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RNA Synthesis (Transcription)

RNA Synthesis (Transcription). Dr. Shumaila Asim Lecture # 2. RNA Synthesis (Transcription). Introduction First step in gene expression RNA is involved in the transfer of genetic information stored in DNA to proteins

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RNA Synthesis (Transcription)

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  1. RNA Synthesis(Transcription) Dr. Shumaila AsimLecture # 2

  2. RNA Synthesis (Transcription) Introduction • First step in gene expression • RNA is involved in the transfer of genetic information stored in DNA to proteins • Process of DNA directed RNA synthesis (copying process) (genes in DNA make RNA copies) • Synthesis of an RNA strand with a base sequence complementary to one strand of DNA (template) and identical in sequence to the other strand

  3. RNA Synthesis (Transcription) • Selective process; one particular gene or groups of genes are transcribed • Specific sequences indicate the beginning and end of DNA segment to be transcribed • Some proteins are also involved in this “selectivity of the process”

  4. RNA Synthesis (Transcription) • Occurs in 5 − 3 direction • Catalyzed by RNA polymerase I, II & III (DNA dependent RNA polymerases (RNAPs) • Each RNAP consists of two large subunits and many small subunits

  5. Numbering -5 -4 -3 -2 -1 +1 +2 +3 +4 +5 +6 DNA A – T – A – C – G – T – T – G – A – C – G ----- RNAA – A – C – U – G – C ----- • Position +1 of a gene is the base that is complementary to the first base of the 5 end of the RNA transcript of that gene (initiation point) • Sequences preceding first base are numbered negatively and are said to be upstream of the initiation point • Sequences following the first base are numbered positively – downstream

  6. Specific Sequences • Promoters – Specific areas or sequences of DNA for initiation (different for different RNAPs) ,these are • Pribnow box (TATA box) (Prokaryotes) • Goldberg-Hogness box or TATA box or consensus sequence (Eukaryotes)

  7. Specific Sequences • Terminators – Sequences for termination • Enhansers & silencers – Sequences for increasing or decreasing rate of transcription • Transcription Unit – Region between promoter and terminator (includes sequences for initiation, elongation & termination)

  8. Requirements • Template = DNA single strand • Substrates = Ribonucleotides(ATP, GTP, UTP, CTP) • Initiation factors • Transcription factors [Tf – II or GTF (General Transcription factors)] (Tf-II include Tf-IIA, Tf-IIB, Tf-IIC, Tf-IID, Tf-IIE, Tf-IIF, Tf-IIH) • Tf-IID contains TATA binding protein (TBP) and 14 TAFs (Transcription activating factors) • Activators, Co-activators and components of PIC (pre-initiation complex)

  9. Requirements • Enzymes = DNA-dependent RNA polymerases [RNA polymerase (RNAP) I, II, III] • RNAP–I synthesizes rRNA • RNAP–II synthesizes mRNA • RNAP–III synthesizes tRNA, 5S rRNA • Each RNAP holoenzyme consists of Core-enzyme + one or more sigma ()factors • Sigma () factors assist the core enzyme to recognize promoter region

  10. Requirements • RNAP is zinc containing metallo-enzyme, requires Mg++ ions for its activity • Core enzyme consists of two identical -subunits, one each ,  and  subunits • -subunit is thought to be the catalytic subunit (the growing chain of RNA remains attached to this subunit)

  11. Generalized Functions of RNAPs • Unwinding the DNA in front, • Synthesis of RNA, • Re-winding it behind, • Dissociating the growing RNA chain from the template and • Perform proofreading.

  12. RNA Synthesis (Transcription) • A cyclic process, involving RNA chain initiation, elongation & termination • RNA polymerase attaches to and initiates transcription at a distinct site, the promoter (a special DNA sequence) • GTFs (Tf-II) facilitate promoter-specific binding of RNAPs to form pre-initiation complex (PIC) • Other proteins (co-activator proteins) regulate the rate of transcription initiation

  13. RNA Synthesis (Transcription)

  14. RNA Synthesis (Transcription Steps) Initiation: • Formation of transcription bubble • Protomer–Polymerase complex (Pre-initiation complex) • PIC formation leads to Structural changes (melting or unwinding of ssDNA) producing “bubble” known as “transcription bubble”

  15. RNA Synthesis (Transcription Steps) • First nucleotide (always purine ATP or GTP) attaches to enzyme (RNAP) through its 5-end • RNAP now catalyzes the formation of a phospho-diester bond between the first nucleotide (free 3-end) and the 5-position of next entering nucleotide

  16. RNA Synthesis (Transcription Steps) • Synthesis of RNA continues in 5 − 3 direction in a sequence dictated by DNA template (base pairing rule) anti-parallel to DNA template • Pyrophosphate (liberated in each addition) is rapidly degraded to two inorganic phosphates making the RNA synthesis irreversible

  17. RNA Synthesis (Transcription Steps) Elongation: • After addition of 10 – 20 ribonucleotides, RNAP undergoes a second structural change • Moves away from the “promoter” • Progresses along DNA molecule – thereby producing “bubble” (DNA unwinding) along DNA template as RNA synthesis proceeds (elongation)

  18. RNA Synthesis (Transcription Steps) • Once the RNA polymerase has synthesized a short stretch of RNA (approximately ten bases), it shifts into the elongation phase. • This phase requires further conformational change in RNA polymerase that leads it to grip the template more firmly.

  19. RNA Synthesis (Transcription Steps) Termination: • Specific sequences or specific signals are recognized by Rho factor ( factor) of RNAP (in Prokaryotes) • Rho factor attaches to DNA; the RNAP cannot move further, • Dissociates from DNA and • Newly synthesized RNA is released.

  20. RNA Synthesis (Transcription Steps) • In eukaryotes, the termination is independent of Rho factor • Completed RNA chain and RNAP are released from template • RNAP holoenzyme reforms, finds another promoter and synthesis of new RNA starts

  21. Modifications (RNA) in primary transcription • Terminal additions, • Base modifications, • Trimming, • Internal segment removal and splicing  lead to    functional RNA molecule

  22. Modifications (RNA) in primary transcription • RNA polymerase I synthesizes large ribosomal RNAs in nucleolus • RNAP–II synthesizes the precursors of mRNAs • RNAP–III synthesizes small RNAs like tRNAs, the small 5S rRNA

  23. 5.8 S rRNA 18 S rRNA 28 S rRNA specific endonucleases 45 S Precursor rRNA Spacer sequences Post-transcriptional RNA Processing A. rRNA Processing 5 S rRNA is directly produced from a 5S separate gene

  24. introns m RNA hn RNA (exons only) (introns + exons) Post-transcriptional RNA Processing B. mRNA – Needs extensive processing • Cap formation (7-methyl-guanylate is added to 5-end) • Poly-adenylation at 3-end (Poly A polymerase) • Splicing

  25. Sequences removed ii. Precursor molecule t RNA(s) Post-transcriptional RNA Processing C. tRNA a. i. One precursor may contain one or moretRNAs b. CCA sequence is added to 3-end c. Some bases may be modified (i.e. some of A, U, G & C bases are methylated, reduced & deaminated) before tRNA can adopt final functional structure

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