Transcription: Mechanisms and Regulation in Molecular Biology

1. 沈湯龍 助理教授Tang-Long Shen, PhD (Department of Molecular Medicine, Cornell University) 細胞生物學，訊息傳遞，癌症生物學 一號館315/317室Tel: 3366-4998 (Office) 3366-4602 (Lab) E-mail: shentl@ntu.edu.tw

2. Molecular Biology (Spring, 2009) What is transcription? How transcription works? Stages Machinery Molecular mechanism How transcription is regulated? Operon Mechanisms Examples of transcriptional regulation RNA silencing Phage strategy Ch. 11 Ch. 12 Ch. 13, 14

3. The basis of life Life form (behavior)

4. Transcription in prokaryotes

5. Transcription in Prokaryotes vs. Eukaryotes Eukaryotic Cell Prokaryotic Cell Because there is no nucleus to separate the processes of transcription and translation, when bacterial genes are transcribed, their transcripts can immediately be translated. Transcription and translation are spatially and temporally separated in eukaryotic cells; that is, transcription occurs in the nucleus to produce a pre-mRNA molecule. The pre-mRNA is typically processed to produce the mature mRNA, which exits the nucleus and is translated in the cytoplasm.

6. What is transcription?

7. Central Dogma of Biology: DNA → RNA → protein ☆ Gene Expression: Transcription Transcription = DNA → RNA ☆Gene functions (majority) are expressed as the proteins they encode: Translation Translation = RNA → protein

8. RNA chain is structurally similar to DNA chain But……..

9. Gene Transcription: DNA → RNA Genetic information flows from DNA to RNA by RNA polymerase RNA is identical in sequence with one strand of the DNA (but T→U), called coding strand. • Four stages of transcription: • Promoter recognition and initial melting • (binary complex formation) • 2. Initiation (ternary complex formation) • 3. Elongation • 4. Termination

10. Transcription Unit RNA polymerase Transcription unit binding May include more than one gene release A transcription unitis the distance between sites of initiation and termination by RNA polymerase; may include more than one gene (particularly in prokaryotes). , 3’ 5’ (Primary transcript) (polycistron) mRNA no number 0 A relative location on a linear sequence

11. How transcription works?

12. Basic principles of transcription Process Participants Initiation: polymerase and promoters Elongation: RNA polymerase Termination: terminator

13. Stages of transcription: follow a polarity along the template strand (5’) Promoter : closed complex Terminator (3’) : open complex : tertiary complex Promoter clearance Bubble moves on Abortive initiation: to ensure the initiation in a right way. (before the 10th base is added on nascent RNA chain within the bubble) Movement models • Sliding • inchworm Extending RNA chain is accomplished with RNA poly (bubble) moves along DNA. The bases after 9th enable added on the growing RNA chain. move Recognize termination signal Release RNA chain (by disrupt RNA:DNA hybrid) Dissociation of RNA pol

14. Initiation • Binding of an RNA polymerase to the dsDNA • (Sliding) to find the promoter • Unwind the DNA helix (Tx bubble) • Synthesis of the RNA strand at thestart site (initiation site),this position called position +1

15. Transcription Bubble To fulfill the principle process of transcription, that is complementary base pairing, hence a transient bubblehas to be created. Two strands of DNA are separated (about 12~14 bp in length). Template strand is used to synthesize a complementary sequence of RNA. The length of RNA-DNA hybrid within the bubble is about 8~9 bp. As RNA polymerase moves along the DNA, the transient bubble moves along with and the RNA chain grows continuously.

16. Progression of transcription bubble is association with RNA polymerase movement on DNA RNA extension 5’ 3’ DNA rewind behind DNA unwind ahead nascent RNA

17. Reaction in Transcription (RNA polymerization) RNA polymerization DNA replication 5’ → 3’ ~800 bp/sec Direction 5’ to 3’ NTP γ 5 ~40 nt/sec β Substrates:dATP, dTTP, dGTP, dCTP 4 3 2 Substrates ATP, UTP, GTP, CTP Phosphate α,β,γ Nucleotide Ribose 5C -- 1,2,3,4, 5 α Protein translation N → C termini ~15 aa/sec NTP 5 1 4 3 2 tRNA-amino acids NTP 5 γ 3 β α NTP 5 3

18. Summary- Transcription Bubble RNA-DNA hybrid length Ternary Complex: Polymerase-DNA-RNA ~ 8 to 9 bases, it is short and transient Functions of a RNA polymerase Unwinding and Rewind DNA NTPs polymerized to a RNA chain Moving on the DNA About 25-base RNA molecule associated with the ternary complex at any moment.

