Assignment

1. Assignment Part 3 Expression Of The Genome Chapter 12 Mechanisms Of Transcription 崔品 200331060129

5. OUTLINE • RNA Polymerases and the Transcription Cycle • The difference of Transcription Cycle in Bacteria and Transcription in Eukaryotes

6. RNA PLOYMERASES AND THETRANSCRIPTION CYCLE • Transcription involves synthesis of an RNA chain representing one strand of a DNA duplex. • RNA synthesis is catalyzed by the enzyme RNA polymerase • The mechanisms in Eukaryotes and Prokaryotes are similar though there are some • differences.

7. RNA Polymerases Come in Different Forms, but Share Many Features • RNA polymerases performs essentially the same reaction in all cells, from bacteria to humans. • Enzymes from these organisms share many features, especially in those parts that directly involved with catalyzing the synthesis of RNA • The cellular RNA polymerases are made up of multiple subunits.

8. TABLE 12-1 The Subunits of RNA polymerases

9. From the table above ,we can see: • Eukaryotes have three different polymerases each ;bacteria have just one .The three eukaryotic enzymes are called RNA PolⅠ, Ⅱ and Ⅲ . • In this chapter we focused primarily on Pol Ⅱ ,as it is the enzyme that transcribes the vast majority of genes in the cell and all the protein coding genes.

10. The structure of the bacterial and yeast Pol Ⅱ enzyme are similar. Both resemble a crab claw in shape ,the pincers being made up of the largest subunits , β and β‘ in the case of the bacterial enzyme. The active site is at the base of the pincers, and access to and from the active site is afforded through five channels: • One allows double-stranded DNA to enter between the pincers; • Two others allow the single strands to leave the enzyme behind the active site; • Another channel provides the route NTPs entering the active site ; • The RNA product exits the enzyme through the fifth channel.

11. Pol Ⅱ differs from the bacteria enzyme in one important way .The former has a so-called “tail” at the C-terminal end of the large subunit, and this is absent from the bacteria enzyme .This tail is made up of multiple repeats of heptapeptide sequence.

12. Comparison of the crystal structures of prokaryotic and eukaryotic RNA polymerase • prokaryotic RNA polymerase • eukaryotic RNA polymerase

13. Transcription by RNA Polymerases Proceeds in a Series of Steps • To transcribe a gene, RNA polymerases proceeds through a series of well-defined steps which are grouped into three phases: • Initiation; • Elongation; • Termination; • (Both in eukaryotes and prokaryotes)

14. Three Steps of initiation • 1:Formation of a closed complex • RNA polymerases binds to a region of DNA called a promoter. In bacteria, this step involves an initiation factor called sigma, which recognizes various sequences within promoters. The RNA polymerase with sigma attached binds the promoter in a defined orientation, so the same DNA strand is always transcribed from a given promoter. The RNA polymerase and promoter form a closed complex.

15. Step 2 Transition to an open complex • The closed complex undergoes a transition to the open complex. The pincers at the front of the enzyme clamp down tightly on the downstream DNA. Sigma also changes conformation, and the DNA strands separate, forming a bubble of single-stranded DNA.

16. Step 3 Promoter escape • A region of sigma partially blocks the RNA exit channel. Once this region has been ejected and an RNA chain longer than 10 nucleotides has been synthesized, the elongation phase begins. Transition to the elongation phase is called promoter escape.

17. Steps of elongation • Unwinding of DNA in front of the enzyme • Synthesis of RNA • RNA proofreading • Dissociation of RNA • Re-annealing of DNA behind the enzyme

18. Termination • Once the polymerase has transcribed the length of the gene ,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 ;in others it is less clear what instructs the enzyme to cease transcribing and dissociate from the template.

19. RNA proofreading • The proofreading activity of RNA polymerase is less efficient than that of DNA polymerase. • The proofreading activity of DNA polymerase only allows about 1mistake per 10 thousand nucleotides. • Two mechanism : • Pyrophosphorolytic editing • Hydrolytic editing

20. initiation • elongation • termination

21. Difference between eukaryotic and prokaryotic transcription • Transcription in both eukaryotes and prokaryotes follows the above steps ,but there are differences in many cases: • Initiation • Termination

22. Isomerization to the open complex occurs spontaneouslyand does not require ATP hydrolysis In eukaryotes this step does require ATP hydrolysis Initiation in bacteria

23. The bacteria promoters vary in strength and sequence,but have certain defining feaures • It is the addition of an initiation factor called σ that converts core enzyme into the form that initiates only at promotors. • That form of the enzyne is called the RNA polymerase holoenzyme.

