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Transcription

Transcription. Gene Expression Part 1. Gene Expression. There are 4 major events that occur durin the process of gene expression Transcription RNA processing Translation Protein processing. A Gene is a Transcription Unit. Promoter. Terminator. Regulatory sequence. DNA. Transcription.

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  1. Transcription Gene Expression Part 1

  2. Gene Expression • There are 4 major events that occur durin the process of gene expression • Transcription • RNA processing • Translation • Protein processing

  3. A Gene is a Transcription Unit Promoter Terminator Regulatory sequence DNA Transcription 3¢ mRNA 5¢ Many codons Start codon Stop codon Ribosomal- binding site

  4. Prokaryotic Gene Structure Shine-Delgarnobox Shine-Delgarnobox Stop CodonTAA, TAG, TGA Stop Codon +1 ATG ATG Cis-RegulatoryElements Coding Sequence = ORF 1 Coding Sequence = ORF 2 Protein B Spacer = 5’UTR of 2nd cistron USE/Promoter/Operator Protein A 5’ UTR = Leadersequence Cistron 1 Cistron 2 Terminationsequence Regulatory Sequences Structural or Coding Sequences Regulatory and Coding Sequence Unit = Operon DNA

  5. Prokaryotic Gene Structure ATG ATG TRANSCRIPTION Cistron 2 Cistron 1 5’ UTR = Leadersequence Spacer PolycistronicmRNA ORF Protein A ORF Protein B +1 AUG AUG Stop CodonUAA, UAG, UGA Stop CodonUAA, UAG, UGA Shine-Delgarnobox Shine-Delgarnobox Shine-Delgarnobox Stop CodonTAA, TAG, TGA Shine-Delgarnobox Stop CodonTAA, TAG, TGA +1 DNA Cis-RegulatoryElements Coding Sequence= ORF Coding Sequence= ORF Protein B USE/Promoter/Operator Protein A Terminatorsequence 5’ UTR = Leadersequence Spacer = 5’UTR of 2nd cistron

  6. Eukaryotic Gene Structure Stop CodonTAA, TAG, TGA Start Codon ATG Cis-Regulatory Elements Exon1 Exon2 Exon3 Promoter/Enhancer 5’ UTR 3’ UTR TRANSCRIPTION Exon1 Exon2 Exon3 RNA Processing Start Codon Stop Codon polyA tail Exon1 AAAAAAAA Exon2 Exon3 3’ UTR 5’ UTR

  7. Transcription factors & RNA polymerase recognize & bind the promoter DNA adjacent to the promoter is denatured forming the open promoter complex Transcription Proceeds Through 3 Steps Initiation Elongation • RNA polymerase moves along the DNA in synthesizing a RNA transcript. Synthesis is 5’3’ – Only 1 strand of DNA is read as a template. Termination • A termination signal is reached causing RNA polymerase to dissociated from the DNA Figure 12.2

  8. A Prokaryotic Promoter Coding strand Transcriptional start site Promoter –35 sequence –10 sequence +1 5¢ 3¢ C T T T T T A A A A G A A A A A A G A T T T T C T T 3¢ 5¢ A Template strand 5¢ 3¢ RNA Transcription

  9. Reaching A Consensus +1 Transcribed –10 region –35 region N6 A lac operon N17 TTTACA TATGTT N7 A N17 lacI GCGCAA CATGAT N7 A trp operon N17 TTGACA TTAACT N7 A rrnX N16 TTGTCT TAATAT recA N7 A N16 TTGATA TATAAT lexA A N6 N17 TTCCAA TATACT A N7 N16 TTTACA tRNAtyr TATGAT Consensus TTGACA TATAAT

  10. RNA Polymerases Structure of a bacterial RNA polymerase Structure of a eukaryotic RNA polymerase II

  11. RNAPolymerases • Differences between eukaryotes & prokaryotes • Prokaryotes • 1 enzyme with 4 subunits • 2 α’s, 1 , & 1 ’ • actual polymerase function • Sigma factors (σ ) • recognize & bind promoter DNA sequence • Eukaryotes • 3 separate holoenzymes – each has ~12 subunits • RNA Pol I – 28S, 18S, 5.8S rRNA • RNA Pol II – mRNA, snRNA • RNA Pol III – tRNA, 5S rRNA • 3 sets of basal transcription factors • recognize promoter DNA sequences

  12. The Process of Transcription • Initiation • Where/when most regulation of gene expression occurs • Different between proks & euks • Elongation • Essentially same between prokaryotes & eukaryotes • Some regulation, more in proks than euks • Termination • Different between proks & euks • Some regulation

  13. Prokaryotic Transcription Initiation Figure 12.6

  14. Rho Dependent Termination in Prokaryotes Figure 12.8 r-dependent termination rho utilization site Rho protein is a helicase

  15. Rho Dependent Termination in Prokaryotes Figure 12.8 r-dependent termination

  16. r-independent termination requires two sequences in the RNA A stem-loop structure upstream of 7-9 U residues Rho Independent Termination in Prokaryotes NusA Figure 12.9 r-independent termination Stabilizes the RNA pol pausing

  17. Eukaryotic Promoters Pol I UBF SL1 45S rRNA precursor UCE Core Pol II GSTFs TFIID pre-mRNA UAS TATAbox TFIIIB Pol III TFIIIC A B tRNA precursor • RNA Pol I • rRNA precursor • RNA Pol II • mRNAs, U6 snRNA • RNA Pol III • tRNA, 5S rRNA, U1-U5 snRNAs

  18. The Pol II Transcription Initiation Complex Figure 12.12

  19. TFIIH regulates formation of the open complex Pol II Initiation Complex Figure 12.12 • Has a helicase function that promotes melting of DNA • Phosphorylates the carboxyl terminal domain domain of RNA pol II • RNA pol II releases TFIIB • Promoter clearance occurs RNA pol II proceeds to the elongation stage

  20. Chromatin Structure Affects Promoter Access Core histone proteins COCH3 Histone acetyltransferase COCH3 COCH3 COCH3 COCH3 COCH3 COCH3 COCH3 COCH3 Histone deacetylase COCH3 COCH3 Acetyl group Histone acetylation ATP-dependent chromatin remodeling complex Change in the relative positions of a few nucleosomes Change in the spacing of nucleosomes over a long distance Chromatin remodeling

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