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Chapter12 Mechanisms of Transcription

Chapter12 Mechanisms of Transcription. 胡红霞 04 级生物科学 200431060178. The Central Dogma: transcription translation DNA RNA PROTEIN Transcription is the first step of the expression of the genome !. mRNA transcript.

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Chapter12 Mechanisms of Transcription

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  1. Chapter12Mechanisms of Transcription 胡红霞 04级生物科学 200431060178

  2. The Central Dogma: transcriptiontranslation DNA RNA PROTEIN Transcription is the firststep of the expression of the genome!

  3. mRNA transcript

  4. Transcription and replication: 1. A new chain synthesized upon a DNA template 2. In a 5’ to 3’ direction Transcription : Ribonucleotides Use RNA polymerases 3. Needs no primer (de novo) 4. Does not remain base-paired to the template all the time 5. Less accurate (10-5 vs 10-8) 6. Selectively copies certain parts of the genome and makes anything from one to several hundred , or even thousand, copies of any given section (Recall replication) Similarities vs Differences Transcription & Replication

  5. Outlines • RNA Polymerases and the Transcription Cycle • The Transcription Cycle in Bacteria • Transcription in Eukaryotes All these topics are very important !!!

  6. RNA polymerases and the transcription cycle

  7. §1.1 RNA Polymerases RNA Polymerases Come in Different Forms , but Share Many Features RNA Polymerase performs essentially the same reaction in all cells, so RNA polymerases from bacteria to humans are highly conserved, especially in those parts of the enzyme directly involved with catalyzing the synthesis of RNA.

  8. The Subunits of RNA Polymerases

  9. Eukaryotic cells have three RNA polymerases • RNA Pol I : transcribes the large ribosomal RNA precursor gene • RNA Pol II : most studied, transcribes most genes – essentially all protein-encoding genes ( focus) • RNA Pol III : transcribes tRNA genes, some small nuclear RNA genes and the 5S rRNA gene

  10. Comparison of the crystal structures of prokaryotic and eukaryotic RNA polymerase

  11. The bacterial RNA polymerase The core enzyme alone can synthesize RNA The massive RNA holoenzyme contains 6 subunits: the б subunit, β’ subunit, β subunit , ω subunit, and two α dimer subunits.

  12. crab claw active center cleft (can bind two Mg2+) the two pincers of the crab claw -- β’ and β subunits

  13. RNA polymerase holoenzyme

  14. §1.2 The transcription cycle RNA polymerases proceeds through a series of well-defined steps : • Initiation • Elongation • Termination

  15. Initiation Important points : A promoter is the DNA sequence that initially binds the RNA polymerases. The promoter- polymerase complex undergoes structural changes required for initiation to proceed. The new ribonucleotide is added to the 3’ end of the growing chain, so transcription always occurs in a 5’ to 3’ direction. The choice of promoter is the main regulation.

  16. Transcription initiation involves three defined steps : • Closed complex : form when initially bind to a promoter • Open complex : the DNA strands separate and the transcription bubble forms • Stableternary complex : form after an enzyme gets further than 10 bp (that is when the enzyme has escaped the promoter)

  17. Initiation The phases of the transcription cycle Elongation Termination

  18. Elongation The functions of RNA polymerase during this elongation phase : • The catalysis of RNA synthesis; • Unwinds the DNA in front; • Re-anneals the DNA behind; • Dissociates the growing RNA chain from the template; • Moves along the DNA template; • RNA proofreading.

  19. Transcription Cycle

  20. Termination Stops and releases the RNA product once the polymerase has transcribed the length of the gene (or genes) . In some cells there are specific, well-characterized sequences that trigger it. In others it is less clear what instructs the termination.

  21. The Transcription Cycle in Bacteria

  22. б factor The RNA polymerase initiate transcription at any point on a DNA molecule; yet the б factor converts core enzyme into the form that initiates only at promoters. (б70 is predominant in E.coli ) RNA polymerase holoenzyme = RNA polymerase + б factor

  23. The б subunit is composed of α helices connected by turns and loops. These elements organize into four domains : N-terminal domain 1 N-terminal domain 2 Linker domain C-terminal domain After synthesis of a 9-12 nucleotide RNA, the б subunit dissociates from the core polymerase, and the core begins the elongation of the RNA transcript.

  24. Bacteria Promoters Vary in strength and sequence , but have certain defining features.

  25. Characteristic structure of promoters recognized by polymerase containing б70 : Two conserved sequences: each of 6 nucleotides, centered at about 10 and 35 base pairs upstream of the site where RNA synthesis starts. -10 and -35 regions, or elements; A nonspecific stretch of 17-19 nucleotides between.

