1. Chapter12 Mechanisms of Transcription 胡红霞 04 级生物科学 胡红霞 04 级生物科学 200431060178 200431060178

2. 2 The Central Dogma: transcription translation DNA RNA PROTEIN Transcription is the first step of the expression of the genome !

3. 3 mRNA transcript

4. 4 & Similarities vs Differences Transcription & Replication Transcription and replication: 1. A new chain synthesized upon a DNA template 2. In a 5’ to 3’ direction Transcription : 1.Ribonucleotides 2.Use RNA polymerases 3. Needs no primer (de novo) 4. Does not remain base-paired to the template all the time 5. Less accurate (10 -5 vs 10 -8 ) 6. Selectively copies certain parts of the genome and makes anything from one to several hundred, or even thousand, copies of any given section (Recall replication)

5. 5 Outlines 1.RNA Polymerases and the Transcription Cycle 2.The Transcription Cycle in Bacteria 3.Transcription in Eukaryotes All these topics are very important !!!

6. 6 RNA polymerases and the transcription cycle

7. 7 § 1.1 RNA Polymerases RNA Polymerases Come in Different Forms, but Share Many Features RNA Polymerase performs essentially the same reaction in all cells, so RNA polymerases from bacteria to humans are highly conserved, especially in those parts of the enzyme directly involved with catalyzing the synthesis of RNA.

8. 8 The Subunits of RNA Polymerases

9. 9 Eukaryotic cells have three RNA polymerases RNA Pol I : transcribes the large ribosomal RNA precursor gene RNA Pol II : most studied, transcribes most genes – essentially all protein- encoding genes ( focus) RNA Pol III : transcribes tRNA genes, some small nuclear RNA genes and the 5S rRNA gene

10. 10 Comparison of the crystal structures of prokaryotic and eukaryotic RNA polymerase

11. 11 The bacterial RNA polymerase The core enzyme alone can synthesize RNA The massive RNA holoenzyme contains 6 subunits: the б subunit, β’ subunit, β subunit, ω subunit, and two α dimer subunits.

12. 12 crab claw the two pincers of the crab claw -- β’ and β subunits active center cleft (can bind two Mg 2+ )

13. 13 RNA polymerase holoenzyme

14. 14 § 1.2 The transcription cycle RNA polymerases proceeds through a series of well- defined steps : Initiation Elongation Termination

15. 15 Initiation Important points : A promoter is the DNA sequence that initially binds the RNA polymerases. The promoter- polymerase complex undergoes structural changes required for initiation to proceed. The new ribonucleotide is added to the 3’ end of the growing chain, so transcription always occurs in a 5’ to 3’ direction. The choice of promoter is the main regulation.

16. 16 Closed complex : form when initially bind to a promoter Open complex : the DNA strands separate and the transcription bubble forms Stable ternary complex : form after an enzyme gets further than 10 bp (that is when the enzyme has escaped the promoter) Transcription initiation involves three defined steps :

17. 17 The phases of the transcription cycle Initiation Elongation Termination

18. 18 Elongation The functions of RNA polymerase during this elongation phase : 1.The catalysis of RNA synthesis; 2.Unwinds the DNA in front; 3.Re-anneals the DNA behind; 4.Dissociates the growing RNA chain from the template; 5.Moves along the DNA template; 6.RNA proofreading.

19. 19 Transcription Cycle

20. 20 Termination Stops and releases the RNA product once the polymerase has transcribed the length of the gene (or genes). In some cells there are specific, well- characterized sequences that trigger it. In others it is less clear what instructs the termination.

21. 21 The Transcription Cycle in Bacteria

22. 22 б factor The RNA polymerase initiate transcription at any point on a DNA molecule; yet the б factor converts core enzyme into the form that initiates only at promoters. (б 70 is predominant in E.coli ) RNA polymerase holoenzyme = RNA polymerase + б factor

23. 23 The б subunit is composed of α helices connected by turns and loops. These elements organize into four domains : N-terminal domain 1 N-terminal domain 2 Linker domain C-terminal domain After synthesis of a 9-12 nucleotide RNA, the б subunit dissociates from the core polymerase, and the core begins the elongation of the RNA transcript.

