2. Transcription Gene Expression Part 1

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

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

5. Cistron 1Cistron 2 Regulatory and Coding Sequence Unit = Operon ATG Shine- Delgarno box +1 5’ UTR = Leader sequence Spacer = 5’UTR of 2 nd cistron Stop Codon TAA, TAG, TGA ATG Shine- Delgarno box Stop Codon Cis-Regulatory Elements USE/Promoter /Operator DNA Termination sequence Coding Sequence = ORF 1 Coding Sequence = ORF 2 Protein A Protein B Structural or Coding Sequences Regulatory Sequences Prokaryotic Gene Structure

6. Polycistronic mRNA 5’ UTR = Leader sequence ORF Protein A +1 AUG Shine-Delgarno box AUG Shine-Delgarno box ORF Protein B Stop Codon UAA, UAG, UGA Spacer Shine-Delgarno box Cis-Regulatory Elements Cistron 1 Coding Sequence= ORF +1 ATG 5’ UTR = Leader sequence Spacer = 5’UTR of 2 nd cistron Stop Codon TAA, TAG, TGA Shine-Delgarno box ATG Coding Sequence= ORF Cistron 2 USE/Promoter /Operator DNA Terminator sequence Protein A Protein B Stop Codon TAA, TAG, TGA Prokaryotic Gene Structure

7. Eukaryotic Gene Structure Promoter/ Enhancer 5’ UTR Cis- Regulatory Elements Start Codon ATG Exon1 Exon2Exon3 Stop Codon TAA, TAG, TGA 3’ UTR Exon1 Exon2Exon3 AAAAAAAAExon1 Exon2Exon3 5’ UTR 3’ UTR Start CodonStop Codon polyA tail

8. Transcription Proceeds Through 3 Steps Transcription factors & RNA polymerase recognize & bind the promoter DNA adjacent to the promoter is denatured forming the open promoter complex 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

9. Template strand Transcription Coding strandTranscriptional start site +1 –35 sequence–10 sequence Promoter G T C A T A C G A T A T T A T A T A T A A T A T A T 35 5 53 3 RNA A A Prokaryotic Promoter

10. –35 region –10 region lac operon Consensus lacI trp operon rrnX recA lexA tRNA tyr Transcribed +1 TTTACATATGTT AN6N6 GCGCAACATGAT AN7N7 TTGACATTAACT AN7N7 TTGTCTTAATAT AN7N7 TTGATATATAAT A TTCCAATATACT A TATGAT TTGACATATAAT A N 16 TTTACA N 16 N 17 N 16 N 17 N7N7 N7N7 N6N6 Reaching A Consensus

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

12. 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

13. 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

14. Figure 12.6 Prokaryotic Transcription Initiation

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

16.  -dependent termination Figure 12.8 Rho Dependent Termination in Prokaryotes

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

18. Eukaryotic Promoters RNA Pol I –rRNA precursor RNA Pol II –mRNAs, U6 snRNA RNA Pol III –tRNA, 5S rRNA, U1-U5 snRNAs Pol I UBFSL1 UCECore 45S rRNA precursor Pol II GSTFsTFIID UASTATA box pre-mRNA Pol III TFIIIB tRNA precursor AB TFIIIC

19. Figure 12.12 The Pol II Transcription Initiation Complex

20. Figure 12.12 Pol II Initiation Complex TFIIH regulates formation of the open complex 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

21. Histone acetylation Chromatin remodeling Core histone proteins Histone acetyltransferase Histone deacetylase Acetyl group COCH 3 ATP-dependent chromatin remodeling complex Change in the spacing of nucleosomes over a long distance Change in the relative positions of a few nucleosomes Chromatin Structure Affects Promoter Access