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Chapter 5 Transcription

Chapter 5 Transcription. A. Transcription in prokaryotes. 5.1 Basic principles of transcription. An overview, the process of RNA synthesis ( initiation, elongation, termination). 5.2 Escherichia coli RNA polymerase. Properties, a subunit, b subunit, b ’ subunit, sigma ( s ) factor.

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Chapter 5 Transcription

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  1. Chapter 5 Transcription

  2. A. Transcription in prokaryotes

  3. 5.1 Basic principles of transcription An overview, the process of RNA synthesis ( initiation, elongation, termination) 5.2 Escherichia coli RNA polymerase Properties, a subunit, b subunit, b’ subunit, sigma (s) factor 5.3 The E. colis70 promoter Promoter, s70 size, -10 sequence, -35 sequence, transcription start site, promoter efficiency 5.4 transcription process. Promoter binding, unwinding, RNA chain initiation, elongation, termination (r factor)

  4. 5.1: Basic principles of transcription • Transcription: an overview (comparison with replication) • The process of RNA synthesis: initiation, elongation, termination

  5. 5.1-1: Transcription: an overview

  6. Key terms defined in this section (Gene VII) +1 Gene X upstream downstream Primary transcript m7Gppp mRNA AAAAAn Coding strand of DNA has the same sequence as mRNA.Downstream identifies sequences proceeding further in the direction of expression; for example, the coding region is downstream of the initiation codon.

  7. Upstream identifies sequences proceeding in the opposite direction from expression; for example, the bacterial promoter is upstream from the transcription unit, the initiation codon is upstream of the coding region. Transcription unit is the distance between sites of initiation and termination by RNA polymerase; may include more than one gene. Promoteris a region of DNA involved in binding of RNA polymerase to initiate transcription

  8. RNA Terminator is a sequence of DNA, represented at the end of the transcript, that causes RNA polymerase to terminate transcription. RNA polymerases are enzymes that synthesize RNA using a DNA template (formally described as DNA-dependent RNA polymerases). Primary transcript is the original unmodified RNA product corresponding to a transcription unit.

  9. Replication: synthesis of two DNA molecules using both parental DNA strands as templates. Duplication of a DNA molecule. 1 DNA molecule  2 DNA molecules Transcription:synthesis of one RNA molecule using one of the two DNA strands as a template. 1 DNA molecule  1 RNA molecule

  10. Replication-synthesis of the leading strand the same direction as the replication fork moves

  11. Replication- Synthesis of the Okazaki fragments Opposite to the replication fork movement

  12. Coupling the synthesis of leading and lagging strands with a dimeric DNA pol III (E. coli)

  13. Transcription

  14. RNA synthesis occurs in the 5’3’ direction and its sequence corresponds to the sense strand (coding strand). • The template of RNA synthesis is the antisense strand (template strand). • Phosphodiester bonds: same as in DNA • Necessary components: RNA polymerase, transcription factors, rNTPs,promoter &terminator/template

  15. DNA 非模板链（编码链）, （+）正链 或正义链） DNA 双螺旋 5’-CGCTATAGCGTTTGCAGGCGTTCACGGC-3’ 3’-GCGATATCGCAAACGTCCGCAAGTGCCG-5’ 转录 模板链,负链（-）或反义链 mRNA （RNA transcript） 5’-CGCUAUAGCGUUUGCAGGCGUUCACGGC-3’

  16. 5.1-2: The process of RNA synthesis • initiation • elongation • termination

  17. +1 Promoter Terminator Sense strand DNA Transcribed region Antisense strand Transcription RNA Fig. 2. Structure of a typical transcription unit

  18. Before initiation: RNA pol recognizes promoter (RNA聚合酶识别启动子) • RNA聚合酶结合到启动子上游附近的双链DNA模板； • 沿双链DNA滑动，找到启动子(promoter)序列； • 负责识别启动子的是RNA聚合酶的σ亚基； • 真核生物的转录在启动子(promoter)序列处首先结合转录起始因子； 8-18

