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Promoter

Promoter. Promoter is a region of DNA that facilitates the transcription of a particular gene. Promoters are located near the genes they regulate, on the same strand and typically upstream (towards the 5' region of the sense strand). Promoter.

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Promoter

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  1. Promoter Promoter is a region of DNA that facilitates the transcription of a particular gene. Promoters are located near the genes they regulate, on the same strand and typically upstream (towards the 5' region of the sense strand).

  2. Promoter • In bacteria the promoter is recognized by RNA polymerase and sigma factor, • In eukaryotes • at least seven different factors are necessary for the binding of an RNA polymerase II to the promoter.

  3. Prokaryotic promoters Consists of two short sequences: -10 element or the Pribnow box, (TATAAT) T A T A A T 77% 76% 60% 61% 56% 82% . -35 element (TTGACAT). T T G A C A 69% 79% 61% 56% 54% 54%

  4. Promoter Clearance Closed binary complex Open binary complex Abortive initiation Initiation

  5. Types of Eucaryotic Promoters Promoter may be strong or weak

  6. Eucaryotic Promoters type II

  7. Eucaryotic Promoters’ Elements • Promoter elements • Core promoter • Transcription Start Site (TSS) • A binding site for RNA polymerase • TATA box • Proximal promoter - • Specific sites CAAT, GC • Distal promoter • Specific transcription factor binding sites

  8. Eucaryotic Promoters • These are classified as follows: • Constitutive promoters, which induce the expression of the downstream-located coding region in all tissues irrespective of environmental or developmental factors. • Synthetic promoters, which comprise consensus DNA sequences of common elements of natural promoter regions. • Inducible promoters, which are only expressed under the presence of factors/compounds. They may be Chemically or Physically regulated • Tissue-specific promoters, which operate in particular tissues and at certain developmental stages of a plant. They may be induced by endogenous and exogenous factors, so they may be also classified as inducible.

  9. Constitutive promoters • Plant pathogen/Dicot promoters • Opine promoters • CaMV 35S promoter • Monocot promoters • Plant ubiquitin promoter (Ubi) • Rice actin 1 promoter (Act-1) • Maize alcoh dehydrogenase 1 promoter (Adh-1)

  10. Inducible promoters • Chemically induced • Alcohol-regulated: • Tetracycline-regulated: • Steroid-regulated: • Metal-regulated: • Pathogenesis-related • Physically induced • Temperature-regulated promoters • Light-regulated promoters

  11. Tissue Specific promoters • Root promoters • Fruit promoters • Seed promoters

  12. Transcription Factors for Class II promoters (RNAP II) • Basal factors Required for initiation at nearly all promoters; determine site of initiation; interact with TATA box. • Upstream factors DNA binding proteins that recognize consensus elements upstream of TATA box. Ubiquitous. Increase efficiency of initiation. Interact with proximal promoter elements (e.g., CCAAT box). • Inducible (regulated) factors Work like upstream factors but are regulatory. Made or active only at specific times or in specific tissues. Interact with enhancers or silencers.

  13. Basal Factors Required to form the Pre-Initiation Complex 6 factors + RNAP II = Pre-Init. Complex 6 Factors are TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH First binds TFIIDIt contains TBP and 8-10 TBP associated factors - TAFs

  14. Class II pre-initiation complex Six class II factors: TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH. Preinitiation complex is assembled in a orderly manner D DA DAB DAB-PolF DAB-PolF-EH

  15. Promoters of monocot & DicotConstitutive promoters • Monocot promoters • Plant ubiquitin promoter (Ubi) • Rice actin 1 promoter (Act-1) • Maize alcoh dehydrogenase 1 promoter (Adh-1) • Dicot promoters (from pathogens) • Opine promoters • CaMV 35S promoter • introns are normally present in promoters of monocots

  16. Monocot: Plant ubiquitin promoter (Ubi) from Maize Extends from -899 bp 5' of the transcription start site (+1) to about 1093 bp 3' of the transcription start site (~2Kb). It comprises of: • a TATA box sequence located at -30, • two overlapping heat shock element located at the -214 and -204 position (enhance the expression of the ubiquitin protein in response to temperature stress.) • an 83 bp untranslated exon sequence 3' of and adjacent to the transcription start site and • an intron of around 1 kb, which extends from 84 to 1093 position.

  17. Monocot: Rice actin 1 promoter (Act-1) • The portion of the rice Act-1 gene used in vectors for monocot transformation normally contains: • ~1 kb of regulatory sequences located 5' of the transcribed region, • the 5' non-coding exon 1, • the intron 1, and • the coding exon 2 of the Act-1 gene. used for expressing diverse genes of cereals, i.e. maize, rice, barley, wheat and rice.

  18. Cauliflower mosaic virus: CaMV 35S promoter • Cauliflower mosaic virus (CaMV) replicate through reverse transcription just like retroviruses, but the viral particles contain DNA instead of RNA. The DNA is transcribed into a full length, terminally redundant 35S RNA and a subgenomic 19S RNA. • The promoter of the 35S RNA is a very strong constitutive promoter responsible for the transcription of the whole CaMV genome.

  19. Cauliflower mosaic virus (CaMV) 35s promoter: some features • CaMV promoter has 350 basepairs upstream of the 35S transcript (–343 to +8)

  20. Cauliflower mosaic virus (CaMV) 35s promoter: some features • Region A is mainly required for expression in roots, and region B for expression in leaves.

  21. Cauliflower mosaic virus (CaMV) 35s promoter: some features • When both domain A and domain B are present, expression is detectable in most tissues at all stages of development.

  22. Cauliflower mosaic virus (CaMV) 35s promoter: some features • The domain A extends from the -90 to +1 (minimal promoter), with TATA box (–46 to +8).

  23. Cauliflower mosaic virus (CaMV) 35s promoter: some features • The A region also contains a least three CAAT-like boxes. These CAAT-like boxes drive the expression of transgenes.

  24. Cauliflower mosaic virus (CaMV) 35s promoter: some features • Subdomains of the B region (B1 to B5) interact with various transcription factors. Complete B region allows a more general constitutive expression.

  25. Cauliflower mosaic virus (CaMV) 35s promoter: some features • In subdomain B, two separate enhancer domains for infectivity were identified, –207 to –150 and –95 to –56, only one of which is necessary.

  26. Cauliflower mosaic virus (CaMV) 35s promoter: some features • The 19bp palindrome at the 3' end of the CaMV 35S promoter containing the TATA box and surrounding DNA sequences were purine rich where recombinase proteins binds (recombination hotspots). Thus CaMV 35S promoter may take part in horizontal gene transfer and recombination (supervirus? a recipe for disaster).

  27. Cauliflower mosaic virus (CaMV) 35s promoter: some features • The 25bp border repeats of the Agrobacterium T-DNA also show remarkable similarities to the recombination hotspot of the CaMV 35S promoter.

  28. Cauliflower mosaic virus (CaMV) 35s promoter: some features • Duplication of CaMV 35S Promoter Sequences Creates a Strong Enhancer for Plant Genes (increase tenfold expression)

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