  26. The б factor mediates binding of polymerase to the promoter The regions that recognize the -10 and -35 elements of the promoter are region 2 and 4, respectively. ( helix-turn-helix ) бregion 2 recognizes -10 element бregion 3 recognizes the extended -10 element б region 4 recognizes -35 element

  27. Regions of б

  28. UP-element An additional DNA elements that binds RNA polymerase and increases binding by providing an additional specific interaction between the enzyme and the DNA. Is recognized by αCTD of the polymerase б and α subunits recruit RNA polymerase core enzyme to the promoter

  29. “Extended –10” element Another class of б70-promoters lacks a –35 region and has an “extended –10” element. And this element compensates for the absence of a -35 region. This element is recognized by an α helix in б region 3 through two specific base pairs.

  30. Transition to the open complex involves structural changes in RNA polymerase and in the promoter DNA

  31. isomerization Closed complex Open complex Structural changes: • DNA around the transcription start site is unwound, forming a bubble of single-stranded DNA • The enzyme also changes (this “melting” occurs between positions -11 and +3) Occurs spontaneously; not require ATP to provide energy.

  32. The five channels: NTP-uptake RNA-exit Downstream DNA Nontemplate-strand (NT) Template- Strand (T) channels into and out of the open complex

  33. Upon isomerization there are two striking structural changes in the polymerase • The pincers at the front clamp down tightly on the downstream DNA. • There is a major shift occurs in the N-terminal region of s (region 1.1) .In the closed complex, s region 1.1 lies within the active center while in the open complex, it shifts to the outside of the enzyme, allowing the DNA access to the cleft

  34. Transcription is initiated without a primer • RNA polymerase can initiate a new RNA chain on a DNA template. Difficulty : RNA polymerase starts most transcripts with an A, but A-T pair has only two hydrogen bonds. Various parts of polymerase holoenzyme , including part of б provide specific interactions with the initiating ribonucleotide.

  35. RNA polymerase synthesizes several short RNAs before entering the elongation phase • Abortive initiation: the enzyme synthesizes short RNA molecules less than 10 nucleotides and then releases the transcript. • Promoter escape : Once the polymerase manages to make an RNA longer than 10bp, a stable ternary complex containing the enzyme , the DNA template and a growing chain is formed and the elongation starts. Elongation

  36. The elongation polymerase is a processive machine that synthesizes and proofreads RNA Processes DNA: RNA polymerase: • Enter between the pincers • The strands separate • Reform a double helix behind • Adds new ribonucleotides • Releases the RNA product

  37. Attentions : Only 8 – 9 nucleotides of the growing RNA chain remain base-paired to the DNA template at any given time; The remainder of the RNA chain is peeled off and directed out of the enzyme through the RNA exit channel

  38. Proofreading mechanisms Pyrophosphorolytic editing • The enzyme catalyzes the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi, using its active site.

  39. Proofreading mechanisms Hydrolytic editing • The enzyme backtracks by one or more nucleotides and cleaves the error-containing sequence. It is stimulated by Gre factor. Termination

  40. Transcription is terminated by signals within the RNA sequence • Terminators : sequences that trigger the elongating polymerase to dissociate from the DNA and release the RNA chain it has made. • Two types: Rho-independent terminators Rho-dependent terminators

  41. Rho-independent terminators Sequence of a rho-independent terminator: 1.A short inverted repeat (20 bp) 2.A stretch of about 8 A:T base pairs

  42. Rho-independent terminators Require A:U base pairs for they are the weakest of all base pairs, then the RNA will more readily dissociate from the template.

  43. Polymerase transcribes an inverted repeat ; Form a stem loop within RNA ; Cause termination by disrupting the elongation complex . Transcription termination

  44. Rho-dependent terminators Require the action of Rho factor. Rho (r) A ring-shaped protein with six identical subunits; Binds to single-stranded RNA as it exits; Has an ATPase activity The Rho transcription terminator

  45. Rho is directed to a particular RNA molecule Rho Binding Specificity : • In the binding sites which consist of stretches of about 40 nucleotides that remain largely single-stranded and are rich in C residues; • Only binds those transcripts still being transcribed beyond the end of a gene or operon.

  46. Transcription in Eukaryotes

  47. Transcription in eukaryotes is undertaken by polymerases closely related to RNA polymerases found in bacteria. • However, there are some differences between the two cases.

  48. Differences • Eukaryotes have Pol I, II and III , whereas bacteria has only one; • Eukaryotes require several initiation factors, whereas bacteria require only one --- s factor; • Isomerization to the open complex in eukaryotes require ATP hydrolysis, whereas in bacteria it occurs spontaneously; • In eukaryotes promoter escape is regulated by the phosphorylation state if the CTD tail; • Elongation factors and proofreading mechanism; • Termination (some of these will be discussed later)

  49. 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 Pol II

  50. Regulatory sequence • Beyond –-typically upstream of –- the core promoter and required for efficient transcription in vivo. • Categories: • Promoter proximal elements • Upstream activator sequences, UASs • Enhancers • Silencers • Boundary elements • Insulators • All these DNA elements bind regulatory proteins (activators and repressors)

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