1. GENE: RNA polymerases and transcription factors

2. Structure of genes Prokaryotic and eukaryotic genes differ in their structure, however there are a number of common features between the two. Eukaryotic genes are monocistronic, code for only one protein Prokaryotic genes are polycistronic.

3. 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

4. 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

5. 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

6. 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 dissociate from the DNA

7. 7 RNA polymerases come in different forms, but share many features RNA polymerases perform essentially the same reaction in all cells. Bacteria have only a single RNA polymerases while in eukaryotic cells there are three: RNA Pol I, II and III.

8. RNA Polymerases 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 have ~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

9. 9 RNA Pol II is the focus of eukaryotic transcription, because it is the most studied polymerase, and is also responsible for transcribing most genes-indeed, essentially all protein-encoding genes RNA Pol I transcribes the large ribosomal RNA precursor gene. RNA Pol III transcribes tRNA gene, some small nuclear RNA genes and the 5S rRNA genes. Eukaryotic RNA polymerases

10. 10 “Crab claw” shape of RNAP Active center cleft

11. 11 There are various channels allowing DNA, RNA and ribonucleotides (rNTPs) into and out of the enzyme’s active center cleft.

12. 12 Transcription by RNA polymerase proceeds in a series of steps Initiation Elongation Termination

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. 15 Initiation Promoter: the DNA sequence that initially binds the RNA polymerase The structure of promoter-polymerase complex undergoes structural changes to proceed transcription DNA at the transcription site unwinds and a “ bubble ” forms Direction of RNA synthesis occurs in a 5 ’ -3 ’ direction (3 ’ - end growing)