2. Lecture 3 RNA Biosynthesis (Transcription)

3. The synthesis of RNA molecules using DNA strands as the templates so that the genetic information can be transferred from DNA to RNA. Transcription RNA DNA

4. Template: both processes use DNA as the template. Bond: phosphodiester bonds are formed in both cases. Direction: both synthesis directions are from 5´ to 3´. Similarity between replication and transcription

5. ReplicationTranscription templatedouble strandssingle strand substratedNTPNTP primer yesno EnzymeDNA polymeraseRNA polymerase productdsDNA ssRNA(mRNA,tRNA, rRNA) base pairA-T, G-CA-U, T-A, G-C Differences between replication and transcription

6. Section 1 Template and Enzymes

7. The whole genome of DNA needs to be replicated, but only small portion of genome is transcribed in response to the development requirement, physiological need and environmental changes. Structural genes: DNA regions that can be transcribed into RNA. § 1.1 Template

8. The template strand: the strand from which the RNA is actually transcribed. It is also termed as antisense strand. The coding strand: its sequence is the same as the newly created RNA transcript (except for the substitution of uracil for thymine). It is also called as sense strand. Transcription The template strand and the coding strand

9. Asymmetric transcription Only the template strand is used for the transcription, but the coding strand is not. Both strands can be used as the templates. The transcription direction on different strands is opposite.

10. § 1.2 RNA Polymerase The enzyme responsible for the RNA synthesis is DNA-dependent RNA polymerase(DDRP). –The prokaryotic RNA polymerase is a multiple- subunit protein of ~480kD. –The holoenzyme of RNA-pol in E.coli consists of 5 different subunits:  2    . holoenzyme Core enzyme      

11. subunitMWfunction  36,512 Determine the DNA to be transcribed  150,618Catalyze polymerization  155,613Bind & open DNA template  70,263 Recognize the promoter for synthesis initiation RNA-pol of E. Coli Rifampicin, a bactericidal antibiotic drug, typically used to treat Mycobacterium infections, including tuberculosis and leprosy. It can bind specifically to the  subunit of RNA- pol, and inhibit the RNA synthesis.

12. Promoter –T–The promoter is the DNA sequence that RNA-pol can bind. It is the key point for the transcription control. § 1.3 Recognition of Origins

13. Prokaryotic promoter Consensus sequence The -10 region of TATAAT is the region at which a stable complex of DNA and RNA-pol is formed. The -35 region of TTGACA sequence is the recognition site and the binding site of RNA-pol.

14. Prokaryotic promoter TTGACA TATAAT start   -10-35UP ß’ ß   DNA

15.  subunit: recognize the promoter. Holoenzyme bind to the promoter.

16. Section 2 Transcription Process

17. General concepts Three phases: initiation, elongation, and termination. The prokaryotic RNA-pol can bind to the DNA template directly in the transcription process. The eukaryotic RNA-pol requires co- factors to bind to the DNA template together in the transcription process.

18. § 2.1 Transcription of Prokaryotes Initiation: RNA-pol recognizes the promoter and starts the transcription. Elongation: the RNA strand is continuously growing. Termination: the RNA-pol stops synthesis and the nascent RNA is separated from the DNA template.

19. a. Initiation RNA-pol recognizes the TTGACA region, and slides to the TATAAT region, then opens the DNA duplex (open complex). The unwound region is about 17  1 bp.

21. The first nucleotide on RNA transcript is always purine triphosphate. GTP is more often than ATP. The pppGpN-OH structure remains on the RNA transcript until the RNA synthesis is completed. The three molecules form a transcription initiation complex. RNA-pol (  2  ) - DNA - pppGpN- OH 3

22. No primer is needed for RNA synthesis. The  subunit falls off from the RNA-pol once the first 3,5 phosphodiester bond is formed. The core enzyme moves along the DNA template to enter the elongation phase.

24. The σcycle The σfactor dissociates and joins with a new core polymerase to initiate another RNA chain.

25. RNApol (  2  ) - DNA - pppGpN- OH 3 Transcription initiation complex

26. b. Elongation The release of the  subunit causes the conformational change of the core enzyme. The core enzyme slides on the DNA template toward the 3 end. Free NTPs are added sequentially to the 3 -OH of the nascent RNA strand.

