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- DNA specify all kinds of proteins in the cell

DNA Transcription. - DNA specify all kinds of proteins in the cell - DNA is NOT the direct template for the protein synthesis. There must be intermediate specified by the DNA *The flow of the genetic information DNA transcription RNA translation protein.

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- DNA specify all kinds of proteins in the cell

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  1. DNA Transcription • - DNA specify all kinds of proteins in the cell • - DNA is NOT the direct template for the protein synthesis. • There must be intermediate specified by the DNA • *The flow of the genetic information • DNA transcription RNA translation protein. • • Structure of RNA • Large, unbranched macromolecule, but smaller than DNA • Ribose sugar instead deoxy ribose • Uracil instead Thymine. • Single stranded in most cases.%G ≠ %C • - Complementary to DNA template. • - Double helical regions “hairpin loop”

  2. *Types of RNA • Ribosomal RNA (rRNA) 80% • - Component of ribosomes / protein synthesis • - Three different sizes of rRNA: • 23 S, 16 S, 5 S  Prokaryotic cell , eukaryotic mitochondria • - Four different sizes of rRNA: • 28 S, 18 S, 5.8 S, 5 S  Eukaryotic cell. • t-RNA • - Smallest RNA (74 – 95 nucleotide) • - 15% of total RNA, contains unusual bases. • - Act as “adaptor” carries the specific a.a to the site of protein synthesis. • m-RNA • - 5% • - Carries the genetic information from DNA to the cytosol where it used as a template for protein synthesis. • snRNA • - Plays role in RNA modification after the transcription in eukaryotic cell

  3. Types of RNA Ribonucleases

  4. • t-RNA, m-RNA, rRNA, snRNA are synthesized by RNA polymerases. • • RNA synthesis • Needs DNA template: RNA polymerase takes instruction from DNA template. • Activated precursors ATP, GTP, UTP, CTP. • Divalent metallic ion mg+2 • RNAn + ribonucleoside triphosphate  RNAn+1 + PPi • 5’ 3’ synthesis • - 3’ – OH makes nucleophilic attack to α-phosphate. • - The reaction is driven by hydrolysis of PPi • - No need for primer. • - No exo- or endo- nuclease activity.

  5. • t-RNA, m-RNA, rRNA, snRNA are synthesized by RNA polymerases. • • RNA synthesis • Needs DNA template: RNA polymerase takes instruction from DNA template. • Activated precursors ATP, GTP, UTP, CTP. • Divelent metalic ion mg+2 • RNAn + ribonucleoside triphosphate  RNAn+1 + PPi • 5’ 3’ synthesis

  6. - 3’ OH makes nucleophilic attack to α-phosphate. - The reaction is driven by hydrolysis of PPi - No need for primer. - No exo- or endo- nuclease activity.

  7. * Transcription in prokaryotes • - One type of RNA polymerase. • - DNA gene contains promotor sites that specifically bind RNA polymerase and determine where the transcription begins. • Strong promotor  Frequent transcription • Mutation in the promotor  Impair transcription • RNA polymerase is multisubunit enzyme. • - Core enzyme 2αββ’ units • 5’ 3’ synthesis of RNA. but, lacks the specificty, cannot recognize the promotor region on the DNA template. • - Holo enzyme = Core enzyme + σ unit • σ Factor enables the polymerase to recognize the promotor region.  (it will be released when transcription starts)

  8. DNA Transcription

  9. Coding (sense) strand Transcription unit - 35 - 10 5’ 3’ 3’ 5’ +1 Template strand (anti sense) Up stream Down stream Termination Promotor regions in bacterial gene • 10:5’TATAAT3’(pribnow box) • -35:5’TTGACA 3’ • +1:start site:the first nucleotide of the DNA sequence to be transcripted. • eg. • Coding template:5’ GCG ATA TAA TAG 3’ • Template strand:3’ CGC TAT ATT ATC 5’ • mRNA : 5’ GCG AUA UAA UAG 3’