24. RNA polymerase holoenzyme T.aquaticus

25. Features of bacterial promoters

26. The σ factor mediates binding of polymerase to the promoter • The σ factor can be divided into four regions called σ region 1 through σ region 4 .The region that recognize the -10and -35 elements of the promoter are region 2 and 4 , respectively. • Two helices within region 4 form a common DNA –binding motif called a helix-turn-helix.

27. Regions of σ

28. Transition to the open complex involves structural changes in RNA polymerase and in the promoter DNA • The closed complex then undergoes isomerization to the open complex • Isomerization to the open complex occurs spontaneously and does not require ATP hydrolysis

29. Σ and α subunits recruit RNA polymerase core enzyme to tne promoter

30. Channels into and out of the open complex

31. Transcription is initiated by RNA polymerase without the need for a primer • RNA polymerase synthesize several short RNAs before entering the elongation phase • The elongation polymerase Is a processive machine that synthesizes and proofreads RNA

32. Transcription is terminated by signals within the RNA sequence • Sequences called terminators trigger the elongating polymerase to dissociate from the DNA and release the RNA chain it has made . • In bacteria ,terminators come in two types: • Rho-indepedent • Rho-depedent

33. Rho-independent terminators • Rho-independent terminators,also called intrinsic terminators,cosist of two sequence elements:a short inverted repeat of about 20 nucleotides, and a stretch of about eight A:T base pairs. • The RNA that results from the inverted repeat sequence is able to base pair with itself ,and forms a hairpin structure thatdisrupts the elongationcomplex .A:U base pairs the weakest of all base pair allowing the RNA to easily dissociate from the DNA

34. Rho-dependent terminators • Rho-dependent terminators requires the action • Of the Rho factor ,a ring –shaped hexameric • Protein • The Rho factor binds to the transcribed RNA as it exits the polymeras.Using the energy derived from ATP hydrolysis, theRho factor pulls the RNAfromthe template and RNA polymerase.

35. Sequence of a Rho-independent terminators

36. Transcription termination • shomn is a model • for how the • Rho-independent • terminatorsmight work.

37. the rho • Xc transcription • Aaa termination • e factor

38. Transcription in eukaryotes • Transcription in eukaryotes is undertaken by polymerase closely related to RNA polymerase found in bacteria.

39. RNA polymerase Ⅱ core promoters are made up of combinations of four different sequence elements

40. Initiation in Eukaryotes • Promoter escape is regulated by the phosphorylation state of the CTD tail. Thus, the form of Pol Ⅱ that binds the promoter in the pre-initiation complex has an unphosphorylated CTD. This domain becomes phosphorylated by one or more kinases, including one that is part of one of the general transcription factors, TFIIH. • The TATA element is recognized by the general transcription factor called TFIID. • The component of TFIID that binds to the TATA DNA sequence is called TBP

41. Transcriptioninitiation by RNA polymerase Ⅱ

42. TBP binds to and distorts DNA using a β sheet inserted into the minor groove

43. In vivo ,transcription requires additional proteins, including the mediator complex

44. Mediator consists of many subunits ,some conserved from Yeast to Human

45. A new set of factors stimulate Pol Ⅱ elongtion and RNA proofreading

46. Elongating polymerase is associated with a new set of protein factors required for various types of RNA processing

47. Eukaryotic Transcription Termination • Polyadenylation • Models of termination

48. Introduction of polyadenylation • Eukaryotic mRNA has to be processed in various ways before being exported from the nucleus for translation. • One such process takes place during the terminationphase of the transcription cycle, • Where a long chain of adeniness is added to the 3’end of the mRNA transcript .This process Is called polyadenylation .

49. Polyadenylation involves several factors • The extended C-terminal domain of the largest subunit of RNA polymerase----C-terminal domain (CTD) tail • Cleavage and polyadenylation specificity factor (CPSF) • Cleavage stimulation factor(CstF) • Additional cleavage factors • Poly-A polymerase(PAP) • Poly-A-binding proteins

50. Steps of polyadenylation • Transcription of poly-A signal sequence • Cleavage of the RNA • Polyadenyation by poly-A polymerase