Chapter 12: Mechanisms of Transcription

1. Chapter 12:Mechanisms of Transcription ◆ RNA Polymerases and the Transcription Cycle ◆ The Transcription Cycle in Bacteria ◆ Transcription in Eukaryotes

2. Similarity Both involve enzymes that synthesize a new strand of nucleic acid complementary to a DNA template strand. Differences 1.RNA is made from ribonucleotides. 2.RNA polymerase catalyzes the reaction. 3.RNA polymerase does not need a primer. 4.The RNA product does not remain base-paired to the template DNA strand. (Figure12-1) 5.Transcription is less accurate than replication. 6.Transcription selectively copies only certain parts of the genome and makes one to several hundred, or even thousand, copies of any given section of the genome. Replication must copy the entire genome and do so only once every cell division. Transcription is, chemically and enzymatically, very similar to DNA replication. However, there are some important differences.

3. Part 1:RNA Polymerases and the Transcription Cycle

4. RNA Polymerases Come in Different Forms,but Share Many Features • RNA Polymerase performs essentially the same reaction in all cells. • From bacteria to mammals, the cellular RNA Polymerases are made up of multiple subunits. (Table 12-1) • Bacteria have only a single RNA polymerase, while in eukaryotic cells there are three: RNA Pol I, II and III. • Pol II is the most studied of these enzymes, and is responsible for transcribing most genes----indeed, essentially all protein-encoding genes. • Pol I transcribes the large ribosomal RNA precursor gene. • Pol III transcribes tRNA genes, some small nuclear RNA genes, and the 5S rRNA gene.

5. The bacterial RNA polymerase core enzyme alone is capable of synthesizing RNA and comprises two copies of the αsubunit and one each of theβ, β’andωsubunits. b’ a b a w

6. Figure 12-2 Comparison of the crystal structures of prokaryotic and eukaryotic RNA polymerases The same colorindicate the homologous subunits of the two enzymes

7. Overall ,the shape of each enzyme resembles a crab claw pincer Active center cleft pincer

8. Transcription by RNA Polymerase Proceeds in a Series of Steps • To transcribe a gene, RNA polymerase proceeds through a series of well-defined steps which are grouped into three phases: Initiation Elongation Termination

9. Initiation • A promoter is the DNA sequence that initially binds the RNA polymerase. Once formed, thepromoter-polymerase complex undergoes structural changes required for initiation to proceed. • DNA at the transcription site unwinds and a “bubble” forms. • Like replication, transcription occurs in a 5’ to 3’ direction. • Only one of the DNA stands acts as a template. • The choice of promoter determines which stretch of DNA is transcribed and is the main step at which regulation is imposed.

10. Elongation • Once the RNA polymerase has synthesized a short stretch of RNA (approximately 10 bases), transcription shifts into the elongation phase. • This transition requires further conformational change in polymerase that leads it to grip the template more firmly. • Additional tasks of the RNA polymerase: ☺Unwinds the DNA in front and re-anneals it behind; ☺Dissociates the growing RNA chain from the template; ☺Performs proofreading functions.

11. 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 ; in others it is less clear what instructs the enzyme to cease transcribing and dissociate from the template.

12. Figure 12-3 The phases of the transcription cycle: initiation, elongation and termination elongation initiation Termination

13. Transcription Initiation Involves Three Defined Steps • The first step is the initial binding of polymerase to a promoter to form a closed complex. • In the second step, the closed complex undergoes a transition to the open complex in which the DNA strands separate over a distance of some 14bp around the start site to form the transcription bubble. • Once an enzyme gets further than the 10bp, it is said to have escaped the promoter. At this point it has formed a stable ternary complex, containing enzyme, DNA, and RNA. This is the transition to elongation .

14. Part 2:The Transcription Cycle in Bacteria

15. BacterialPromoters Vary in Strength and Sequence, but Have Certain Defining Features • In cells, polymerase initiates transcription only at promoters. It is the addition of an initiation factor called σ that converts core enzyme into the form that initiates only at promoters. That form of the enzyme is called the RNA polymerase holoenzyme. holoenzyme = σfactor + core enzyme

16. Figure 12-4RNA polymerase holoenzyme T.aquaticus.

17. The predominant σfactor in E.coli is s70 • Promoter recognized by s70 contains two conserved sequences (-35 and –10 regions/elements) separated by a non-specific stretch of 17-19 nucleotides. • The “+1” position is designated as the transcription start site.

18. Figure 12-5 Features of bacterial promoters (a) • σ70promoters contain recognizable –35 and –10 regions, but the sequences are not identical. • Comparison of many different promoters derives theconsensus sequencesreflecting preferred –10 and –35 regions. • Promoters with sequences closer to the consensus are generally “stronger” than those match less well. • The strength of the promoter describes how many transcripts it initiates in a given time.

19. Figure 12-5 Features of bacterial promoters (b) • UP-element is an additional DNA elements that increases polymerase binding by providing an additional specific interaction between the RNA polymerase and the DNA.

20. Figure 12-5 Features of bacterial promoters (c) Another class of σ70promoter lacks a –35 region and has an “extended –10 element” compensating for the absence of –35 region

21. The σFactor Mediates Binding Polymerase to the Promoter • Theσ70 factorcan be divided into four regions called σregion 1 through σregion 4. Figure 12-6 Regions of σ

22. Region 2 recognizes -10 element Region 4 recognizes -35 element • Region 3 recognizes the extended -10 element

23. Two helices within region 4 form a common DNA-binding motif called ahelix-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 contacts with DNA backbone.

