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RNA synthesis, processing and modification.pptx

  1. WELCOME TO PRESENTATION PRESENTED BY Dr. Md. Ariful Karim MD (Biochemistry) Phase- A Resident, Year- ii Sir Salimullah Medical College Mitfort Hospital
  3. Gene is the functional unit of DNA, composed of coding region and it’s regulatory sequence that carry genetic information encoded within the base sequence of coding region.
  4. Coding region of gene Coding region of a gene is a mosaic of exons and introns.  Exons are discrete unit of DNA within the coding segments which are expressed.  Introns are interposed between exons within the coding segments of DNA that are transcribed but not expressed.
  5. Regulatory Sequences Coding region of a gene is flanked by regulatory sequence.  Promoter sequence  Terminator sequence  Enhancer  Silencer
  6. Regulatory sequences of gene  Promoter: It consist of TATA box, CAT box, GC box. It lies towards the 5’ end of gene. It initiates transcription.  Terminator: It lies towards the 3’ end of gene. It terminates the transcription.
  7. Regulatory sequences of gene  Enhancer It lies in upstream or downstream or within the coding region. It accelerates the transcription.  Silencer: It lies in upstream or downstream or within the coding region. It suppresses the transcription process.
  8. Transcription
  9. Transcription  Synthesis of RNA according to the base sequence of template DNA.  Transfer of genetic information from DNA to RNA.  All types of RNAs are copied from DNA by transcription.
  11. Requirements  Activated nucleotide triphosphate.  DNA template  RNA polymerase  Initiating nucleotide  RNAP associated proteins and enzymes  Magnesium ion ,manganese.
  12.  TATA, or Hogness box and different core promoter elements such as Inr (initiator) or DPE(downstream promoter element) are present as promoter sequences. 
  13.  These sequences serve as binding sites for proteins known as general transcription factors (GTFs). Which in turn interact with each other and with RNA pol II.
  14. General transcription factors  Recognition of the promoter  Recruitment of RNA pol II to the promoter  Initiation of transcription
  15. General transcription factors  TFIID, TATA-binding protein and TATA-associated factors, recognizes and binds the TATA box (and other core promoter elements).  TFIIF,another GTF, brings the polymerase to the promoter.  The helicase activity of TFIIH melts the DNA, and its kinase activity phosphorylates polymerase
  16. Criteria of transcription  Totally conservative process  Asymmetric  Highly selective process  No primer is needed.  DNA template is read from 3’ to 5’ direction and synthesized from 5’ to 3’ direction.
  17.  RNA polymerase has no proof reading property.  It is a process of less fidelity.  Primary transcript needs extensive post transcriptional modification. Criteria of transcription
  18. Types of RNA polymerase  RNA polymerase I :trascribes rRNA (28s,18s,5.8s)  RNA polymerase II :hn mRNA,Sn RNA, Sc RNA  RNA polymeraseIII :tRNA, 5s rRNA
  19.  Transcription unit It is the segment of gene between promoter sequence and terminator sequence. Transcription unit includes signal for transcription initiation, elongation, termination.
  20.  Primary transcript Initial linear copy of a transcription unit. Primary transcript of all RNA are modified to mature RNA.
  21.  Transcription bubble It is the transiently melted DNA that exposes the single strand template for bonding of RNAP. As RNAP moves, transcription bubble also moves.
  22.  TSS(Transcription start site) it is the first nucleotide of a gene to be transcribed. It is designated as +1.
  23. Initiation of transcription  TFIID containing TBP and TATA-associated factors recognizes and binds the TATA box  TFIIF brings the polymerase to the promoter  TFIIH melts the DNA, and its kinase activity phosphorylates polymerase  Binding of initiating nucleotide with with RNAP
  24. Elongation 5’ 3’ 5; 3’ Ribonucleotides
  25. Termination  Rho-independent termination forming a hairpin like loop rich in C-G facilitating separation of newly synthesized RNA.  Rho dependent termination ATP dependent helicase activity of Rho-protein separates RNA- DNA hybrid helix.
  26. Post transcriptional modification Primary transcript is the initial linear RNA copy of a transcription unit. The primary transcripts of tRNA and rRNA are post transcriptionally modified by cleavage of the original transcripts by ribonucleases. tRNAs are then further modified to give it’s unique identity.
  27. Post transcriptional modification of rRNA  rRNAs of eukaryotic cells are generated from a single long precursor molecules called pre- rRNAs.28S, 18S, and 5.8S rRNA of eukaryotes are produced from a single pre-rRNA molecule.Eukaryotic 5S rRNA is synthesized by RNA pol III.
  28. Post transcriptional modification of rRNA
  29.  Sequences at both ends of the molecule are removed.  Intron is removed from the anticodon loop by nucleases.  addition of a –CCA sequence to the 3’ end.  modification of bases at specific positions to produce the “unusual bases” characteristic of tRNA Post transcriptional modification of tRNA
  30. Post transcriptional modification of tRNA
  31. Post transcriptional modification mRNA  Primary transcripts synthesized in the nucleus by RNA pol II is known as heterogeneous nuclear RNA (hnRNA). The pre-mRNA components of hnRNA undergo extensive co- and post transcriptional modification in the nucleus.
  32. 5’ “Capping”  The cap is a 7-methylguanosine attached to the 5 - terminal end of the mRNA.  Addition of guanosine monophosphate by the nuclear enzyme guanylyltransferase.  Methylation of this terminal guanine occurs in the cytosol and is catalyzed by guanine-7 methyltransferase. Importance:The addition of this 7-methylguanosine cap helps stabilize the mRNA and permits efficient initiation of translation
  33. Addition of poly-A tail  It is done by trimming at 3’ end followed by addition of 40-250 Adenine nucleotide catalyzed by poly-A polymerase. Importance:  Stabilize the mRNA  Facilitates the exit from nucluus.  And aid in translation.
  34. Splicing (Removal of introns and joining the exons)  Mature mRNA consist of exons only by grace of splicing.
  35. Mechanism of splicing  SnRNP=Protein+SnRNA  ScRNP=Protein+ScRNA  SnRNP+ScRNP=Ribozyme  Ribozyme+hn mRNA= Spliceosome  Spliceosome removes the introns from hn mRNA and joins the exons.
  36. Importance of splicing  Mature mRNA consist of exons only by grace of splicing.  Make uninterrupted genetic code.  After splicing, mature mRNA molecules leave the nucleus and pass into the cytosol.
  37. Reverse Transcription  Reverse transcription is the process in cells by which an enzyme makes a copy of DNA from RNA. The enzyme reverse transcriptase is found in retroviruses, such as the human immunodeficiency virus (HIV).
  38. Thank you
  39. Questions on RNA synthesis, processing and modification  Define transcription. Give the features of transcription.  What is transcription factor?  Write in short about post transcriptional modification.  Briefly state the post transcriptional modification of mRNA and tRNA.  Briefly discuss the importance of post transcriptional modification.  Enumerate the regulatory sequence of a gene with their functions. What is enhancer, promotor ?  What is reverse transcription?