19. Machinery in transcription

20. RNA Pol I rRNA RNA Pol II mRNA RNA Pol III tRNA, 5S rRNA Transcription in Prokaryotes RNA polymerase Prokaryotes have a single RNA polymerase enzyme--synthesizes mRNAs, rRNAs, and tRNAs Transcribe over > 1000 transcription units. The complexity is modified by interacting with diverse regulatory factors. Eukaryotes have three RNA polymerase Enzymes:

21. E. coli RNA polymerase RNA polymerase binds to the promoter Core enzyme + sigma factor = holoenzyme 155 KD 36.5 KD 11 KD 36.5 KD 70 KD Initiation only 151 KD 465kD Both initiation & elongation

22. 2 a subunits Enzyme assembly Promoter recognition b subunit Catalytic Center b' subunit Catalytic Center Template-binding s subunit Promoter specificity Structure of E. coli RNA Polymerase:

23. Eubacteria RNA polymerase (Pol) About 7000 RNA polymerase molecules are present in an E.. coli cell. Most of them are engaged in transcription. In a short period of time, 2000-5000 Pol molecules can be synthesized.

24. E. coli Polymerase:α subunit • Encoded by the rpoA gene • Two identical subunits in the core enzyme • Required for core protein assembly • May play a role in promoter recognitionandregulatory factors interaction • ADP-ribosylation on an arginine upon T4 infection

25. E. coli polymerase: b subunit • Encoded by rpoB gene. • The catalytic center of the RNA polymerase • Rifampicin(used for anti-tuberculosis): bind to the β subunit (12A away from active site), and inhibit transcription initiation. Blocking the path for extending RNA chain beyond 2-3 nts. Mutation in rpoB gene can result in rifampicin resistance. • Streptolydigins：resistant mutations are mapped to rpoB gene as well. Inhibits transcription elongation but not initiation. 3. b subunit may contain two domains responsible for transcription initiation and elongation

26. E. coli polymerase: b’ subunit • Encoded by the rpoC gene . • Binds two Zn 2+/Mg 2+ ions and may participate in the catalytic function of the polymerase • Heparin:binds to the b’ subunit and inhibits transcription in vitro due to itcompetes with DNA for binding to the polymerase. 3. b’ subunit may be responsible for binding to the template DNA .

27. E. coli polymerase: s factor • Many prokaryotes contain multiple s factors to recognize different promoters. The most common s factor in E. coli is s70. (differential specificity) • Binding of the s factor converts the core RNA pol into the holoenzyme. • s factor is critical in promoter recognition, by decreasing the affinity of the core enzyme for non-specific DNA sites (104) and increasing the affinity for the corresponding promoter • s factor is released from the RNA pol after initiation (RNA chain is 8-9 nt) • Less amount of s factor is required in cells than that of the other subunits of the RNA pol.

28. Holoenzyme on promoter recognition (Core enzyme + sigma factor = holoenzyme) Core enzyme has the ability to synthesize RNA on a DNA template, but cannot initiate transcription at the proper sites. Holoenzyme has ~104-fold lower affinity for loose binding complexes than core. About 60 min half-life reduce to <1 sec. Holoenzyme has ~103-fold higher affinity for specific binding to promoters than core with a half life of several hours. Totally, sigma factor can result in 107 increase in DNA binding specificity. Core enzyme does not distinguish between promoters and other sequences of DNA.

29. Sigma factor is required only for initiation reversible Wide range Faster Tight binding Fastest Less than 10 bases Slow Beyond 10 bases leads to elongation

30. Recycle of sigma factor for the utilization of core enzyme Sigma factor is much less in number than core enzyme Evidence: 1/3 of sigma factors are not associated with core enzyme while elongation recycled Immediately after initiation

31. Molecular structure of RNA polymerases in functioning

32. Architecture of RNA polymerases (prokaryotes) (<100 kD) Bacterial RNA polymerase (465kD) T7 RNA polymerase Multiple subunits: 2α+β+β’+(σ) 25A wide Enzyme movement ~200 nts/sec ~40nts/sec Specificity recognition between enzyme and DNA bases (upstream of startpoint +1) A channel/groove on the surface ~25A wide forms a path for DNA. Path holds for 16 bp in prokaryotes 25 bp in eukaryotes More DNA bp can reside inside the enzyme Further crystal structure will provide more direct and detailed view in a molecular level.

34. Crystal structure of yeast RNA polymerase II

35. Architecture of RNA polymerases (eukaryotes) Yeast RNA polymerase contains 12 subunits (10 are shown here) Nevertheless, it shares similar organization as bacterial one. Cleft between two large subunits forms as an active center A channel/groove on the surface forms a path for DNA. 25 bp DNA can be held in the path.

