1. Chapter 12: Mechanisms of Transcription

2. RNA polymerase and transcription cycle
The transcription cycle in bacteria
Transcription in eukaryotes

3. Mechanistic features of transcription is very similar to DNA replication but there are some important differences:
RNA polymerase does not need a primer.
The RNA product does not remain base-paired to the template DNA strand.
Transcription, though very accurate , is less accurate than replication.

4. RNA Polymerase and The Transcription Cycle

5. RNA polymerases come in different forms, but share many features
Transcription by RNA polymerase proceeds in a series of steps
Transcription initiation involves 3 defined steps

6. 1.RNA polymerases performs essentially the same reaction in all cells
Table 12-1: The subunits of RNA polymerases

7. 2.Bacteria have only a single RNA polymerases while in eukaryotic cells there are three: RNA Pol I, II and III

8. b’ prokaryotic a b a w eukaryotic
RPB2
RPB3
RPB1
RPB11
RPB6
eukaryotic
Figure 12-2 Comparison of the crystal structures of prokaryotic and eukaryotic RNA polymerases

9. 3.To transcribe a gene , RNA polymerase proceeds through a series of well-defined steps which are grouped into three phases: Initiation Elongation Termination

10. initiation
Elongation
Termination

11. Initiation
Promoter.
promoter-polymerase complex undergoes structural changes required for initiation to proceed.
DNA at the transcription site unwinds and a “bubble” forms
Direction of RNA synthesis occurs in a 5’-3’ direction (3’-end growing)

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

13. Elongation
Once the RNA polymerase has synthesized a short stretch of RNA ,it shifts into the elongation phase.
During elongation , the enzyme performs an impressive range of tasks in addition to the catalysis of RNA synthesis, it unwinds the DNA in front and re-anneals it behind, it dissociates the growing RNA chain from the template as it moves along ,and it performs proofreading functions.

14. Termination
Once the polymerase has transcribed the length of the gene (or genes), it must stop and release the RNA product. This step is called Termination
In some cells there are specific, well-characterized, sequences that trigger termination; but in others it is less clear.

15. Figure 12-3-Elongation and termination

16. 4.The first phase in the transcription cycle-initiation-can itself be broken down into a series of defined steps:
Forming closed complex
Forming open complex
Promoter escape

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

18. The transcription cycle in bacteria

19. 1.Bacterial promoters vary in strength and sequences, but have certain defining features
In cells polymerase initiates transcription only at promoters. It is the addition of an initiation factor called s that converts core enzyme into the form that initiates only at promoters. That form of the enzyme is called the RNA polymerase holoenzyme (Figure 12-4)

20. Figure 12-4 RNA polymerase holoenzyme T.aquaticus.,

21. 2. In the case of E. coli ,the predominant s factor is called s70
2.In the case of E. coli ,the predominant s factor is called s70. They share the following characteristic structure:
Two conserved sequences each of six nucleotides, are separated by a non-specific stretch of nucleotides (Figure 12-5) .
The two defined sequences are centered, respectively, at about 10 base pairs and at about 35 base pairs upstream of the site where RNA synthesis starts.
Position +1 is the transcription start site. The sequences are thus called -35(minus 35) and –10(minus 10) regions, or elements.

22. Figure 12-5: Features of bacterial promoters

23. An additional DNA element that binds RNA polymerase is found in some strong promoters, for example those directing expression of the ribosomal RNA (rRNA) genes This is called an UP-element (Figure 12-5b)and increases polymerase binding by providing an additional specific interaction between the enzyme and the DNA.

24. Another class of s70 promoter lacks a –35 region and instead has an “extended –10 element” compensating for the absence of –35 region. (Figure 12-5c)

25. 3.The s factor mediates binding of polymerase to the promoter
The s70 factor comprises four regions called s region 1 to s region 4.
The regions that recognize the -10 and -35 elements of the promoter are region 2 and 4, respectively.

26. Figure 12-6: regions of s

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

28. The -10 region is also recognized by an a helix.
The region of s that interacts with the -10 region is doing more than simply binding DNA.
The a helix involved in recognition of the -10 region contains several essential aromatic amino acids that can interacts with bases on the non-template strand to stabilize the melted DNA.