24. 24 Bacteria Promoters Vary in strength and sequence, but have certain defining features.

25. 25 Characteristic structure of promoters recognized by polymerase containing б 70 : Two conserved sequences: each of 6 nucleotides, centered at about 10 and 35 base pairs upstream of the site where RNA synthesis starts. -10 and -35 regions, or elements; A nonspecific stretch of 17-19 nucleotides between.

26. 26 The б factor mediates binding of polymerase to the promoter The regions that recognize the -10 and -35 elements of the promoter are region 2 and 4, respectively. ( helix-turn-helix ) б region 2 recognizes -10 element б region 3 recognizes the extended -10 element б region 4 recognizes -35 element

27. 27 Regions of б

28. 28 UP-element An additional DNA elements that binds RNA polymerase and increases binding by providing an additional specific interaction between the enzyme and the DNA. Is recognized by αCTD of the polymerase б and α subunits recruit RNA polymerase core enzyme to the promoter

29. 29 “Extended –10” element Another class of б 70 -promoters lacks a –35 region and has an “extended –10” element. And this element compensates for the absence of a -35 region. This element is recognized by an α helix in б region 3 through two specific base pairs.

30. 30 structural changes Transition to the open complex involves structural changes in RNA polymerase and in the promoter DNA

31. 31 Closed complex Open complex Structural changes: DNA around the transcription start site is unwound, forming a bubble of single-stranded DNA The enzyme also changes (this “melting” occurs between positions -11 and +3) Occurs spontaneously; not require ATP to provide energy. isomerization

32. 32 channels into and out of the open complex The five channels: NTP-uptake RNA-exit Downstream DNA Nontemplate- strand (NT) Template- Strand (T)

33. 33 Upon isomerization there are two striking structural changes in the polymerase 1.The pincers at the front clamp down tightly on the downstream DNA. 2.There is a major shift occurs in the N- terminal region of  (region 1.1).In the closed complex,  region 1.1 lies within the active center while in the open complex, it shifts to the outside of the enzyme, allowing the DNA access to the cleft

34. 34 Transcription is initiated without a primer RNA polymerase can initiate a new RNA chain on a DNA template. Difficulty : RNA polymerase starts most transcripts with an A, but A-T pair has only two hydrogen bonds. Various parts of polymerase holoenzyme, including part of б provide specific interactions with the initiating ribonucleotide.

35. 35 RNA polymerase synthesizes several short RNAs before entering the elongation phase Abortive initiation: the enzyme synthesizes short RNA molecules less than 10 nucleotides and then releases the transcript. Promoter escape : Once the polymerase manages to make an RNA longer than 10bp, a stable ternary complex containing the enzyme, the DNA template and a growing chain is formed and the elongation starts. Elongation

36. 36 The elongation polymerase is a processive machine that synthesizes and proofreads RNA Processes DNA: Enter between the pincers The strands separate Reform a double helix behind RNA polymerase: Adds new ribonucleotides Releases the RNA product

37. 37 Attentions : Only 8 – 9 nucleotides of the growing RNA chain remain base-paired to the DNA template at any given time; The remainder of the RNA chain is peeled off and directed out of the enzyme through the RNA exit channel

38. 38 Proofreading mechanisms The enzyme catalyzes the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi, using its active site. Pyrophosphorolytic editing

39. 39 Proofreading mechanisms Hydrolytic editing The enzyme backtracks by one or more nucleotides and cleaves the error- containing sequence. It is stimulated by Gre factor. Termination

40. 40 Transcription is terminated by signals within the RNA sequence Terminators : sequences that trigger the elongating polymerase to dissociate from the DNA and release the RNA chain it has made. Two types: Rho-independent terminators Rho-dependent terminators

41. 41 Rho-independent terminators Sequence of a rho- independent terminator: 1.A short inverted repeat (20 bp) 2.A stretch of about 8 A:T base pairs

42. 42 Rho-independent terminators Require A:U base pairs for they are the weakest of all base pairs, then the RNA will more readily dissociate from the template.

