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What is transcription ? How transcription works ? Stages Machinery Molecular mechanism

Molecular Biology (3/30~4/25, 2007). What is transcription ? How transcription works ? Stages Machinery Molecular mechanism How transcription is regulated ? Regulators Mechanisms Examples of transcriptional regulation Phage strategy RNA silencing. Ch. 9. Ch. 10,11.

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What is transcription ? How transcription works ? Stages Machinery Molecular mechanism

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  1. Molecular Biology (3/30~4/25, 2007) What is transcription? How transcription works? Stages Machinery Molecular mechanism How transcription is regulated? Regulators Mechanisms Examples of transcriptional regulation Phage strategy RNA silencing Ch. 9 Ch. 10,11 Ch. 12, 11 沈湯龍 (Tang-Long Shen) 助理教授細胞生物學一號館315室Tel: 3366-4998; E-mail: shentl@ntu.edu.tw

  2. 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.

  3. Transcription in prokaryotes

  4. The basis of life

  5. What is transcription?

  6. 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

  7. RNA is structurally similar to DNA But……..

  8. 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

  9. Transcription Unit RNA polymerase Transcription unit binding release May include more than one gene 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) no number 0 mRNA 3’ 5’ A relative location on a linear sequence

  10. How transcription works?

  11. Basic principles of transcription Template recognition: polymerase and duplex DNA Initiation: polymerase* and promoters Elongation: RNA polymerase Termination: terminator abortive initiation

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

  13. Transcription Bubble To fulfill the principle process of transcription, that is complementary base pairing, 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.

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

  15. Elongation Progression of transcription bubble is association with RNA polymerase movement on DNA RNA extension 5’ 3’ Movement models • Sliding: • inchworm DNA rewind behind DNA unwind ahead RNA

  16. 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 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 NTP 5 γ 3 β α NTP 5 3

  17. Stages of transcription (5’) Promoter : closed complex Terminator (3’) : open complex Binary Promoter clears Bubble moves on Ternary Abortive initiation: to ensure the initiation in a right way. Movement models (before the 10th base is added on nascent RNA chain within the bubble) • Sliding: common • 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

  18. Machinery in transcription

  19. 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:

  20. 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

  21. 2 a subunits Enzyme assembly, Promoter recognition, factor binding b subunit Catalytic Center b' subunit Catalytic Center Template-binding s subunit Promoter specificity Structure and functions of E. coli RNA Polymerase

  22. 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.

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

  24. 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

  25. 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 it competes with DNA for binding to the polymerase. 3. b’ subunit may be responsible for binding to the template DNA .

  26. 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.

  27. 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.

  28. 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

  29. 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

  30. Molecular structure of RNA polymerases in functioning

  31. Architecture of RNA polymerases (prokaryotes) (<100 kD) Bacterial RNA polymerase (465kD) T7 RNA polymerase Multiple subunits: 2α+β+β’+(σ) 25A wide Enzyme movement ~40nts/sec ~200 nts/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 on the enzyme Further crystal structure will provide more direct and detailed view in a molecular level.

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

  33. Ternary Complex Channel within RNA polymerase Active center + Enzyme movement

  34. DNA in and out DNA out rudder RNA dissociated RNA flipped out Flexible ss DNA DNA turns Rigid straight duplex DNA DNA in entry (control by bridge protein) How many bp(s) in the bubble?

  35. Contact among the ternary structure in the active site These contacts can stabilize the single strand nucleic acid chains.

  36. Cycle of making and breaking bonds between enzyme and nucleic acids nt enters, adds, and interacts with the bridge protein straight nt still interacts with the bridge protein, which leads the protein to bending due to Pol moves one bp forward. bent Meanwhile, bridge blocks free nt enters. straight Finally, bridge releases Its interaction with newly added nt on RNA chain. Change in conformation of “bridge” protein is closely related to translocation of the enzyme along the nucleic acid.

  37. How does RNA polymerase find promoter sequences? Directed walk vs. Random walk Random diffusion (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. Transitions in shape and size of RNA polymerase during transcription Covered DNA length 75-80 bp (-55 to +20) 60 bp (-35 to +20s) 30-40 bp (interact w/ RNA pol)

  39. 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 DNA/RNA binding polymerize RNA

  40. Sequence elements in Transcription Promoter Coding sequence Terminator

  41. 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?

  42. AT has only 2 H-bonds, which is easier to be broken (Open binary complex formation) (recognition domain (Closed binary complex formation) (i.e. the distance of separation between -10 and -35; intermediate sequence is irrelevant) Pribnow, D.: Nucleotide Sequence of an RNA Polymerase Binding Site at an Early T7 Promoter. PNAS 72, 784 (1975). Pribnow, D.: Bacteriophage T7 early promoters: nucleotide sequences of two RNA polymerase binding sites. J. Mol. Biol. 99, 419 (1975). Schaller, H. et al.: Nucleotide Sequence of an RNA Polymerase Binding Site from the DNA of Bacteriophage fd. PNAS 72, 737 (1975).

  43. The sequence comparison of five E. coli promoters TTGACA TATAAT Consensus Consensus: the most common base sequence to appear at such points on the DNA helix; there may be variationsin various organisms

  44. 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.) 5. UP element. TA rich sequence upstream of promoter.

  45. Functions of promoter domains -35 recognition domain Closed binary complex formation -10 unwinding domain: due to A-T pairs need 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.

  46. Other structures may exist in a promoter A-T rich sequence It interacts with the α subunit of the RNA polymerase, which to ensure the higher gene expression. 100-fold variation in vitro Down mutation: mutations are tend to be concentrated in the most highly conserved positions. Up mutation: less cases happen within promoters

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