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

  20. Elongation • Covalently adds ribonucleotides to the 3’-end of the growing RNA chain. • The RNA polymerase extend the growing RNA chain in the direction of 5’ 3’ • The enzyme itself moves in 3’ to 5’ along the antisense DNA strand. Link

  21. Termination • Ending of RNA synthesis: the dissociation of the RNA polymerase and RNA chain from the template DNA at the terminator site. • Terminator: often contains self-complementary regions which can form a stem-loop or hairpin structure in the RNA products

  22. Terminator structure

  23. 5.2 Escherichia coli RNA polymerase • E. coli RNA polymerase • a subunit • b subunit • b’ subunit • sigma (s) factor

  24. 5.2-1 E. coli RNA polymerase Synthesis of single-stranded RNA from DNA template.

  25. RNA polymerase (NMP)n + NTP  (NMP)n+1 + PPi • Requires no primer for polymerization • Requires DNA for activity and is most active with a double-stranded DNA as template. • 5’  3’ synthesis • Require Mg2+ for RNA synthesis activity • lacks 3’  5’ exonuclease activity, and the error rate of nucleotides incorporation is 10-4 to 10-5. Is this accuracy good enough for gene expression?? • 6.usually are multisubunit enzyme.

  26. E. coli polymerase • E. coli has a single DNA-directed RNA polymerase that synthesizes all types of RNA. • One of the largest enzyme in the cells • Consists of at least 5 subunits in the holoenzyme, 2 alpha (a), and 1 of beta (b), beta prime (b’), omega (w) and sigma (s) subunits • Shaped as a cylindrical channel that can bind directly to 16 bp of DNA. The whole polymerase binds over 60 bp. • RNA synthesis rate: 40 nt per second at 37oC

  27. E. coli RNA polymerase 155 KD 36.5 KD 11 KD 36.5 KD 70 KD Initiation only 151 KD Both initiation & elongation

  28. E.coli RNA 聚合酶的亚基性质和功能 亚基 基因 相对分子量 亚基数 功能  rpoA 4.0104 2 core core assemble, promoter recognition  rpoB 1.51105 1 core  and ’combined together to form catalyzed center ’ rpoC 1.55105 1 core  ? 11104 1 core unknown  rpoD 7.0104 1  factor different factors recognize different promoters 8-28

  29. 可解离的sigma亚基赋予RNA聚合酶对原核启动子的特异性可解离的sigma亚基赋予RNA聚合酶对原核启动子的特异性   +  ’        ’ 核心酶 Core enzyme 全酶Holoenzyme 非特异性结合启动子，并且结合紧密 特异性结合启动子，结合程度较弱 8-29

  30. 全酶 RNA聚合酶起始转录 “扫描” 启动子 -35 -10 封闭复合物          rNTPs PPi    ’ ’ ’ 开放复合物; 起始 5’pppA mRNA 核心酶 8-30

  31. RNA聚合酶的主要功能 ①识别和结合DNA链上的启动子； ②能沿DNA双链作单向运动； ③解开DNA双螺旋，转录后又恢复双螺旋； ④能同时结合单链DNA和转录产物RNA； ⑤按DNA反义链为模板选择正确的底物NTP，以5’ → 3’方向催化磷酸二酯键的形成，合成RNA链； ⑥识别转录的终止信号； ⑦能够与转录因子相互作用，调节转录； ⑧能在转录受到阻遏时进行自我调整，借助辅助因子，恢复和维持RNA的合成。 8-31

  32. The polymerases of bacteriophage T3 and T7 are smaller single polypeptide chains, they synthesize RNA rapidly (200 nt/sec) and recognize their own promoters which are different from E. coli promoters. RNA polymerase differs from organism to organism

  33. 5.2-2: a subunit

  34. E. coli polymerase: a subunit Two identical subunits in the core enzyme Encoded by the rpoA gene Required for assembly of the core enzyme Plays a role in promoter recognition. Experiment: When phage T4 infects E. coli, the α subunit is modified by ADP-ribosylation of an arginine. The modification is associated with a reduced affinity for the promoters formerly recognized by the holoenzyme. plays a role in the interaction of RNA polymerase with some regulatory factors