24. The interaction with -10 region is less well-characterized and is more complicated. Reasons: • The -10 region is within that element that DNA melting is initiated in the transition from the closed to open complex. • The a helix recognizing –10 can interact with bases on the nontemplate strand to stabilize the melted DNA.

25. The extended -10 element, where present, is recognized by an a helix in σregion 3. • The helix makes contact with the two specific base pairs that constitute that element.

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

27. Transition to the Open Complex Involves Structural Changes in RNA Polymerase and in the Promoter DNA • In the case of the bacterial enzyme bearing σ70 ,this transition, often calledisomerization, does not require energy, but is the result of a spontaneous conformational change in the DNA-enzyme complex to a more energetically favorable form.

28. There are five channels into the RNA polymerase holoenzyme. Figure 12-8 Channels into and out of the open complex

29. Within the active center cleft, the DNA strands separate from position +3. • The nontemplate strand goes through the nontemplate-strand channel and travels across the surface of the enzyme. • The template strand goes through the template-strand channel. • The double helix re-forms at -11 in the upstream DNA behind the enzyme.

30. Two striking structural changes in the enzyme upon isomerization • First, the pincers at the front of the enzyme clamp down tightly on the downstream DNA. • Second, there is a major shift in the position of the N-terminal region of σ. In the closed complex, σ region 1.1 is in the active center; in the open complex, the region 1.1 shift to the outside of the center, allowing DNA access to the cleft.

31. Transcription is Initiated by RNA Polymerase without the Need for a Primer • The initiation requires that the initiating ribonucleotide (usually an A) be brought into the active site and held stably on the template while the next NTP is presented with correct geometry. • Thus 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.

32. RNA Polymerase Synthesizes Several Short RNAs before Entering the Elongation Phase • Once ribonucleotides enter the active center cleft and RNA synthesis begins, there follows a period called abortive initiation. • Abortive initiation: the enzyme synthesizes and releases short RNA molecules less than 10 nucleotides in length. • Once a polymerase manages to make an RNA longer than 10bp, a stable ternary complex is formed. This is the start of the elongation phase.

33. Structural barrier for the abortive initiation • The 3.2 region of s factor lies in the middle of the RNA exit channel in the open complex. • Ejection of this region from the channel (1) is necessary for further RNA elongation; (2) takes the enzyme several attempts

35. The Elongating Polymerase Is a Processive Machine that Synthesizes and Proofreads RNA Synthesizing by RNA polymerase 1. DNA enters the polymerase between the pincers. 2. Strand separation in the catalytic cleft. 3. NTP addition. 4. RNA product spooling out (Only 8-9 nts of the growing RNA remain base-paired with the DNA template at any given time). 5. DNA strand annealing in behind.

36. Proofreading by RNA polymerase • Pyrophosphorolytic editing: the enzyme catalyzes the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi. • Hydrolytic editing: the enzyme backtracks by one or more nucleotides and removes the error-containing sequence. This is stimulated by Gre factor, a elongation stimulation factor.

37. Transcripyion 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-independent and Rho-dependent.

38. Rho-independent terminators, also called intrinsic terminators, consist of two sequence elements: a short inverted repeat (of about 20 nucleotides) & a stretch of about 8 A:T base pairs.

39. Figure 12-9 Sequence of a rho-independent terminator

40. When polymerase transcribes an inverted repeat sequence, the resulting RNA can form a stem-loop structure (often called a “hairpin”) by base-pairing with itself. • The hairpin is believed to cause termination by disrupting the elongation complex. • The hairpin only works as an efficient terminator when it is followed by a stretch of A:U base pairs. • A:U base pairs are the weakest of all base pairs, so they can make the dissociation more easier.

41. Figure 12-10 Transcription termination

42. Rho-dependent terminators • Have less well-characterized RNA elements, and requires Rho protein for termination. • Rho is a ring-shaped single-stranded RNA binding protein, like SSB, and has six identical subunits. • Rho binding can wrest the RNA from the polymerase-template complex using the energy from ATP hydrolysis. • Rho binds to rut sites (for Rho Utilization) and does not bind the transcripts that are being translated.

43. Figure 12-11 The ρtranscription termination factor

44. Part 3: Transcription in Eukaryotes

45. A little comparison

46. In addition to the RNAP and GTFs, in vivo transcription also requires • Mediator complex • DNA-binding regulatory proteins • Chromatin-modifying enzymes

47. RNA Polymerase II Core Promoters Are Made up of Combinations of four Different Sequence Elements • The eukaryotic core promoter : the minimal set of sequence elements required for accurate transcription initiation by the Pol II machinery in vitro. • A core promoter is typically about 40 nucleotides long, extending either upstream or downstream of the transcription start site. • Four elements in Pol II core promoters: The TFIIB recognition element (BRE) The TATA element (or box) The initiator (Inr) The downstream promoter element (DPE)

48. Figure 12-12 Pol II core promoter The figure shows the position of various DNA elements relative to the transcription start site. Below are the consensus sequence for each element and above are the names of the general transcription factors that recognize them.

49. Regulatory sequences are also required for efficient transcription in vivo besides the core promoter. • These elements include: ☼ Promoter proximal elements ☼ Upstream activator sequences (UASs) ☼ Enhancers ☼ A series of repressing elements called: silencers, boundary elements, and insulators. All these DNA elements bind regulatory proteins, which help or hinder transcription from the core promoter.

50. RNA Polymerase II Forms a Pre-Initiation Complex with General Transcription Factors at the Promoter • Pre-initiation complex:The complete set of general transcription factors and polymerase bound together at the promoter and poised for initiation. • The TATA element where pre-initiation complex formation begins is recognized by the the general transcription factor called TFIID. • TFIID = TBP + TAFs TBP associated factors TATA binding protein