36. DNA in and out DNA out rudder RNA dissociated RNA flipped out Flexible ss DNA DNA turns DNA in Rigid straight duplex DNA entry (control by bridge protein)

37. How does RNA polymerase find promoter sequences? Directed walk Diffusion Random walk (Direct displacement) No DNA protein is known to work in this way RNA polymerase found promoters is very faster. Diffusion in the whole genome cannot support this fast. Enzyme moves preferentially from a weak site to a strong site

38. Cycle of making and breaking bonds between enzyme and nucleic acids straight bent straight Change in conformation of “bridge” protein is closely related to translocation of the enzyme along the nucleic acid.

39. Transitions in shape and size of RNA polymerase during transcription (and compositions) Covered DNA length 75-80 bp (-55 to +20) 60 bp (-35 to +20s) 30-40 bp (interact w/ RNA pol)

40. How to resume the stalled/pausing RNA polymerase? Cleavage 3’ end of RNA chain Backtracks of RNA polymerase as a whole (Create a 3’-OH for further polymerization) A constant distance between active site and frond end To correct mispositioned template during stall Accessory factors are needed such as: GreA and GreB for E. coli RNA polymerase TFIIS for eukaryotic RNA polymerase II One more function of RNA polymerase: * cleavage activity is from RNA polymerase itself. unwind Rewind polymerize RNA Moving on the DNA

41. Sequence elements in Transcription Promoter Coding sequence Terminator

42. What is a promoter? • The sequence of DNA needed for RNA polymerase to bind to the template and accomplish the initiation reaction. • Its structure (not transcribed) is the signal (others are needed to be converted into RNAs or proteins). • It is a cis-actingsite. • Different from sequences whose role is to be transcribed or translated. What signal (structure) of a promoter provides?

43. The sequence comparison of five E. coli promoters TTGACA TATAAT Consensus Consensus sequences : they show which residues are conserved and which residues are variable (homology)

44. (Open binary complex formation) (recognition domain (Closed binary complex formation) (i.e. the distance of separation between -10 and -35; intermediate sequence is irrelevant)

45. Prokaryotic promoters display four conserved features: 1. Startpoint: >90% PURINE (A or G) 2. -10 consensus sequence (Pribnow box)--TAtAaT T80 A95 t45 A60 a50 T96 3. -35 consensus sequence--TTGACa T82 T84 G78 A65 C54 a45 4. Distance (spacing) between the -10 and -35 sequences (The distance is critical in holding the two sites at the appropriate separation for the geometry of RNA polymerase.)

46. Transcription startsite The sequence around the start site influences initiation • A purine(A or G) in 90% of all genes • Often, there are C and T bases on either side of the start site nucleotide (i.e. CGT or CAT)

47. Functions of promoter domains -35 recognition domain Closed binary complex formation -10 unwinding domain: due to A-T pairs require lower energy to disrupt (melt) Open binary complex formation Sequence around the startpoint (+1 to +30): influences the initiation event. Rate of promoter clearance Other ancillary proteins may help RNA polymerase to recognize deficient promoters.

48. Promoter efficiency • There is considerable variation in sequence between different promoters, and the transcription efficiency can vary by up to 1000-fold . • The –35 sequence, -10 sequence, and sequence around the start sites all influence initiation efficiency. • The sequence of the first 30 bases to be transcribed controls the rate at which the RNA polymerase clears the promoter, hence influences the rate of the transcription and the overall promoter strength . • Strand separation in the initiation reaction (see below) • Some promoter sequence are not strong enough to initiate transcription under normal condition, activating factor is required for initiation. For example, Lac promoter Plac requires cAMP receptor protein (CRP )

49. Supercoiling during transcription At initiation ∵ Supercoiled structure requires less free energy for the initial melting of DNA ∴ it enhances the efficiency of transcription in vitro After initiation DNA is rotated during RNA pol movement; front is overwound and behind is released. A twin domain on transcribing DNA formed

50. RNA polymerase-promoter interactions A promoter with consensus sequences for the -10 and -35 regions (boxed) is shown; the sequences of actual promoters deviate from those shown here. The "jaws" of RNA polymerase are shown on the right of the molecule. This region of the RNA polymerase would grasp the DNA downstream of the catalytic site. Contacts between RNA polymerase and promoter DNA are shown by the solid lines. Not all contacts occur in every RNA polymerase-promoter interaction, but in all known cases (including promoters activated by regulator proteins), at a minimum, some contacts between the 35 and the 10 region appear to be required. J Bacteriol, June 1998, p. 3019-3025, Vol. 180, No. 12