Welcome to Chapter 12

1. Welcome to Chapter 12 Mechanisms of transcription

2. Introduction • Up to this point we have been considering maintenance to the genome ,that is ,how the genetic material is organized ,protected, and replicated. • In the next parts ,we will describe the basicprocessed responsible for gene expression. • First let us go into the world of transcription

3. Transcription Vs Replication Transcription is chemically and enzymatically, very similar to DNA replication.Both involve enzymes that synthesize a new strand of nucleic acid complementary to DNA template strand.Moreover ,there are many differences between them.

4. The differences go as follows: • RNA is made of ribonucleotides • RNA polymerase ,which catalyzes the reaction,needs no primer • The newly synthesized RNA does not remain base-paired to the template DNA strand • Less accurate ,one mistake occurs in 10,000 • Because of different purpose ,transcription selectively copies only certain parts of the genome and makes anything from one to several hundred,or even thousand.

5. Question :why transcription is less accurate than replication? I think the difference makes good sense if we associate it with the results of the mistakes. DNA is the molecule in which the genetic material is stored,and DNA replication si the process by which that genetic material is passed on.Any mistake can easily be catastrophic:it becomes permanent in the genome of that individual and also gets passed on to subsequent generations.

6. Transcription ,in contrast,produces only transient copies and normally several from each transcribed region. • Thus ,a mistake during transcription will rarely do more harm than render one out of many transient transcripts defective.

7. Outline • 1. RNA polymerase & Transcription cycle • 2. The transcription cycle in bacteria • 3.Transcription in eukaryotes

8. Topic 1: RNA polymerase & The transcription cycle

9. RNA polymerase • RNA pol come in different forms ,but share many features,especially in those parts of the enzyme directly involved with catalyzing the synthesis of RNA • RNA pol performs essentially the same reaction in all cells,from bacteria to humans.

10. 1 The structure of RNA pol • From bacteria to mammals ,the cellular RNA polymerase are made up of multiple subunits . • Bacteria have only a single RNA pol ,which is the core enzyme capable of synthesizing RNA • Eukaryotic cells have three, namely RNA pol I ,II ,and III .They are responsible for synthesis of different kinds of RNA

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

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

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. RNA pol II is the focus ,which is responsible for transcribing most genes-indeed,essentially all protein-encoding genes. • RNA Pol I transcribes the large ribosomal RNA precursor gene. • RNA Pol III transcribes tRNA genes,some small nuclear RNA genes,and the 5S rRNA gene

15. Since the structure of RNA Pol is this,there come the question:How do they function? Or how do they realize the process of transcription?

16. Transcription by RNA Pol proceeds in a series of steps • Initiation • Elongation • Termination Let us go deep into the details

17. Process 1: Initiation (1)Promoter :the DNA sequence that initially binds the RNA pol (2)Promoter-polymerase complex undergoes structural changes (3)The DNA around the point where transcription unwinds,forming a “bubble”( similar to DNA replication) (4)Again like DNA replication,the direction of transcription is from 5’ to 3’

18. Additionally ,unlike replication,only one of the DNA strands acts as a template on which the RNA strand is built.

19. Transcription Initiation Invoves 3 Defined Steps • Form closed complex • Form open complex • Form stable ternaty complex

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

21. Closed complex • Initial binding of pol to a promoter In this form ,DNA remains double-stranded,and the enzyme is bound to one face of the helix.

22. Open complex • DNA strands separate around the transcription site • The transcription bubble forms

23. Stable ternary complex • Enzyme escape the promoter once it gets further than the 10 bp • Stable ternary complex contains enzyme,DNA and RNA • Then the elongation phase comes

24. Process 2 : Elongation • Begins when the enzyme has synthesized a short stretch of RNA (about 10 bp) • The RNA pol undergoes further comformational changes to grip the template more firmly • The enzyme functions:RNA synthesis ,unwind the DNA chains in front,re-anneal it behind,dissociate the growing RNA chain from the template

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

26. Process 3: Termination • Once the length of the gene has been transcribed ,the RNA pol must stop and release the product • In some cells ,there are specific,well-characterized sequences.In other cells,it remains to be seen what instructs the termination