43. 43 Transcription termination Polymerase transcribes an inverted repeat ; Form a stem loop within RNA ; Cause termination by disrupting the elongation complex.

44. 44 Rho-dependent terminators Require the action of Rho factor. Rho (  ) A ring-shaped protein with six identical subunits; Binds to single-stranded RNA as it exits; Has an ATPase activity The Rho transcription terminator

45. 45 Rho is directed to a particular RNA molecule Rho Binding Specificity : In the binding sites which consist of stretches of about 40 nucleotides that remain largely single-stranded and are rich in C residues; Only binds those transcripts still being transcribed beyond the end of a gene or operon.

46. 46 Transcription in Eukaryotes

47. 47 Transcription in eukaryotes is undertaken by polymerases closely related to RNA polymerases found in bacteria. However, there are some differences between the two cases.

48. 48 Differences 1.Eukaryotes have Pol I, II and III, whereas bacteria has only one; Eukaryotes require several initiation factors, whereas bacteria require only one ---  factor; Isomerization to the open complex in eukaryotes require ATP hydrolysis, whereas in bacteria it occurs spontaneously; In eukaryotes promoter escape is regulated by the phosphorylation state if the CTD tail; Elongation factors and proofreading mechanism; Termination (some of these will be discussed later)

49. 49 RNA polymerase II core promoters are made up of combinations of four different sequence elements RNA polymerase II core promoters are made up of combinations of four different sequence elements II The eukaryotic core promoter : the minimal set of sequence elements required for accurate transcription initiation by Pol II

50. 50 Regulatory sequence Beyond –-typically upstream of –- the core promoter and required for efficient transcription in vivo. Categories: 1.Promoter proximal elements 2.Upstream activator sequences, UASs 3.Enhancers 4.Silencers 5.Boundary elements 6.Insulators All these DNA elements bind regulatory proteins (activators and repressors)

51. 51 RNA Pol II forms a pre- initiation complex with GTFs at the promoter Pre-initiation complex Pre-initiation complex = GTFs + polymerase+ promoter Functions of the general transcription factors (GTFs): help polymerase 1.Bind to the promoter ; 2.Melt the DNA ; 3.Escape from the promoter ; 4.Begin the elongation phase.

52. 52 IID =TBP + TAFs TFIID =TBP + TAFs Resulting II Transcription initiation by RNA polymerase II

53. 53 II Transcription initiation by RNA polymerase II The C-terminal domain (CTD) contains repeats of heptapeptide and its phosphorylation helps the promoter escape

54. 54 TBP binds to and distorts DNA using a  sheet inserted into the minor groove Unusual compared with a helices using by most proteins. TBP relies on the ability of TATA sequence to undergo the distortion.

55. 55 The other GTFs also have specific roles in initiation TAFs (TBP associated factors) About 10 TAFs are associated with TBP. Two bind DNA elements at the promoter; Several bind DNA in the way like histone proteins; Another regulates the binding of TBP to DNA. TFIIB A single polypeptide chain, enters the pre-initiation complex after TBP. TFIIB binds to the TBP-TATA complex asymmetrically. Thus the transcription has an fixed orientation.

56. 56 TFIIF Two-subunit factor; Binding of Pol II– TFIIF stabilizes the DNA-TBP- TFIIF complex; Required for others. TFIIE Two subunits; Has roles in recruitment and regulation of TFIIH The other GTFs also have specific roles in initiation & TFIIF & TFIIE

57. 57 Nine subunits –- the largest and most complex of the GTFs. Function : pre-initiation complex to the open complex; Controls the ATP-dependent transition of the pre-initiation complex to the open complex; Two have ATPase activity Two have ATPase activity Another is a protein kinase( promoter melting and escape) Another is a protein kinase( promoter melting and escape) The other GTFs also have specific roles in initiation TFIIH

58. 58 In vivo, transcription initiation requires additional proteins Reason for requirement: DNA is packaged into chromatin, not linear Different activators interact with different Mediator subunits to bring polymerase to different genes.