27. Transcription bubble: RNA-pol, DNA segment of ~40nt and the nascent RNA form a complex called the transcription bubble. The 3 segment of the nascent RNA hybridizes with the DNA template, and its 5 end extends out the transcription bubble as the synthesis is processing.

28. RNA-pol of E. Coli

29. Simultaneous transcriptions and translation

30. c. Termination The RNA-pol stops moving on the DNA template. The RNA transcript falls off from the transcription complex. Two different strategies for transcription termination –  -dependent or  -independent manner.

31.  -dependent termination A protein factor,  factor, destabilizes the interaction between the template and the mRNA, releasing the newly synthesized mRNA from the elongation complex. The  factor, a hexamer, is a ATPase and a helicase.

33.  -independent termination The termination signal is a stretch of 30-40 nucleotides on the RNA transcript, consisting of G-C rich hairpin loop followed by a series of U.

34. 不依赖   因子的终 止子：含富 GC 的回文 序列和寡聚 U 序列。

35. The stem-loop structure alters the conformation of RNA-pol, leading to the pause of the RNA-pol moving. Then the competition of the RNA-RNA hybrid and the DNA- DNA hybrid reduces the DNA- RNA hybrid stability, and causes the transcription complex dissociated. Among all the base pairings, the most unstable one is rU:dA. Stem-loop disruption

36. Transcription Cycle Promoter sigma factor Terminator

37. The eukaryotic RNA-pol requires co- factors to bind to the DNA template together in the transcription process. Eukaryotic systems have three kinds of RNA polymerases, each of which is a multiple-subunit protein and responsible for transcription of different RNAs. § 2.2 Transcription of Eukaryotes

38. RNA-polIIIIII products45S rRNAhnRNA 5S rRNA tRNA snRNA Sensitivity to Amanitin Nohighmoderate RNA-pol of eukaryotes hnRNA: heterogeneous nuclear RNA Amanitin is a specific inhibitor of RNA-pol.

39. Amanita phalloides,or death cap α-amanitin α-amanitin is a cyclic peptide of eight amino acids. It is possibly the most deadly of all the amatoxins. It is an inhibitor of RNA polymerase II. This mechanism makes it a deadly toxin. Be careful of this deadly mushroom!

40. Transcription of Eukaryotes Transcription initiation needs promoter and upstream regulatory regions. Cis-acting elements: the specific sequences on the DNA template that regulate the transcription of one or more genes. a. Initiation

41. Cis-acting element -30 Enhancer: DNA sequences that increase the rate of initiation of transcription by RNA pol II

42. RNA-pol does not bind the promoter directly. RNA-pol II associates with six transcription factors, TFII A - TFII H. Trans-acting factors: the proteins that recognize and bind directly or indirectly cis-acting elements and regulate its activity. Transcription factors(TF)

43. TF for eukaryotic transcription

44. TBP of TFII D binds TATA TFII A and TFII B bind TFII D TFII F-RNA-pol complex binds TFII B TFII F and TFII E open the dsDNA (helicase and ATPase) TFII H: completion of PIC Pre-initiation complex (PIC)

45. Order of binding is: IID + IIB + RNA poly. II + IIF +IIE +IIH TBP: TATA binding protein.TBP: TATA binding protein.

46. Pre-initiation complex (PIC) TBP TAF TF II H

47. The elongation is similar to that of prokaryotes. The transcription and translation do not take place simultaneously since they are separated by nuclear membrane. b. Elongation

48. RNA-Pol nucleosome moving direction

49. The termination sequence is AATAAA. The termination is closely related to the post-transcriptional modification. c. Termination

50. The importance of RNA polymerase II tail The “CTD” is the carboxyl- terminal domain of the largest subunit of RNA polymerase II. This domain is central to the mechanism by which post-transcriptional events are coupled to transcription. CTD tail

51. RNA pol can be selectively inhibited Rifampicin: a bactericidal antibiotic drug –It can bind specifically to the  subunit of the bacterial RNA-pol. α-amanitin: a deadly toxin. –It is an inhibitor of RNA polymerase II. Actinomycin D: commonly used in treatment of a variety of cancers. it can bind DNA duplexes, and prevent elongation by RNA polymerase in both bacteria and eukaryotes.