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

30. 5.Transition to the open complex involves structural changes in RNA polymerase and in the promoter DNA
The initial binding of RNA polymerase to the promoter DNA in the closed complex leaves the DNA in double –stranded form .
In the case of the bacterial enzyme bearing s 70,this transition, often called isomerization, does not require energy derived from ATP hydrolysis.

31. 6.The structure of holoenzyme in more detail
A channel runs between the pincers of the claw-shaped enzyme.
The active site of the enzyme, which is made up of regions from both the b and b’ subunits, is found at the base of the pincers within the “active center cleft.”
There are five channels into the enzyme, as shown in the picture of the open complex in Figure 12-8.

32. Figure 12-8 channels into and out of the open complex
The NTP-uptake channel allows ribonucleotides to enter the active center . The RNA-exit channel allows the growing RNA chain to leave the enzyme as it is synthesized during elongation. The remaining three channels allow DNA entry and exit from the enzyme, as follows.
Figure 12-8 channels into and out of the open complex

33. 7.Two striking structural change in the polymerase
First, the pincers at the front of the enzyme clamp down tightly on the downstream;
Second, there is a major shift 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 some 50 Å to the outside of the center, allowing DNA access to the cleft

34. 8.Transcription is initiated by RNA polymerase without the need for a primer
RNA polymerase can initiate a new RNA chain on a DNA template and thus does not need a primer.
The enzyme has to make specific interactions with the initiating ribonucleotide, holding it rigidly in the correct orientation to allow chemical attack on the incoming NTP.

35. 9.RNA polymerase synthesizes several short RNAs before entering the elongation phase
Abortive initiation: In this phase, the enzyme synthesizes short RNA molecules of less than ten nucleotides in length. Instead of being elongated further, these transcripts are released from the polymerase, and the enzyme, without disassociating from the template, begins RNA synthesis again.

36. It is not clear why RNA polymerase undergoes this period of abortive initiation. but once again a region of the s factor appears to be involved, acting as a molecular mimic.

37. 10.The elongating polymerase is a processive machine that synthesizes and proofreads RNA
At the opening of the catalytic cleft, the strands separate to follow different paths through the enzyme before exiting via their respective channels and reforming a double helix behind the elongating polymerase.
Ribonucleotides enter the active site through their defined channel and are DNA strand.

38. Proofreading by RNA polymerase
Pyrohosphorolytic editing （焦磷酸化编辑): the enzyme uses its active site, to catalyze the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi. Then it incorporate another ribonucleotide in its place in the growing RNA chain.
Hydrolytic （水解）editing: the polymerase backtracks by one or more nucleotides and cleaves the RNA product, removing the error-containing sequence.

39. 11.Transcription is terminated by signals within the RNA sequence
Sequences called terminators trigger the elongation polymerase to dissociate from the DNA and release the RNA chain it has made.
There are two types of terminators in bacteria:
Rho-dependent
Rho-independent (intrinsic terminator)

40. Figure 12-9 Sequence of a rho-independent terminator
Rho-independent terminator contains a short inverted repeat followed by a stretch of about 8 A:T base pairs.
Figure 12-9 Sequence of a rho-independent terminator

41. Shown is a model for how the rho-independent terminator might work.
Figure transcription termination
Shown is a model for how the rho-independent terminator might work.

42. 12.Once attached to the transcript, Rho uses the energy derived from ATP hydrolysis to wrest the RNA from the template and from polymerase.
Rho is directed to a particular RNA molecule:
There is some specificity in the sites it binds. These sites are rich in C residues.
Rho fails to bind any transcript that is being translated.

43. Figure 12-11 the r transcription terminator
The crystal structure of the rho termination factor is shown in a top down view.

44. transcription in eukaryotes

45. 1.RNA polymerase II core promoters are made up of combinations of 4 different sequence elements
Figure 12-2 shows the location, relative to the transcription start site, of four elements found in PolⅡ core promoters:
TFIIB recognition element (BRE)
TATA element (or box)
Initiator (Inr)
Downstream promoter element (DPE)

46. Figure 12-12: Pol II core promoter

47. Pre-initiation complex: The complete set of general transcription factors and polymerase, bound together at the promoter and poised for initiation

48. Some TAFs help bind the DNA at certain promoters, and others control the DNA-binding activity of TBP.
Upon binding DNA, TBP extensively distorts the TATA sequence.
TFIIA, TFIIB, TFIIF together with polymerase, and then TFIIE and TFIIH, which bind upstream of PolⅡ.