27. Topic 2 :The Transcription Cycle In Bacteria

28. 2-1 Bacterial promoters vary in strength & sequence,but have certain defining features • The bacterial core RNA pol can ,in principle ,initiate transcription at any point on a DNA molecule .In cells,polymerase initiates transcription only at promoters. • It is the addition of initiation factor called σthat converts core enzyme into the form that initiates only at promoters. • That form of the enzyme is called holoenzyme ,which is made up of core enzyme and σfactor

29. 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

30. The details of σ factor • Structure : composed of 4 regions called σregion 1 through σregion 4 • Function :recognize the site of promoter, mediates binding of polymerase to the promoter

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

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

33. Take E.coli as a example In the case of E.coli ,the predominant σfactor is calledσ70 factor . Promoters recognized by σ70 factor share the following characteristic structure:two conserved sequences,each of six nucleotides,are separated by a nonspecific stretch of 17-19nucleotides. The two defined sequences are centered,respectively,at about 10 bp and at about 35 bp upstream of the site where RNA synthesis starts. The sequences are thus called the –35 and –10 regions,or elements. Position +1is the transcription start site.

34. Consensus sequence • Although the vast majority of σ70 promoters contain recognizable –35 and –10 regions,the sequences are not identical. • Comparison of many different sequences reflecting preferred –10 and –35 regions • Promoters with sequences closer to the consensus are generally “stronger” than those that match less well. • By the strength of a promoter,we mean how many transcripts it initiates in a given time.

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

36. Up-element • An additional DNA element that binds RNA polymerase is found in some strong promoters • Up-element can increases polymerase binding by providing an additional specific interaction between the enzyme and DNA • The magnificence is this : another class of σ70–promoters lacks a –35region and instead gas a so called “extended-10” element,which compensates for the absence of –35 region.

37. 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

38. 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

39. 2-2 The features of transcription in bacteria • 1.Transition to the open complex involves structural changes in RNA pol and in the promoter DNA (melting , isomerization, the active center cleft) • 2.Transcription is initiated by RNA pol without the need for a primer • 3.RNA pol synthesizes several short RNAs before entering the elongation phase. (Abortive initiation)

40. 4.The elongating pol is a processive machine that synthesizes and proofreads RNA.(pyrophosphorolytic editing & hydrolytic editing) • 5.transcription is terminated by signals within the TNA sequence (Rho-independent Vs Rho-dependent, intrinsic terminators.)

41. Rho-independent terminatorcontains a short inverted repeat (~20 bp) and a stretch of ~8 A:T base pairs. Fig 12-9

42. Fig 12-11 the r transcription terminator RNA tread trough the “ring” Hexamer, Open ring

43. Topic 3 : transcription in eukaryotes

44. Transcription in bacteria Vs in eukaryotes • Eukaruotes have three different pol (I,II,III), whereas bacteria have only one. • Bacteria require only one additional initiation factor(σfactor ) , but several initiation factors are required for efficient and promoter-specific initiation in eukaryotes,which is called the general transcription factors(GTFs)

45. The factors needed for transcription in vivo • GTFs • Polymerase • Mediator complex • DNA-binding regulatory proteins • Chromatin-modifying enzymes

46. However ,in vitro, the general transcription factors are all that is required,together with pol II . One reason for the difference is that the DNA template in vivo is packaged into nucleosomes and chromatin .This condition complicates binding to the promoter of pol and its associated factors.

47. Core promoter • Core promoter refers to the minimal set of sequence elements required for accurate transcription initiation by the pol II machinery. • A core promoter is typically about 40 nucleotides long, extending either upstream or downstream of the transcription start site.

48. Fig 12-12: Pol II core promoter • TFIIB recognition element (BRE) • The TATA element/box • Initiator (Inr) • The downstream promoter element (DPE)

49. Fore elements in core promoter • BRE : the TFIIB recognition element • The TATA element • Inr : the initiator • DPE: the downstream promoter Generally , a promoter includes only two or three of these four elements .

50. Regulatory sequences • Beyond the core promoter, there are other sequence elements required for efficient transcription in vivo.These elements constitute the regulatory sequences. • They can be grouped into varous categories, reflecting their location, and the organism in question ,as much as their function