1. ©2000 Timothy G. Standish Ecclesiastes 3:1 1To every thing there is a season, and a time to every purpose under the heaven:

2. ©2000 Timothy G. Standish Transcription: Concentrating on Prokaryotes Timothy G. Standish, Ph. D.

3. ©2000 Timothy G. Standish All Genes Can’t be Expressed At The Same Time Some gene products are needed by all cells all the time. These constitutive genes are expressed by all cells. Other genes are only needed by certain cells or at specific times, expression of these inducible genes is tightly controlled in most cells. For example, pancreatic  cells make insulin by expressing the insulin gene. If neurons expressed insulin, problems would result.

4. ©2000 Timothy G. Standish Logical Expression Control Points DNA packaging Transcription RNA processing mRNA Export mRNA masking/unmasking and/or modification mRNA degradation Translation Protein modification Protein transport Protein degradation Increasing cost The logical place to control expression is before the gene is transcribed

5. mRNA Transcription Introduction The Central Dogma of Molecular Biology Cell Polypeptide (protein) Translation Ribosome Reverse tanscription DNA

6. ©2000 Timothy G. Standish Stages of Transcription Transcription can be logically divided into Four distinct stages: 1. Template recognition 2. Initiation 3. Elongation 4. Termination l Each stage may participate in regulation, but template recognition and termination appear to be major players

7. ©2000 Timothy G. Standish  General Model For Transcription     RNA Polymerase Core Enzyme Holoenzyme

8. ©2000 Timothy G. Standish General Model For Transcription     

9. ©2000 Timothy G. Standish General Model For Transcription     

10. ©2000 Timothy G. Standish General Model For Transcription     

11. ©2000 Timothy G. Standish NusA General Model For Transcription      Top- Isomerase I Gyrase

12. ©2000 Timothy G. Standish NusA General Model For Transcription     Top- Isomerase I Gyrase  Ribosome Exo- nuclease

13. ©2000 Timothy G. Standish Ribosome NusA General Model For Transcription      Exo- nuclease

14. ©2000 Timothy G. Standish Ribosome NusA General Model For Transcription      Exo- nuclease

15. ©2000 Timothy G. Standish Ribosome NusA General Model For Transcription      Exo- nuclease

16. ©2000 Timothy G. Standish NusA General Model For Transcription      Exo- nuclease

17. ©2000 Timothy G. Standish NusA General Model For Transcription      Exo- nuclease

18. ©2000 Timothy G. Standish  NusA General Model For Transcription     Exo- nuclease

19. ©2000 Timothy G. Standish NusA General Model For Transcription     

20. ©2000 Timothy G. Standish RNA Polymerase RNA Polymerase is a spectacular enzyme, it performs the following functions: 1. Recognition of the promoter region 2. Melting of DNA (Helicase + Topisomerase) 3. RNA Priming (Primase) 4. RNA Polymerization 5. Recognition of terminator sequence

21. ©2000 Timothy G. Standish Prokaryotic Transcription Initiation The  subunit of prokaryotic RNA polymerase is necessary for promoter recognition and binding of RNA polymerase to the promotor Different  subunits allow recognition of different types of promoters thus the type of genes transcribed can be modulated by altering the types of  subunits which attach to RNA polymerase

22. ©2000 Timothy G. Standish Prokaryotic Transcription Initiation Constitutive GeneHeat Shock Gene P1P1 P2P2 Different promoters RNA Pol. 

23. ©2000 Timothy G. Standish Prokaryotic Transcription Initiation Constitutive GeneHeat Shock Gene P2P2 P1P1 Different promoters RNA Pol. 

