Chapter 12

1. Chapter 12 Mechanismsof Transcription 04级生物学基地班 200431060048　钱振宇

2. The Central Dogma DNA RNA Protein transcription translation replication

3. We have discussed about the maintenance of the genome , which is the replication of the DNA. then it comes to the gene expression.

4. Gene expression is the process by which the information in the DNA double helix is converted into the RNA and proteins. and transcription is the first step involves copying DNA into RNA.

5. Compared with replication, transcription has something in common, that is: ＊ DNA template is needed to synthesized a new chain of nucleotide. ＊ the direction is from 5′ to 3′.

6. Also there several important differences: ＊ only some regions of the genome are transcribed instead of the entire genome. ＊ the nucleotides used to build a new chain is ribonucleotides instead of deoxyribonucleotides. ＊ transcription does not require a primer.

7. ＊ The RNA product dose not remain base-paired to the DNA templates. ＊ less accurate than replication.

8. Fig 1 transcription

9. Topic 1 RNA Polymerases and the Transcription cycle

10. RNA Polymerases RNA polymerases performs the same reaction in all cells, they are highly conceived , thus the enzymes from these organisms share many features.

11. Bacteria have just one polymerase, as in E.coli, the basic enzyme called the core enzyme has 5 subunits: one copy of each three subunits--- β, β’ ,and ω. and 2 copies of α.

12. Fig 2 the crystal structure of prokaryotic

13. In eukaryotic cells, there are three polymerase, RNA Pol Ⅰ, Ⅱ, and Ⅲ. Pol Ⅰand Ⅲ are involved in transcribing specialized, RNA-encoding genes. the former is for large ribosomal RNA precursor gene, the latter is for some nuclear RNA gene and the 5S rRNA.

14. RNA Pol Ⅱ is the enzyme we will focus on, for it is the most studied of the three. Active center cleft Fig 3 the crystal structure of eukaryotic

15. Table 1 the subunits of RNA Polymerase

16. There are 5 channels ,each allows double-stranded DNA, template DNA, non-template DNA, rNTP, and RNA products into or out of the enzyme’s active center cleft.

17. Transcription by RNA Polymerase Proceeds in a Series of Steps • Initiation • Elongation • Termination

18. Fig 4 initiation of transcription

19. initiation • Promoter: the DNA sequence which initially binds the RNA polymerase. • The promoter-polymerase complex undergoes structure changes to proceed transcription. • DNA at transcription site unwinds and forms a “bubble”. • 3’-end growing.

20. Fig 5 elongation and termination of transcription

21. Elongation • Once a short stretch of RNA about 10 bases synthesized, transcription turns into elongation phase. • Further conformational change in polymerase required for gripping the template more firmly. • functions: synthesis RNA, unwinds the DNA and reseals behind, dissociates the growing RNA chain from template, proofreads.

22. Termination • Stop and release the RNA products. • In some cells, specific and well-characterized sequences trigger termination, in others there are no such sequence.

23. three defined steps in initiation • Form closed complex. polymerase binds to a promoter, DNA helix remains double-stranded. • Form open complex. DNA strands separate and form the transcription bubble

24. Promoter escape the transition to the elongation phase. form stable ternary complex. (containing polymerase, DNA, and RNA)

25. Topic 2 the Transcription Cycle in Bacteria

26. Bacterial promoters vary in strength and sequence, but have certain defining features.

27. the bacterial core RNA polymerase can initiate transcription at any point of a DNA molecule. However, in cells, the transcription only start at promoters. The point is the δfactor.

28. δ factor Core enzyme Fig 6 RNA polymerase holoenzyme

29. It is the δ factor that converts core enzyme into the form that initiates only at promoters.

30. Promoters share the characteristic structure: • Two conserved sequence, each of six nucleotides. • Nonspecific stretch. 17~19 nucleotides. Fig 7 features of bacterial promoters

31. The δ factor has two conserved sequence that recognize the promoters(-35 and -10 elements). Between them is a non-specific stretch about 17bp.

32. consensus sequence Fig 8 regions of δ

33. The sequence of -35 and -10 elements are not identical. The closer to the consensus sequence, the stronger the transcription will initiate. • By the strength of a promoter, we mean how many transcripts it initiated in a given time. • An Up-element is found in some strong promoters, which increases the polymerase binding by providing an additional interaction site.

34. The δfactor mediates binding of polymerase to the promoter As shown in fig 8, the δ factor can be divided into 4 regions: Region 2--- recognize the -10 element Region 4--- recognize the -35 element Region 3--- recognize the extended -10 element

35. Two helices with the region 4 form a common DNA-binding motif called a helix-turn-helix: ▼One helix inserts into the major groove and interacts with bases in the -35 region. ▼The other lies across the top of the groove, making contact with the DNA backbone.

36. The -10 region is recognized by an α helix. this interaction is less well-characterized and is more complicated: ▼within the element, DNA melting is initiated from the closed to open complex. ▼the helix has to interact with bases on the nontemplate strand in a manner that stabilizes the melted DNA.

37. The UP-element is recognized by a carboxyl terminal domain of the α subunit, called the αCTD. FIG 9 δ andαsubunits recruit RNA core enzyme to the promoter

38. Transition from the closed complex to the open complex. This process is called isomerization. Which involves structure change of the enzyme and the opening of the DNA double helix. It is a spontaneous conformational change , as a result, it need not energy from ATP hydrolysis and is irreversible .

39. NTP entering channel is back DNA entering channel Fig 10 channels into and out of the open complex

40. Two striking structure changes: the β and the β’ pincers clamp down tightly on the downstream DNA. a major shift of the δ region 1.1---when not bound to DNA, it blocks the pathway. In the open complex, it shift an angle out of the enzyme.

41. There is no need of primer for transcriptionbecause RNA polymerase can initiate an new RNA chain on template without a primer . In stead , the initiating ATP is held tightly in the correct orientation by extensive interaction with the holoenzyme.

42. RNA polymerase synthesis several short RNAs before entering the elongation phase RNA synthesis begins an RNA an RNA less than 10 bp longer than 10bp Abortive initiation elongation phase

43. Abortive initiation . the transcripts are released from the polymerase, and the enzyme, without leaving the template, begins synthesis again

44. Structure barrier for the abortive initiation. the δ region 3.2 lies in the middle of the RNA exit channel in the open complex. ejection of this region from the channel is necessary for elongation, and also takes the enzyme several attemps.

45. The elongation polymerase is a processive machine that synthesizes and proofreads RNA. • Two proofreading functions: Pyrophosphorolytic editing remove the incorrect ribonucleotide by reincorporation of PPi. Hydrolytic editing stimulated by Gre factors (the elongation factor ), draw back one or more ribonucleotides and cleaves them.

46. Transcription is terminated by the signals within the RNA sequence • Terminator: the sequence trigger the elongation polymerase to dissociate from the DNA and release the RNA products.

47. Rho-independent intrinsic terminator, requires no factor. • Rho-dependent requires the Rho factor, and need ATP to wrest RNA from DNA and enzyme.

48. a stretch of 8 A-T base Fig 11 sequence of a rho-independent terminator

49. The hairpin cause the termination by disrupting the elongation complex. This is achieved either by forcing open the RNA exit channel in polymerase, or, by disrupting RNA-template interaction.

50. Week base pairing---easy to dissociate Fig 12 transcription termination