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Molecular Biology

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  1. Molecular Biology Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods

  2. Part III: Expression of the Genome Ch 12: Mechanisms of transcription Ch 13: RNA splicing Ch 14: Translation Ch 15: The genetic code

  3. Chapter 12: Mechanisms of Transcription • Molecular Biology Course • RNA polymerase and transcription cycle • The transcription cycle in bacteria • Transcription in eukaryotes

  4. The Central Dogma Transcription Translation DNA RNA PROTEIN replication

  5. Transcription is very similar to DNA replication but there are some important differences: RNA is made of ribonucleotides RNA polymerase catalyzes the reaction The synthesized RNA does not remain base-paired to the template DNA strand Less accurate (error rate: 10-4)

  6. Transcription selectively copies only certain parts of the genome and makes one to several hundred, or even thousand, copies of any given section of the genome. (Replication?)

  7. Transcription bubble Fig 12-1 Transcription of DNA into RNA

  8. CHAPTER12: Mechanisms of Transcription Topic 1: RNA Polymerase and The Transcription Cycle See the interactive animation

  9. RNA polymerase and the transcription cycle RNA polymerases come in different forms, but share many features • RNA polymerases performs essentially the same reaction in all cells • Bacteria have only a single RNA polymerases while in eukaryotic cells there are three: RNA Pol I, II and III

  10. RNA Pol II is the focus of eukaryotic transcription, because it is the most studied polymerase, and is also responsible for transcribing most genes-indeed, essentially all protein-encoding genes • RNA Pol I transcribe the large ribosomal RNA precursor gene • RNA Pol II transcribe tRNA gene, some small nuclear RNA genes and the 5S rRNA genes

  11. Table 12-1: The subunits of RNA polymerases

  12. The bacterial RNA polymerase The core enzyme alone synthesizes RNA b’ a b a w

  13. prokaryotic b’ Fig 12-2 RNAP Comparison a b The same color indicate the homologous of the two enzymes a w eukaryotic RPB2 RPB3 RPB1 RPB11 RPB6

  14. “Crab claw” shape of RNAP (The shape of DNA pol is__) Active center cleft

  15. There are various channels allowing DNA, RNA and ribonucleotides (rNTPs) into and out of the enzyme’s active center cleft

  16. RNA polymerase and the transcription cycle Transcription by RNA polymerase proceeds in a series of steps • Initiation • Elongation • Termination

  17. Initiation • Promoter: the DNA sequence that initially binds the RNA polymerase • The structure of promoter-polymerase complex undergoes structural changes to proceed transcription • DNA at the transcription site unwinds and a “bubble” forms • Direction of RNA synthesis occurs in a 5’-3’ direction (3’-end growing)

  18. Fig 12-3-initiation Binding (closed complex) Promoter “melting” (open complex) Initial transcription

  19. Elongation • Once the RNA polymerase has synthesized a short stretch of RNA (~ 10 nt), transcription shifts into the elongation phase. • This transition requires further conformational change in polymerase that leads it to grip the template more firmly. • Functions: synthesis RNA, unwinds the DNA in front, re-anneals it behind, dissociates the growing RNA chain

  20. Termination • After the polymerase transcribes the length of the gene (or genes), it will stop and release the RNA transcript. • In some cells, termination occurs at the specific and well-defined DNA sequences called terminators. Somecells lack such termination sequences.

  21. Fig 12-3-Elongation and termination Elongation Termination

  22. RNA polymerase and the transcription cycle Transcription initiation involves 3 defined steps • Forming closed complex • Forming open complex • Promoter escape

  23. Closed complex • The initial binding of polymerase to a promoter • DNA remains double stranded • The enzyme is bound to one face of the helix

  24. Open complex • the DNA strand separate over a distance of ~14 bp(-11 to +3 ) around the start site (+1 site) • Replication bubble forms

  25. RNA polymerase and the transcription cycle Stable ternary complex • The enzyme escapes from the promoter • The transition to the elongation phase • Stable ternary complex =DNA +RNA + enzyme

  26. Topic 2The transcription cycle in bacteria CHAPTER12: Mechanisms of Transcription

  27. The transcription cycle in bacteria 2-1 Bacterial promotersvary in strength and sequences, but have certain defining features

  28. Holoenzyme= • factor + core enzyme Figure 12-4 In cell, RNA polymerase initiates transcription only at promoters. Who confers the polymerase binding specificity? ,

  29. Promoters recognized by E. colis factor • The predominant s factor in E. coli is s70. • Promoter recognized by s70 contains two conserved sequences (-35 and –10regions/elements) separated by a non-specific stretch of 17-19 nt. • Position +1 is the transcription start site.