24. ©2000 Timothy G. Standish Transcription Termination There are two types of termination: Rho dependent requires a protein called Rho, that binds to and slides along the RNA transcript. The terminator sequence slows down the elongation complex, Rho catches up and knocks it off the DNA Rho independent termination depends on both slowing down the elongation complex with a hairpin and a U rich region that destabilizes the elongation complex

25. ©2000 Timothy G. Standish RNA Pol. 5’ RNA Pol. 5’ RNA Termination Rho Independent Terminator

26. ©2000 Timothy G. Standish RNA Pol. 5’ RNA Pol. 5’ RNA Termination Rho Independent Terminator

27. ©2000 Timothy G. Standish RNA Pol. 5’ RNA Termination Rho Dependent Terminator  RNA Pol. 5’ RNA  The terminator sequence slows RNA polymerase

28. ©2000 Timothy G. Standish RNA Pol. 5’ RNA Termination Rho Dependent Terminator  Help, rho hit me! RNA Pol. 5’ RNA  Rho catches up with RNA polymerase

29. ©2000 Timothy G. Standish RNA Pol. 5’ RNA Termination Rho Dependent Terminator  RNA Pol. 5’ RNA  The elongation complex disintegrates

30. ©2000 Timothy G. Standish

31. Transcription 5’ 3’ 5’ Template (antisense) strand Coding (sense) strand

32. ©2000 Timothy G. Standish Transcription 5’ 3’ 5’ Template (antisense) strand Coding (sense) strand 5’ RNA Pol.

33. ©2000 Timothy G. Standish Transcription 5’ 3’ 5’ RNA Pol. Template (antisense) strand Coding (sense) strand

34. ©2000 Timothy G. Standish Products of Transcription Transcription produces three major RNA products: 1Ribosomal RNA (rRNA) - Several rRNAs are vital constituents of ribosomes 2Transfer RNA (tRNA) - The molecule that physically couples nucleic acid codons with specific amino acids 3Messenger RNA (mRNA) - The nucleic acid messenger that carries encoded information from genes on DNA to the protein manufacturing ribosomes

35. ©2000 Timothy G. Standish Transfer RNA (tRNA) Acts as the adapter molecule between the genetic code on mRNA and the protein “language” 75-85 bases long A specific amino acid is covalently linked at the 3’ end Elsewhere on the molecule is an anticodon complimentary to the specific amino acid codon on mRNA that codes for the amino acid carried by the tRNA Contain a number of modified bases

36. ©2000 Timothy G. Standish A “Simple” Gene Protein Coding Region Terminator Sequence Promoter/ Control Region Transcription Start Site 3’ Untranslated Region 5’ Untranslated Region 3’5’ RNA Transcript

37. ©2000 Timothy G. Standish Transcription Initiation Proteins called transcription factors bind to the promoter region of a gene If the appropriate transcription factors are present, RNA polymerase binds to form an initiation complex RNA polymerase melts the DNA at the transcription start site Polymerization of RNA begins

38. ©2000 Timothy G. Standish RNA Pol.Initiation T. F. RNA Pol. 5’ RNA Promoter T. F.

39. ©2000 Timothy G. Standish 3’ 5’ 3’ Transcription And Translation In Prokaryotes Ribosome 5’ mRNA RNA Pol.

40. ©2000 Timothy G. Standish Heat Shock Response Elements Sudden changes in the temperature of cells cause stress in response to which heat shock genes are expressed At least some heat shock genes are thought to be chaperones that help proteins fold correctly Heat shock genes have Heat Shock Elements (HSEs) in their control regions Heat Shock Transcription Factors (HSTFs) bind the HSEs up regulating expression of heat shock gene products

41. ©2000 Timothy G. Standish

42. DNA Cytoplasm Nucleus G AAAAAA Export Degradation etc. G AAAAAA Control of Gene Expression G AAAAAA RNA Processing mRNA RNA Transcription Nuclear pores Ribosome Translation Packaging Modification Transportation Degradation

43. ©2000 Timothy G. Standish 5’3’ RNA Transcript A “Simple” Gene Protein Coding Region Terminator Sequence Promoter/ Control Region 3’ Untranslated Region Transcription Start Site 5’ Untranslated Region

44. ©2000 Timothy G. Standish RNA Pol.Initiation Promoter RNA Pol. T. F. 5’ RNA

45. ©2000 Timothy G. Standish 3’ 5’ 3’ Transcription And Translation In Prokaryotes Ribosome 5’ mRNA RNA Pol.