF-Tu The tRNA-ETu occupies a hybrid binding site (not quite in A) Correct codon-anticodon pairing triggers ETu to split GTP and fall off, and tRNA moves into the A position The delay caused by the association/dissociation of ETu helps increase accuracy of translation

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