  30. Fig 12-5a: bacterial promoter The distance is conserved • s70 promoters contain recognizable–35 and –10 regions, but the sequences are not identical. • Comparison of many different promoters derives the consensus sequences reflecting preferred –10 and –35 regions

  31. Consensus sequence of the -35 and -10 region BOX 12-1 Figure 1

  32. Promoters with sequences closer to the consensus are generally “stronger” than those match less well. (What does “stronger” mean?) • The strength of the promoter describes how many transcripts it initiates in a given time.

  33. Fig 12-5b bacterial promoter Confers additional specificity UP-element is an additional DNA elements that increases polymerase binding by providing the additional interaction site for RNA polymerase

  34. Fig 12-5c bacterial promoter Another class of s70 promoter lacks a –35 region and has an “extended –10 element” compensating for the absence of –35 region

  35. The transcription cycle in bacteria 2-2 The s factor mediates binding of polymerase to the promoter • The s70 factor comprises four regions called s region 1 to s region 4.

  36. Fig 12-6: regions of s Region 4 recognizes -35 element Region 2 recognizes -10element Region 3 recognizes the extended –10 element

  37. Binding of –35 Two helices within region 4 form a common DNA-binding motif, called a helix-turn-helix motif One helix inserts into the DNA major groove interacting with the bases at the –35 region. The other helix lies across the top of the groove, contacting the DNA backbone Fig 5-20* Helix-turn-helix DNA-binding motif

  38. Interaction with –10 is more elaborate (精细) and less understood • The -10 region is within DNA melting region • The a helix recognizing –10 can interacts with bases on the non-template strand to stabilize the melted DNA.

  39. UP-element is recognized by a carboxyl terminal domain of the a-subunit (aCTD), but not by s factor Fig 12-7 s and a subunits recruit RNA pol core enzyme to the promoter

  40. The transcription cycle in bacteria 2-3 Transition to the open complex involves structural changes in RNA polymerase and in the promoter DNA This transition is called Isomerization (异构化)

  41. For s70–containing RNA polymerase, isomerization is a spontaneous conformational change in the DNA-enzyme complex to a more energetically favorable form. (No extra energy requirement)

  42. Change of the promoter DNA • the opening of the DNA double helix, called “melting”, at positions -11 and +3.

  43. The striking structural change in the polymerase • 1. the b and b’ pincers down tightly on the downstream DNA • 2. A major shift occurs in the N-terminal region of s (region 1.1) shifts. In the closed complex, s region 1.1 is in the active center; in the open complex, the region 1.1 shift to the outside of the center, allowing DNA access to the cleft

  44. NTP uptake channel is in the back Fig 12-8 channels into and out of the open complex DNA entering channel

  45. The transcription cycle in bacteria Transcription is initiated by RNA polymerase without the need for a primer Initiation requires: • The initiating NTP (usually an A) is placed in the active site • The initiating ATP is held tightly in the correct orientation by extensive interactions with the holoenzyme

  46. The transcription cycle in bacteria RNA polymerase synthesizes several short RNAs before entering the elongation phase Abortive initiation: the enzyme synthesizes and releases short RNA molecules less than 10 nt.

  47. Structural barrier for the abortive initiation • The 3.2 region of s factor lies in the middle of the RNA exit channel in the open complex. • Ejection of this region from the channel (1) is necessary for further RNA elongation, (2) takes the enzyme several attempts

  48. NTP uptake channel is in the back Fig 12-8 channels into and out of the open complex DNA entering channel

  49. The transcription cycle in bacteria The elongating polymerase is a processive machine that synthesizes and proofreads RNA

  50. Synthesizing by RNA polymerase DNA enters the polymerase between the pincers Strand separation in the catalytic cleft NTP addition RNA product spooling out (Only 8-9 nts of the growing RNA remain base-paired with the DNA template at any given time) DNA strand annealing in behind