Transcription & post transcriptional modification

Transcription

1 Transcription & post transcriptional modification

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

3 Similarity between replication and transcription Both processes use DNA as the template Phosphodiester bonds are formed in both cases Both synthesis directions are from 5 to 3

4 Differences between replication and transcription replication transcription template double strands single strand substrate dntp NTP primer yes no Enzyme DNA polymerase RNA polymerase product dsdna ssrna base pair A-T, G-C A-U, T-A, G-C

5 Section 1 Template and Enzymes

6 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 DNA regions that can be transcribed into RNA are called structural genes

7 1.1 Template The template strand is the strand from which the RNA is actually transcribed. It is also termed as antisense strand The coding strand is the strand whose base sequence specifies the amino acid sequence of the encoded protein, Therefore, it is also called as sense strand

8 5' G C A G T A C A T G T C 3' coding strand 3' C G T C A T G T A C A G 5' template strand transcription 5' G C A G U A C A U G U C 3' RNA

9 Asymmetric transcription Only the template strand is used for the transcription Either strands can be used as a templates The transcription direction on different strands is opposite This feature is referred to as the asymmetric transcription

10 5' 3' 3' 5'

11 Organization of coding information in the adenovirus genome

12 1.2 RNA Polymerase The enzyme responsible for the RNA synthesis is DNA-dependent RNA polymerase The prokaryotic RNA polymerase is a multiple-subunit protein of ~480kD Eukaryotic systems have three kinds of RNA polymerases, each of which is a multiple-subunit protein and responsible for transcription of different RNAs

13 Holoenzyme The holoenzyme of RNA-pol in E.coli consists of 5 different subunits: α, αʹ β βʹ ωσ. holoenzyme core β σ βʹ ω α α

14 RNA-pol of E. Coli subunit MW function α Determine the DNA to be transcribed β Catalyze polymerization βʹ Bind & open DNA template σ Recognize the promoter for synthesis initiation

15 RNA-pol of E. Coli

16

17 Rifampicin, a therapeutic drug for tuberculosis treatment, can bind specifically to the β subunit of RNA-pol, and inhibit the RNA synthesis RNA-pol of other prokaryotic systems is similar to that of E. coli in structure and functions

18

19 RNA-pol of eukaryotes RNA-pol I II III products 45S rrna hnrna 5S rrna trna snrna Sensitivity to Amanitin No high moderate Amanitin is a specific inhibitor of RNA-pol.

20

21 1.3 Recognition of Origins Each transcriptable region is called operon One operon includes several structural genes and upstream regulatory sequences (or regulatory regions) The promoter is the DNA sequence that RNA-pol can bind. It is the key point for the transcription control

22 Promoter 5' 3' regulatory sequences promotor RNA-pol structural gene 3' 5'

23 Prokaryotic promoter 5' 3' region T T G A C A A A C T G T -10 region T A T A A T A T A T T A (Pribnow box) Consensus sequence start 3' 5'

24 Consensus Sequence

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

26

27 Section 2 Transcription Process

28 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 cofactors to bind to the DNA template together in the transcription process

29 2.1 Transcription of Prokaryotes Initiation phase: RNA-pol recognizes the promoter and starts the transcription Elongation phase: the RNA strand is continuously growing Termination phase: the RNA-pol stops synthesis and the nascent RNA is separated from the DNA template

30 a. Initiation RNA-pol recognizes the TTGACA region, and slides to the TATAAT region, then opens the DNA duplex. Step for initiation Promoter binding DNA Unwinding RNA chain initiation

31

32

33

34

35

36 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 (α 2 ββʹ σ) - DNA - pppgpn- OH 3ʹ

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

38 b. Elongation The release of the σ subunit causes the conformational change of the core enzyme. The core enzyme slides on the DNA template toward the 3ʹ end. Free NTPs are added sequentially to the 3ʹ -OH of the nascent RNA strand. (NMP) n + NTP RNA strand substrate (NMP) n+1 + PPi elongated RNA strand

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

40 Transcription bubble

41 RNA-pol of E. Coli

42 c. Termination The RNA-pol stops moving on the DNA template. The RNA transcript falls off from the transcription complex. The termination occurs in either ρ - dependent or ρ -independent manner.

43 The termination function of ρ factor The ρ factor, a hexamer, is a ATPase and a helicase.

44 ρ-independent termination The termination signal is a stretch of nucleotides on the RNA transcript, consisting of many GC followed by a series of U. The sequence specificity of this nascent RNA transcript will form particular stem-loop structures to terminate the transcription.

45 DNA rpll protein 5ʹ TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGGCACCAGCCTTTTT... 3ʹ 5ʹ TTGCAGCCTGACAAATCAGGCTGATGGCTGGTGACTTTTTAGTCACCAGCCTTTTT... 3ʹ RNA UUUU... UUUU...