  10. DNA Transcription • • RNA synthesis • Initiation • RNA polymerase bind to promoter site  unwinds the double helical DNA (about two turns before starting the synthesis)

  11. DNA Transcription

  12. Transcription Initiation

  13. Elongation • • RNA polymerase catalyzes the formation of phospho diester bond. And it moves unidirectional until the termination signals. • - NO need for primer • - RNA start with purin (A or G) • - σ unit of the holoenzyme is released after the initiation. • Termination • • Elongation continues until a termination process occurs: • P-Independent termination • The transcripted DNA contains stop signals which are palindromic GC rich region followed by AT rich region. • • DNA Palindrome • 5’ CGACTGCAGTCG 3’ • 3’ GCTGACGTCAGC 5’ • - The sequence on one strand reading 5’ – 3’ is the same as the sequence on the complementary strand reading in the same direction.

  14. Palindrome dependent Termination GC palindromic region stabilizes a secondary structure followed by a string of poly U Stable hair pin slow down the transcription process Poly U bonding to the DNA template is weak

  15. Palindrome dependent Termination

  16. RHO (P-factor) protein termination Hexamer protein Break the RNA-DNA Hybrid by pulling the RNA away

  17. DNA Transcription

  18. • Transcription of Eukaryotic genes: • - Three types of RNA polymerase: • RNA polymerase I:large ribosomal RNA (18S, 5.8S, 28S and it’s location in the nucleolus) • RNA polymerase II:mRNA precursors and it takes place in nucleoplasm, and (SnRNA) • RNA polymerase III:the 5S RNA (nucleoplasm) • • Mitochondrial RNA polymerase:like as in prokaryotes • • RNA polymerase in eukaryotes also catalyzes the: • - The nucleophilic addition of 3’-OH of the growing RNA chain to the α-phosphat • - The 5’ – 3’ addition according to the instruction of anti parallel DNA template • - NO need for primer • - Lock the nuclease activity • • RNA polymerase recognize promoter regions in the gene

  19. Transcription of Eukaryotic cell

  20. Transcription unit (CAAT) box (TATA) box -70 -25 5’ 3’ 3’ 5’ +1 Termination Start site Template strand Coding strand Promotor regions in Eukaryotic gene

  21. • Post transcriptional modification • The 1º - transcript is the linear copy of the transcriptional unit. • • rRNAs are produced from longer precursor. In prokaryotes and eukaryotes cells. • - 5S is synthesized by RNA polymerase III • - m-RNA in prokaryotes is identical to the 1º - transcript. • • m-RNA of eukaryotic cells. • 1º - transcript (hnRNA) modificationm-RNA • Capping(translation & stabilizing) • Poly A- tail: by polytransferase • Removal of Introns • Introns: the nucleotide sequences that don’t code for proteins. • Exons:the nucleotide sequences that code for proteins.

  22. Post transcriptional modification: capping and Tailing Tail (stabilize & exit) The cap is Added by Guanylyl transferase

  23. Removal of Introns RNA polymerase II Exon 1 Intron Exon 2 The 1º- transcript unmature m-RNA Splicing • The introns are removed and the remaining exons spliced together to form the mature RNA • SnRNA + proteinssmall nuclear ribonucleo-protein particles -SnRNP- (spliceosomes) Splicing the exon segments

  24. Post transcriptional modification

  25. • RNA inhibitors • - Some antibiotics prevent cell growth by inhibiting RNA synthesis. • Rifampin • Inhibits the initiation of transcription by binding to β-subunit of prokaryotic RNA polymerase. • Actinomycin D • It binds to the DNA template and interferes with movement of RNA polymerase along the DNA (used as anticancer agent) • • Toxins produced by some fungi • e.g. α-Amantin form a complex with RNA polymerase II so inhibits RNA synthesis inhibits protein synthesis.

  26. The End

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