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The differences between Transcription DNA replication

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  1. The main topics1.RNA polymerase and transcription cycle2.The transcription cycle in bacteria3.Transcription in eukaryotes

  2. The differencesbetween Transcription DNA replication • 1.RNA is made of ribonucleotides • 2.RNA polymerase catalyzes the reaction • 3.The synthesized RNA does not remain base paired to the template DNA strand • 4.Transcription is less accurate • 5.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.

  3. Topic 1: RNA Polymerase and The Transcription Cycle

  4. The structure of RNA polymerase • RNA polymerase comes in different forms ,but share many features. • From bacteria to mammals ,the cellular RNA polymerase are made up of multiple subunits

  5. The subunits of RNA polymerase

  6. The difference between bacteria and eukaryotic cells on RNA polymerase • The bacteria have only a single RNA polymerase • Eukaryotic cells have three: RNA polymerase I ,II ,and III .

  7. RNA Pol II is the focus of eukaryotic transcription, because it is the most studied polymerase, and is also responsible for transcribing most genes-indeed, essentially all protein-encoding genes • RNA Pol I transcribe the large ribosomal RNA precursor gene • RNA Pol II transcribe tRNA gene, some small nuclear RNA genes and the 5S rRNA genes

  8. The bacterial RNA polymerase

  9. “Crab claw” shape of RNA polymerase

  10. Transcription by RNA polymerases Proceeds in a Series of steps • Initiation • Elongation • Termination

  11. Initiation • A promoter is the DNA sequence that initially binds the RNA polymerase. • Promoter-polymerase complex undergoes structural changes required for initiation to proceed . • The base at the transcription site unwinds and producing a “bubble” of single-stranded DNA. • Transcription always occurs in a 5’ to 3’ direction.

  12. Transcription Initiation Involves three Defined Steps • The initial binding of polymerase to promoter to form closed complex • The closed complex undergoes a transition to the open complex . • Promoter escape.

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

  14. Closed complex • The initial binding of polymerase to a promoter • DNA remains double stranded • The enzyme is bound to one face of the helix

  15. Open complex • the DNA strand separate over a distance of ~14 bp (-11 to +3 ) around the start site (+1 site) • Replication bubble forms

  16. Stable ternary complex • The enzyme escapes from the promoter • The transition to the elongation phase • Stable ternary complex =DNA +RNA + enzyme

  17. Elongation • Once the RNA polymerase has synthesized a short stretch of RNA (~ 10 bp), transcription shifts into the elongation phase. • This transition requires further conformational change in polymerase that leads it to grip the template more firmly. • Functions: synthesis RNA, unwinds the DNA in front, re-anneals it behind, dissociates the growing RNA chain

  18. Termination • After the polymerase transcribes the length of the gene ,it will stop and release the RNA transcript. • In some cells, termination occurs at the specific and well-defined DNA sequences called terminators. Some cells lack such termination sequences.

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

  20. Topic 2 The Transcription Cycle In Bacteria

  21. The certain defining featuresof Bacterial promoters • The bacterial core RNA polymerases can ,in principle ,initiate transcription at any point on a DNA molecule .In cells, polymerase initiates transcription only at promoters • An initiation factor called σ that converts core enzyme into the form that initiates only at promoter. That form of the enzymes is called the RNA polymerase holoenzyme.

  22. Bacteria promoter • Contain two conserved and recognizable sequences .-35 and -10,but the sequences are not identical • Comparison of many different promoters derives the consensus sequences reflecting preferred –10 and –35 regions

  23. bacterial promoter

  24. Consensus sequence of the -35 and -10 region

  25. Up-element • UP-element is an additional DNA elements that increases polymerase binding by providing the additional interaction site for RNA polymerase.

  26. 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

  27. Extended –10 element • Another class of σ70 promoter lacks a –35 region and has an “extended –10 element” compensating for the absence of –35 region

  28. The structure of σ70 factor

  29. Region 2.recognize the -10 element in promoter. The region -10 is recognized by an α helix. Region3.recognized the extended -10 element by an α helix. Region 4.Recognize -35 element by a structure called a "helix-turn-helix".

  30. Helix-turn-helix DNA-binding motif

  31. Transition to the open complex • involve structural changes in RNA polymerase and in the promoter DNA

  32. Change of the promoter DNA • the opening of the DNA double helix, called “melting”, at positions -11 and +3.

  33. The striking structural change in the polymerase • 1.the b and b’ pincers down tightly on the downstream DNA • 2. A major shift occurs in the N-terminal region of s (region 1.1) shifts. In the closed complex, s 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

  34. Fig 12-8 channels into and out of the open complex

  35. Elongation • The elongating polymerase is a processive machine that synthesizes and proofreads RNA.

  36. Synthesis • DNA enters the polymerase between the pincers • Strand separation in the catalytic cleft • NTP addition • RNA product spooling out (Only 8-9 nts of the growing RNA remain base-paired with the DNA template at any given time) • DNA strand annealing in behind

  37. Proofread • Pyrohosphorolytic (焦磷酸键解)editing: the enzyme catalyzes the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi. • Hydrolytic (水解)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.

  38. Termination • Transcription is terminated by signals within the RNA sequence • Rho-dependent (requires Rho protein) • Rho-independent, also called intrinsic (内在) terminator

  39. Rho-independent terminator Rho-independent terminator contains a short inverted repeat (~20 bp) and a stretch of ~8 A:T base pairs.

  40. Rho -dependent terminator • Have less well-characterized RNA elements, and requires Rho protein for termination • Rho is a ring-shaped single-stranded RNA binding protein, like SSB • Rho binding can wrest (夺取) the RNA from the polymerase-template complex using the energy from ATP hydrolysis • Rho binds to rut (r utilization) RNA sites • Rho does not bind the translating RNA

  41. Fig 12-11 the r transcription terminator

  42. Topic 3 • transcription in eukaryotes

  43. The structure of RNA polymerase II core promoters • RNA polymerase II core promoters are made up of combinations of 4 different sequence elements

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

  45. Initiation • TBP in TFIID binds to the TATA box • TFIIA and TFIIB are recruited with TFIIB binding to the BRE • RNA Pol II-TFIIF complex is then recruited • TFIIE and TFIIH then bind upstream of Pol II to form the pre-initiation complex • Promoter melting using energy from ATP hydrolysis by TFIIH ) • Promoter escapes after the phosphorylation of the CTD tail

  46. TBP binds to and distorts DNA using a b sheet inserted into the minor groove TBP-DNA complex. The TATA binding protein (TBP) is shown here in purple complexed with the DNA TATA sequence (shown in gray) found at the start of many Pol II genes.

  47. The other GTFs also have specific roles in initiation • TAFs: Two of the TAFs bind DNA elements at the promoter ;several are histone-like; another appears to regulate the binding of TBP to DNA, using an inhibitory • TFIIB: a single polypeptide chain, enter the pre-initiation complex after TBP • TFIIF: two subunits ,associating with pol II and recruited to the promoter together with that enzyme • TFIIE: Recruit and regulate TFIIH.

  48. TFIIH: controls the ATP-dependent transition of the pre-initiation complex to the open complex. two functions as ATPase and protein kinase

  49. TFIIB-TBP-promoter complex

  50. In vivo ,transcription requires additional proteins • The mediator complex • Transcriptional regulatory proteins • Nucleosome-modifying enzymes