46

47 Stem-loop disruption 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

48 2.2 Transcription of Eukaryotes a. Initiation Transcription initiation needs promoter and upstream regulatory regions. The cis-acting elements are the specific sequences on the DNA template that regulate the transcription of one or more genes.

49 Cis-acting element cis-acting element GCGC CAAT TATA exon structural gene intron exon start TATA box (Hogness box) enhancer CAAT box GC box

50 TATA box

51 Transcription factors RNA-pol does not bind the promoter directly. RNA-pol II associates with six transcription factors, TFII A - TFII H. The trans-acting factors are the proteins that recognize and bind directly or indirectly cis-acting elements and regulate its activity.

52 TF for eukaryotic transcription

53 Pre-initiation complex (PIC) 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 TFII H: completion of PIC helicase and ATPase kinases (phosphorylation Of CTD)

54 Pre-initiation complex (PIC) RNA pol II TF II A TBP TAF TATA TF II F TF II B TF II E TF II H DNA

55

56

57 Phosphorylation of RNA-pol TF II H is of protein kinase activity to phosphorylate CTD of RNA-pol. (CTD is the C-terminal domain of RNA-pol) Only the RNA-pol can move toward the downstream, starting the elongation phase Most of the TFs fall off from PIC during the elongation phase

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

59 b. Elongation

60 c. Termination The termination sequence is AATAAA followed by GT repeats. The termination is closely related to the post-transcriptional modification.

61

62

63 Section 3 Post-Transcriptional Modification

64

65

66 3.1 Modification of hnrna Primary transcripts of mrna are called as heteronuclear RNA (hnrna). hnrna are larger than matured mrna by many folds. Modification includes Capping at the 5ʹ - end Tailing at the 3ʹ - end mrna splicing RNA edition

67 a. Capping at the 5ʹ - end OH OH O O O H N 2 N N H 2 C O P HN N 5' O O O P O O O P O O 5' CH 2 O N N N NH NH 2 O CH 3 Pi O O P O OH AAAAA-OH 3' O m 7 GpppGp----

68 ppp 5' NpNp pp 5' NpNp Pi removing g phosphate group G 5' ppp 5' NpNp GTP PPi forming 5'-5' triphosphate group methylating at G7 m 7 GpppNpNp m 7 Gpppm 2' Npm 2' Np methylating at C2' of the first and second nucleotides after G

69 The 5ʹ - cap structure is found on hnrna too. The capping process occurs in nuclei. The cap structure of mrna will be recognized by the cap-binding protein required for translation. The capping occurs prior to the splicing.

70 b. Poly-A tailing at 3ʹ - end

71

72 c. mrna splicing mrna DNA The matured mrnas are much shorter than the DNA templates.

73 Split gene The structural genes are composed of coding and non-coding regions that are alternatively separated bp L A B C D E F G A~G no-coding region 1~7 coding region

74 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.

75 mrna splicing

76 Splicing mechanism

77 lariat

78 Twice transesterification intron 5'exon 3'exon 5' U pa G pu 3' pg-oh pgpa 5' UOH first transesterification G pu 3' 5' U pu 3' second transesterification 5' pgpa GOH 3'

79

80

81

82

83

84

85

86 U1 AGGURAGU U2AF 65kDa 35kDa CURACU Y(n)NCAGG SF1 ESE ESE ISE ESS ISS Trans-acting factor Splicing coactivator Cis-acting factor U1 AGGURAGU U2 U2AF 65kDa 35kDa CURACU Y(n)NCAGG SF1 SR ESE Modified from Black 2003, Cartegni 2002, and Patel,AA 2003

87 Cis- ac3ng element Classical splice motif 5 prime splice site 3 prime splice site Branch point Polypyrimidine tract Auxiliary splice motif Exonic sequence enhancer Exonic sequence silencer Intronic sequence enhancer Intronic sequence silencer

88 U1 AGGURAGU U2AF 65kDa 35kDa CURACU Y(n)NCAGG SF1 Splicing coactivator SR U1 AGGURAGU U2 U2AF 65kDa 35kDa CURACU Y(n)NCAGG SF1 ESE Modified from Black 2003, Cartegni 2002, and Patel,AA 2003

89 Splicing coactivator U1 AGGURAGU U2 U2AF 65kDa 35kDa CURACU Y(n)NCAGG SF1 SR ESE Modified from Black 2003, Cartegni 2002, and Patel,AA 2003

90 U1 AGGURAGU U2AF 65kDa 35kDa CURACU Y(n)NCAGG SF1 hnrnp U1 AGGURAGU U2 U2AF 65kDa 35kDa CURACU Y(n)NCAGG SF1 ESS Modified from Black 2003, Cartegni 2002, and Patel,AA 2003

91 U1 AGGURAGU U2 U2AF 65kDa 35kDa CURACU Y(n)NCAGG SF1 hnrnp ESS Modified from Black 2003, Cartegni 2002, and Patel,AA 2003

92

93