  35. 大肠杆菌RNA聚合酶: a亚基 • 在核心酶中两个a亚基是相同的； • a亚基是由rpoA基因编码； • 对于RNA聚合酶核心蛋白的组装是必需的； • 在启动子识别上可能起着重要的作用；

  36. 5.2-3&4: b and b’ subunit

  37. b is encoded by rpoB gene, and b’ is encoded by rpoC gene . Make up the catalytic center of the RNA polymerase Their sequences are related to those of the largest subunits of eukaryotic RNA polymerases, suggesting that there are common features to the actions of all RNA polymerases. The b subunit can be crosslinked to the template DNA, the product RNA, and the substrate ribonucleotides; mutations in rpoB affect all stages of transcription. Mutations in rpoC show that b’ also is involved at all stages.

  38. b subunit may contain two domains responsible for transcription initiation and elongation • Rifampicin (利福平)：has been shown to bind to the β subunit, and inhibit transcription initiation by prokaryotic RNA pol. Mutation in rpoB gene can result in rifampicin resistance. • Streptolydigins(利迪链菌素)：resistant mutations are mapped to rpoB gene as well. Inhibits transcription elongation but not initiation.

  39. b’ subunit Binds two Zn 2+ ions and may participate in the catalytic function of the polymerase Hyparin (肝素)：binds to the b’ subunit and inhibits transcription in vitro. Hyparin competes with DNA for binding to the polymerase. 2. b’ subunit may be responsible for binding to the template DNA .

  40. 大肠杆菌RNA聚合酶:b亚基 1. β亚基由rpoB基因编码； 2. RNA聚合酶的催化中心； Rifampicin(利福平)：与β亚基结合可以抑制转录的起始，rpoB基因突变导致对利福平的抗性； Streptolydigins(利迪链菌素)：抗性突变也定位于rpoB基因，它抑制转录延伸，但不抑制起始； 3. β亚基可能含有两个结构域负责转录的起始和延伸；

  41. 大肠杆菌RNA聚合酶: b’亚基 1. b亚基由rpoC 基因编码； 2. 与两个Zn2+离子结合参与RNA聚合酶的催化功能； • Heparin (肝素)：在体外与β’ 亚基结合并抑制转录； • Heparin与DNA竞争结合RNA聚合酶； 3. β’ 亚基可能是负责与DNA模板的结合；

  42. 5.2-5: Sigma (s) factor

  43. Many prokaryotes contain multiple s factors to recognize different promoters. The most common s factor in E. coli is s70. 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.

  44. 大肠杆菌RNA聚合酶: s亚基 1. 在启动子的识别上σ 因子至关重要，对于非特异性DNA位点核心酶的亲和力会降低（104），而对于相应的启动子亲和力会增加； 2. 当起始后(RNA chain is 8-9 nt) ，σ 因子会从RNA聚合酶中释放出来； 3. 在细胞中σ 因子的需要量比RNA聚合酶其他亚基的需要量少；

  45. 5.3: The E. colis70 promoter • Promoter • s70 size • -10 sequence • -35 sequence • transcription start site • promoter efficiency

  46. 5.3-1: Promoter • The specific short conserved DNA sequences: • upstream from the transcribed sequence, and thus assigned a negative number (location) • required for specific binding of RNA Pol. and transcription initiation (function) • Were first characterized through mutations that enhance or diminish the rate of transcription of gene

  47. +1 Promoter Terminator Sense strand DNA Transcribed region Antisense strand Transcription RNA Different promoters result in differing efficiencies of transcription initiation, which in turn regulate transcription.

  48. 5.3-2,3&4: s70 promoter

  49. Consists of a sequence of between 40 and 60 bp -55 to +20: bound by the polymerase -20 to +20: tightly associated with the polymerase and protected from nuclease digestion by DNaseΙ(see the supplemental) Up to position –40: critical for promoter function (mutagenesis analysis) -10 and –35 sequence: 6 bp each, particularly important for promoter function in E. coli ---5-8 bp--- G C T A TTGACA -----16-18 bp------- TATAAT -35 sequence -10 sequence +1

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