Transcription

1. Transcription

2. Ex Biochem c11-transcription Introduction • Coding strand: identical in sequence with RNA • Template strand: used as template for RNA synthesis • Complimentary to RNA Figure 11.1

3. Ex Biochem c11-transcription 11.1 Introduction • RNA polymerase • Promoter: a special region containing startpoint • Terminator • Upstream: sequences prior to startpoint • Downstream: sequences after startpoint Figure 11.2

4. Ex Biochem c11-transcription 11.2 Transcription Occurs by Base Pairing in a “Bubble” of Unpaired DNA • RNA polymerase separates the two strands of DNA in a transient “bubble.” • When RNA polymerase bind to a promoter • It uses one strand as a template to direct synthesis of a complementary sequence of RNA. • The length of the bubble is ∼12 to 14 bp • The length of RNA-DNA hybrid within it is ∼8 to 9 bp. Figure 11.3

5. Ex Biochem c11-transcription Figure 11.04: The transcription bubble moves along DNA.

6. Ex Biochem c11-transcription Figure 11.05: RNA polymerase surrounds the bubble.

7. Ex Biochem c11-transcription Transcription Reaction Has 3 Stages • Template recognition: bind to promoter • Initiation: RNA polymerase initiates transcription after binding to a promoter site on DNA. • Elongation: During elongation the transcription bubble moves along DNA. • The RNA chain is extended in the 5′–3′ direction. • Termination: When transcription stops: • the DNA duplex reforms • RNA polymerase dissociates at a terminator site Figure 11.6

8. Ex Biochem c11-transcription 11.4 Phage T7 RNA PolymeraseIs a Useful Model System • T3 and T7 phage RNA polymerases are single polypeptides. • They have minimal activities in recognizing a small number of phage promoters. • Crystal structures of T7 RNA polymerase with DNA identify: • the DNA-binding region • the active site Figure 11.7

9. Ex Biochem c11-transcription Figure 11.07: T7 RNA polymerase has a single subunit.

10. Ex Biochem c11-transcription Figure 11.08: RNA polymerase has a channel for DNA. Photo courtesy of Seth Darst, Rockefeller University

11. Ex Biochem c11-transcription Figure 11.09: RNA polymerase surrounds DNA.

12. Ex Biochem c11-transcription Figure 11.10: A top view of RNA polymerase II. Photo courtesy of Roger Kornberg, Stanford University School of Medicine

13. Ex Biochem c11-transcription Figure 11.11: An end view of RNA polymerase II. Photo courtesy of Roger Kornberg, Stanford University School of Medicine

14. Ex Biochem c11-transcription

15. Ex Biochem c11-transcription 11.5 A Model for Enzyme Movement Is Suggested by the Crystal Structure • DNA moves through a groove in yeast RNA polymerase that makes a sharp turn at the active site. Figure 11.12

16. Ex Biochem c11-transcription Figure 11.14: Polymerases must make and break bonds.

17. Ex Biochem c11-transcription • A protein bridge changes conformation to control the entry of nucleotides to the active site. Figure 11.15

18. Ex Biochem c11-transcription 11.6 Bacterial RNA Polymerase Consists of Multiple Subunits • Bacterial RNA core polymerases are ∼500 kD multisubunit complexes with the general structure α2ββ′ • RNA polymerase from E. Coli as typical model • ~7000 in an cell • Complete enzyme (holoenzyme) ~465 kD Figure 11.16

19. Ex Biochem c11-transcription 11.10 How Does RNA Polymerase Find Promoter Sequences? • The rate at which RNA polymerase binds to promoters is too fast to be accounted for by random diffusion. Figure 11.22

20. Ex Biochem c11-transcription • RNA polymerase probably: • binds to random sites on DNA • exchanges them with other sequences very rapidly until a promoter is found Figure 11.23

21. Ex Biochem c11-transcription 11.12 Promoter Recognition Depends On Consensus Sequences • A sequence of DNA whose function is to be recognized by proteins • Usually cis-acting • A promoter is defined by the presence of short consensus sequences at specific locations. • The promoter consensus sequences consist of: • a purine at the startpoint • the hexamer TATAAT centered at –10 • another hexamer centered at –35 • Separation between -10 and -35 • UP element (sometimes), located further upstream • Individual promoters usually differ from the consensus at one or more positions.

22. Ex Biochem c11-transcription Figure 2.16: Proteins bind to cis-acting control sites.

23. Ex Biochem c11-transcription Figure 2.17: Mutations in control sites are cis-acting.

24. Ex Biochem c11-transcription Figure 11.26: The promoter has three components.

25. Ex Biochem c11-transcription 11.13 Promoter Efficiencies Can Be Increased or Decreased by Mutation • Down mutations: to decrease promoter efficiency usually decrease conformance to the consensus sequences. • Up mutations have the opposite effect. • Mutations in the –35 sequence usually affect initial binding of RNA polymerase.

26. Ex Biochem c11-transcription • Mutations in the –10 sequence usually affect the melting reaction that converts a closed to an open complex. Figure 11.27

27. Ex Biochem c11-transcription 11.20 Bacterial RNA Polymerase Terminates at Discrete Sites • Termination may require both: • recognition of the terminator sequence in DNA • formation of a hairpin structure in the RNA product Figure 11.45

28. Ex Biochem c11-transcription 11.21 There Are Two Types of Terminators in E. coli • Intrinsic terminators consist of: • a G-C-rich hairpin in the RNA product • followed by a U-rich region in which termination occurs • Rho-dependent terminators Figure 11.46

29. Ex Biochem c11-transcription 11.22 How Does Rho Factor Work? • Rho factor is a terminator protein that: • binds to a rut site on nascent RNA • tracks along the RNA to release it from the RNA–DNA hybrid structure at the RNA polymerase Figure 11.47

30. Ex Biochem c11-transcription Figure 11.48: A rut site has a biased base composition. rich in C, poor in G, no secondary structure

31. Ex Biochem c11-transcription 11.23 Antitermination Is a Regulatory Event • Termination is prevented when antitermination proteins act on RNA polymerase. • This causes it to read through a specific terminator or terminators. Figure 11.51

32. Ex Biochem c11-transcription • Phage lambda has two antitermination proteins, pN and pQ. • They act on different transcription units. Figure 11.52

33. Ex Biochem c11-transcription Video sources for transcription • http://tw.youtube.com/watch?v=WsofH466lqk • http://tw.youtube.com/watch?v=P6Nyce-4oG4 • Transcription factors

34. Ex Biochem c11-transcription http://www.sabiosciences.com/pathway.php?sn=Transcription_of_mRNA