52. Section 3 Post-Transcriptional Modification

53. The nascent RNA, also known as primary transcript, needs to be modified to become functional tRNAs, rRNAs, and mRNAs. The modification is critical to eukaryotic systems.

55. hnRNA (heteronuclear RNA): primary transcripts of mRNA. hnRNA are larger than matured mRNA by many folds. Modification includes –Capping at the 5- end –Tailing at the 3- end –mRNA splicing –RNA edition § 3.1 Modification of hnRNA

56. a. Capping at the 5- end m 7 GpppGp---- G 5’

57. Capping at the 5- end

58. The 5- cap structure is found on hnRNA too.  The capping process occurs in nuclei. Function: –stabilize the structure of mRNA –recognize by the cap-binding protein required for translation. The capping occurs prior to the splicing.

59. b. Poly-A tailing at 3 - end There is no poly(dT) sequence on the DNA template.  The tailing process dose not depend on the template. The tailing process occurs prior to the splicing. The tailing process takes place in the nuclei.

60. The matured mRNAs are much shorter than the DNA templates. DNA mRNA c. mRNA splicing

61. The structural genes are composed of coding and non-coding regions that are alternatively separated. Split gene EA BCDF G A~G: non-coding region 1~7: coding region

62. Exon and intron Exons are the coding sequences that appear on split genes and primary transcripts, and will be expressed to matured mRNA. Introns are the non-coding sequences that are transcripted into primary mRNAs, and will be cleaved out in the later splicing process. DNA mRNA

64. mRNA splicing

65. lariat Splicing is promoted by the snRNPs (small nuclear ribonucleoproteins) which is a large RNA-protein complex:U1,U2,U4, U5,U6 Spliceosome: represent the snRNP association with hnRNA at the exon-intron junction.

66. Splicing mechanism

67. mRNA splicing

68. Post-transcriptional modifications of mRNA occur in the nucleus. The mature RNA then enters the cytosol to perform its function(translation).

69. d. mRNA editing cytidine deaminase Taking place at the transcription level One gene responsible for more than one proteins

70. tRNA precursor RNA-pol III TGGCNNAGTGCGGTTCGANNCC DNA § 3.2 Modification of tRNA

71. tRNA processing Pre-tRNA requires extensive processing to become a functional tRNA. Four types of modifications are involved: –Removing an extra segment at the 5' end by RNase P. –Removing an intron in the anticodon loop by splicing. –Replacing two U residues at the 3'end by CCA. –Modifying some residues to characteristic bases.

72. RNAase P endonuclease Cleavage ligase

73. tRNA nucleotidyl transferase ATP ADP Addition of CCA-OH

74. Base modification （1）（1） （1）（1） （3）（3） （2）（2） （4）（4） 1.Methylation A→mA, G→mG 2.Reduction U→DHU 3.Transversion U→ψ 4.Deamination A→I

75. § 3.3 Modification of rRNA 45S transcript is the precursor of 3 kinds of rRNAs.

76. Points Ⅰ. Prokaryotic transcription 1. RNA polymerase Sigma (σ) factor +core enzyme = holoenzyme 2. Steps in transcription a. Initiation: promoter: -10 region(TATAAT), -35 region(TTGACA) b. Elongation: No primer, no repair, uses NTPs, transcription bubble c. Termination: 1). Rho-dependent. 2). Rho-independent.

77. Points (continued) Ⅱ. Eukaryotic transcription 1. RNA pol I, II, III. 2. promoter : TATA box, CAAT box, GC box. 3. Transcription factors (TF): TFII Ⅲ. Post-transcription modification of RNA 1. mRNA: 5’ capping; Addition of poly(A) tail; RNA splicing, exon, intron 2. rRNA; tRNA. Ⅳ.Inhibitor –Rifampicin; α-amanitin; Actinomycin D

78. Key terms The central dogma, transcription RNA polymerase, sigma subunit Promoter, TATA box Transcription bubble, open complex Template strand, coding strand RNA processing (or post- transcriptional modification of RNA) Exons, introns, RNA splicing