49. FIGURE 12-13 Transcription initiation by RNA polymerase Ⅱ

50. 2.TBP binds to and distorts DNA using a b sheet inserted into the minor groove
When it binds DNA, TBF causes the minor groove to be widened to an almost flat conformation;
It also bends the DNA by an angle of approximately 80°

51. 3.The other GTFs also have specific roles in initiation
TAFs: TBP is associated with about ten TAFs. Two of the TAFs bind DNA elements at the promoter. Another TAF appears to regulate the binding of TBP to DNA.

52. TFIIB: This protein, a single polypeptide chain, enters the preinitiation complex after TBP
Figure TFIIB-TBP-promoter complex

53. TFIIF: This two-subunit factor associates with that enzyme
TFIIF: This two-subunit factor associates with that enzyme. Binding of Pol Ⅱ-TFIIF stabilizes the DNA-TBP-TFIIB complex and is required before TFIIE and TFIIH are recruited to the pre-initiation complex.
TFIIE and TFIIH: TFIIE, which, like TFIIF, consists if two subunits, binds next, and has roles in the recruitment and regulation of TFIIH.

54. 4.in vivo, transcription initiation requires additional proteins, including the mediator complex
The DNA template in vivo is packaged into nucleosomes and chromatin. This condition complicates binding to the promoter of polymerase and its associated factors.
Different activators are believed to interact with different Mediator aids initiation by regulating the CTD kinase in TFIIH
The need for nucleosome modifiers and remodellers also differs at different promoters or even at the same promoter under different circumstances.

55. Figure 12-17 comparison of the yeast and human mediators
5.Mediator consists of many subunits, some conserved from yeast to human
The yeast and human Mediator each include more than 20 subunits, of which 7show significant sequence homology between the two organisms.
Figure comparison of the yeast and human mediators

56. A new set of factors stimulate Pol II elongation and RNA proofreading
Figure RNA processing enzymes are recruited by the tail of polymerase

57. 6.Some elongation factors
P-TEFb:
phosphorylates CTD
Activates hSPT5
Activates TAT-SF1
TFIIS:
Stimulates the overall rate of elongation by resolving the polymerase pausing
Proofreading

58. 7.Elongation polymerase is associated with a new set of protein factors required for various types of RNA processing
RNA processing:
Capping of the 5’ end of the RNA
Splicing of the introns (most complicated)
Poly adenylation (多聚腺苷化) of the 3’ end

59. Elongation, termination of transcription, and RNA processing are interconnected/ coupled (偶联的) to ensure the coordination (协同性) of these events
Evidence: this is an overlap in proteins involving in those events
The elongation factor hSPT5 also recruits and stimulates the 5’ capping enzyme
The elongation factor TAT-SF1 recruits components for splicing

60. RNA processing 1 5’ end capping
The “cap”: a methylated guanine joined to the RNA transcript by a 5’-5’ linkage
The linkage contains 3 phosphates
3 sequential enzymatic reactions
Occurs early

61. Splicing: joining the protein coding sequences
Dephosphorylation of Ser5 within the CTD tail leads to dissociation of capping machinery
Further phosphorylation of Ser2 recruits the splicing machinery

62. 3’ end polyadenylation Linked with the termination of transcription
The CTD tail is involved in recruiting the polyadenylation enzymes
The transcribed poly-A signal triggers the reactions
Cleavage of the message
Addition of poly-A
Termination of transcription

63. Figure 12-20 polyadenylation and
termination

64. 8.Models to explain the link between polyadenylation and termination
First model: The transfer of the 3’ processing enzymes to RNAP II induces conformational change—RNAP II processivity reduces—spontaneous termination
Second model: absence of a 5’cap on the second RNA molecule—recognized by the RNAP II as improper—terminate transcription

65. 9.RNA Pol I & III recognize distinct promoters , using distinct sets of transcription factors, but still require TBP
Pol I is required for the expression of only one gene, that encoding the rRNA precursor.
Pol III promoters come in various forms, and the vast majority have the unusual feature of being bocated downstream of the transcription start site.

66. Pol I promoter recognition
Fig Pol I promoter region

67. Figure 12-22 Pol III core promoter
Show here is the promoter for a yeast tRNA gene.The order of events leading to transcription initiation is described in the text.

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