59. 59 Additional proteins Assembly of the pre- initiation complex in presence of Mediator, nucleosome modifiers and remodelers, and transcriptional activators

60. 60 Mediator consists of many subunits, some conserved from Yeast to Human Comparison of the yeast and human Mediators Elongation

61. 61 A new set of factors stimulate Pol II elongation and RNA proofreading Another set of factors replaced the initiation factors during the transition from initiation to elongation. These enzymes are recruited to the phosphorylated CTD.

62. 62 Recruitment of enzymes RNA processing enzymes are recruited by the tail of polymerase

63. 63 Various factors stimulating elongation activates stimulates hSPT5 stimulates P- TEFb Elongation by Pol II TAT-SF1 TFIIS RNAaseProofreading

64. 64 Elongation polymerase is associated with a new set of protein factors required for various types of RNA processing The processing events: Capping of the 5’ end of the RNA Splicing (discuss later) Polyadenylation of the 3’ end of the RNA

65. 65 There is an overlap in proteins involved in elongation and those required for RNA processing. Elongation,termination and RNA processing are interconnected to ensure their proper coordination

66. 66 Capping Methylated guanine 5’-5’ linkage The first step: the  phosphate at the 5’ end of the RNA is removed by RNA triphosphatase

67. 67 Capping The second step: the enzyme guanylyl transferase catalyzes the nucleophilic attack

68. 68 Capping The third step: Addition of methyl groups by methyl transferase.

69. 69 Functions of 5’ cap Protection from degradation Increased translational efficiency Transport to cytoplasm Recruitment of the splicing machinery Continues the transcription (possible)

70. 70 Polyadenylation Linked with the termination The polymerase CTD tail is involved in recruiting the enzymes necessary for polyadenylation as well as with capping and splicing

71. 71 Factors involved in Polyadenylation The extended C-terminal domain of the largest subunit of RNA (CTD) Cleavage and polyadenylation specificity factor (CPSF) Cleavage stimulation factor (CstF) Additional cleavage factors Poly-A polymerase (PAP) Poly-A-binding proteins

72. 72 Polyadenylation Transcription of poly-A signal sequence Cleavage of the RNA Polyadenylation by poly-A polymerase

73. 73 Termination RNA polymerase does not terminate immediately when the RNA is cleaved and polyadenylated. Rather, it continues to move along the template, generating a second RNA molecule that can be as long as several hundred nucleotides before terminating. It is clear that the polyadenylation signal is required for termination.

74. 74 Models of termination Spontaneous termination Model 1 Transfer of 3’ processing enzymes from the polymerase CTD tail to the RNA triggers A conformational change in the polymerase Reduces processivity of the enzyme Leading to

75. 75 Transfer of the CPSF and CstF from CTD tail to RNA triggers conformational change, reducing processivity Model 1

76. 76 Models of termination Model 2 The absence of a 5’ cap (for the absence of the capping enzymes on the CTD) on the second RNA molecule is sensed by the polymerase which then recognizes the transcript as improper and terminates.

77. 77 Model 2

78. 78 RNA Polymerase I and III recognize distinct promoters, using distinct sets of transcription factors, but still require TBP RNA Polymerase I and III recognize distinct promoters, using distinct sets of transcription factors, but still require TBP Two other polymerase in eukaryotes – Pol I and Pol III are related to Pol II, but initiates transcription from distinct promoters and transcribes distinct genes

79. 79 RNA polymerase I promoter region Promoter = the core element + the UCE Factors needed: Pol I + SL1+ UBF

80. 80 RNA Pol III core promoter Unusual feature of being located downstream Factors needed: Pol III + TFIIIB + + TFIIIC(tRNA) or TFIIIA(5s rRNA)

81. 81 That ’ s all for mechanisms of transcription !