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RNA Synthesis

Lecture 32. RNA Synthesis. Mukund Modak, Ph.D. U1-U6 <100 nt 1 Splicing RNAs. PROKARYOTIC RNA POLYMERASE. Single enzyme with 5 subunits a, a ’ , b, b ’ s : Holoenzyme (Sigma subunit finds start point)

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RNA Synthesis

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  1. Lecture 32 RNA Synthesis Mukund Modak, Ph.D.

  2. U1-U6 <100 nt 1 Splicing RNAs

  3. PROKARYOTIC RNA POLYMERASE Single enzyme with 5 subunits a, a’, b, b’ s : Holoenzyme (Sigma subunit finds start point) a, a’, b, b’ : Core enzyme (elongation of RNA chain) DNA template and RNA polymerase 4 rNTPS RNA + PPi 5’ 3’ 3’ 5’ 5’ 3’

  4. Figure 30.8 Local unwinding of DNA caused by RNA polymerase

  5. ENZYMATIC SYNTHESIS OF RNA Differences/similarities with DNA polymerases i. Substrate ii. Template conservation (Sense strand = mRNA) iii. Primer need iv. Proof reading Template recognition and copying by base pairing direction of synthesis : 5’ - 3’ Initiation with A or G (Sigma factor also needed) *Termination : U sequence + hairpin + r factor * Prokaryotic system (ATP dependent)

  6. Figure 30.7 Structure of the prokaryotic promoter region

  7. EUKARYOTIC RNA POLYMERASES RNA polymerase I : (rRNA) in Nucleoli RNA polymerase II: (mRNA) RNA polymerase III: (tRNA) Nucleoplasmic EUOKARYOTES PROMOTERS -110 -60 -25 CAAT GCGC TATA (box)

  8. Figure 30.13 A. Eukaryotic general transcription factors bound to the promoter. CTF, SP1, and TFIID are general transcription factors. B. Enhancer stimulation of RNA polymerase II

  9. Figure 30.17 Posttranscriptional modification of mRNA showing the 7-methylguanosine cap and poly-A tail

  10. RNA Synthesis Small RNAs and their functions (all require to form protein complexes) • miRNAs (Micro-RNAs): typically 21 to 25 base long and is produced from stem loop structured precursors RNAs. • Involved in destruction of specific gene transcripts and suppression of translation of specific mRNAs. • SiRNA or RNAi: small interfering RNAs similar to miRNAS, produced by RNAase III enzyme. • Functions in  Silencing of mRNA • 7SL RNA: RNA required in transport (to Golgi) of secretory proteins.  • SN-RNA: (small Nuclear RNAs) form RNPs required in processing of mRNA. • Oligo A: (20-50 nucleotide long) stretch of A nucleotides linked from 2’ to 5’. Aids in interferon action. • tRNAs : Amino acid specific tRNAs required in protein synthesis.

  11. TRANSFER & RIBOSOMAL RNA SYNTHESIS Synthesized as long transcripts 16 S tRNA 23S 5S At arrows - spacer sequences Cleaves at spacer (marked as arrows) sequences with specific RNase CCA ends are added to all. tRNAs Modifications of some bases, e. g. methylation in both tRNA and rRNA

  12. INHIBITORS/DRUGS OF RNA SYNTHESIS 1. Enzyme specific Rifamycin for E. coli enzyme a-amanitin for pol II 2. DNA intercalating agents Actinomycin D Quinacrine 3. Substrate analogs Ara - C Ara - A

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