1. Active from 3:20 today thru midnight on November 25 Homework

2. 37 valid questions Average score – 76.3% Two names did not register R10780801 R11275224 Exam 3

3. Constitutive transcription – continuous expression  usually for genes that perform routine tasks necessary for life Regulated transcription – expression at particular times  for genes that are differentially required under varied conditions Regulated transcription includes control of both initiation and amount of transcription Control is modulated by interactions between proteins and regulatory sequences within the DNA Negative control – binding of a molecule to prevent transcription Works via repressor proteins Positive control – binding of a molecule to encourage/initiate transcription Works via activator proteins Bacterial Gene Regulation

4. Protein-nucleic acid binding Most proteins to be discussed bind specific DNA/RNA sequences Most commonly via α-helix insertion into major groove(s) Bacterial Gene Regulation

5. Protein domains - Regions of a protein that have a particular function DNA binding domains have amino acids that associate with nucleotides of particular DNA sdquences Specificity is dictated by the unique patterns of atoms in these nucleotides Most commonly via α-helix insertion into major groove(s) Bacterial Gene Regulation

6. Protein domains - Regions of a protein that have a particular function Most regulatory proteins have at least two domains DNA binding Allosteric Often have a third Multimerization Bacterial Gene Regulation

7. Allosteric regulation Protein shape is intimately related to function Molecule binding can significantly alter shape Ligand – a molecule binding to a complementary site on a protein to alter its conformation Allostery – “other shape” A ligand binds and the protein changes shape to reveal or conceal another binding site Bacterial Gene Regulation

8. Allosteric regulation Small molecule effectors http://bio156.aznetwork.com/animat ed!/chapter04/videos_animations/al losteric_inhibition.html Bacterial Gene Regulation

9. Negative control Two scenarios Repressor protein has an active DNA-binding domain in the absence of an inducer ligand Repressor protein has an inactive DNA-binding domain in the absence of a co-repressor Bacterial Gene Regulation

10. Positive control Two scenarios Activator protein has an inactive DNA-binding domain in the absence of an effector ligand Repressor protein has an active DNA-binding domain in the absence of a inhibitor Bacterial Gene Regulation

11. The lac operon Operon – a cluster of genes undergoing coordinated regulation Very common in bacteria The lactose (lac) operon is responsible for producing three polypeptides responsible for the metabolism of lactose Glucose is the preferred energy source metabolized via glycolysis a monosaccharide Lactose is an alternative, only metabolized if needed A disaccharide Thus, the lac operon is inducible, turned on when needed but off the rest of the time Bacterial Gene Regulation

12. The lac operon One gene (lacZ) in the lac operon encodes  -galactosidase, which breaks the bond between galactose and glucose in the lactose molecule Glucose and galactose can then be metabolized via glycolysis One intermediate product is allolactose Bacterial Gene Regulation

13. The lac operon structure A multi-part regulatory region and three structural genes Structural genes LacZ   -galactosidase LacY  permease LacA  transacetylase One other gene, LacI (located upstream, not part of the operon), encodes the lac repressor protein Bacterial Gene Regulation

14. Another gene, LacI (located upstream), encodes the lac repressor protein Three domains DNA binding Multimerization Allosteric Forms a homotetramer Allolactose is the ligand that induces conformational change  decreased DNA binding Bacterial Gene Regulation

15. One last gene encodes catabolite activator protein (CAP) Three domains DNA binding Multimerization Allosteric Forms a homodimer Cyclic AMP (cAMP) is the ligand that induces conformational change increased DNA binding cAMP is produced only when glucose is not present Bacterial Gene Regulation

16. The lac operon structure A multi-part regulatory region CAP binding site – bound by catabolite activator protein Promoter – bound by RNA polymerase Operator – bound by lac repressor Bacterial Gene Regulation

17. Let’s put it all together Three scenarios exist Lactose and glucose present Glucose +, lactose – Lactose +, glucose - Bacterial Gene Regulation What does the cell ‘want’ to do in each case and how is it accomplished?

18. Glucose +, lactose =(normal metabolism) The cell ‘wants’: to use glucose, no lactose is available so why bother transcribing genes to metabolize it? The cell accomplishes this by: shutting down the lac operon Bacterial Gene Regulation Negative control No allolactose present  repressor is active  Repressor binds to operator  blocks transcription

19. Glucose -, lactose + =(lactose metabolism) The cell ‘wants’: to use glucose but there isn’t any, lactose is available so it must transcribe genes to metabolize it The cell accomplishes this by: activating the lac operon Bacterial Gene Regulation Positive control Allolactose present  repressor is inactive Repressor cannot bind to operator  transcription CAP binds CAP binding site  recruits RNA pol, increasing transcription

20. Glucose +, lactose + The cell ‘wants’: to use both but no need to expend large amounts of extra energy by specially targeting lactose for use The cell accomplishes this by: mostly metabolizing glucose but allowing the lac operon to be transcribed at a minimal level Bacterial Gene Regulation Allolactose present  repressor is inactive, transcription can happen Glucose is present  no cAMP  no CAP binding  no RNA pol recruitment  minimal lacZ transcription https://www.youtube.com/watch?v=mwkI5VFd1Gg - Watch just for the music http://highered.mheducation.com/olc/dl/120080/bio27.swf

21. Wait a minute?!?! If no lactose is present, transcription is shut down No permease is available to allow lactose in And even if it got in, it wouldn’t be metabolized and no allolactose would be produced to release the repressor from the operator How does transcription EVER start? Leaky transcription Binding of the repressor is reversible Sometimes it just falls off, allowing a very low level of transcription and low levels of permease and  -galactosidase in the cell Bacterial Gene Regulation

22. The trp operon Tryptophan is an essential amino acid that can be synthesized by the cell But, why bother if tryptophan is already present? The trp operon is repressible, meaning it’s usually on but can be turned off Furthermore, it can be fine tuned to match the needs of the cell  a process called attenuation Bacterial Gene Regulation

23. The trp operon structure A multi-part regulatory region and five structural genes Structural genes trpA-E  enzymes involved in the anabolism (building molecules trpR  elsewhere, encodes trp Repressor

24. trp repressor protein has a similar structure but works the opposite way of lac repressor Three domains DNA binding Multimerization Allosteric Forms a homodimer Tryptophan is the ligand (corepressor) that induces conformational change  increased DNA binding Bacterial Gene Regulation

25. The lac operon structure A multi-part regulatory region Promoter – bound by RNA polymerase Operator – bound by trp repressor Attenuator – we’ll get to that Bacterial Gene Regulation

26. Let’s put it all together Three scenarios exist Tryptophan + as needed Tryptophan + but low Tryptophan - Bacterial Gene Regulation What does the cell ‘want’ to do in each case and how is it accomplished?

27. Tryptophan + The cell ‘wants’: to use use the available tryptophan, so why bother transcribing genes to metabolize it? The cell accomplishes this by: shutting down the trp operon Bacterial Gene Regulation

28. Tryptophan - The cell ‘wants’: tryptophan and needs to manufacture it for itself The cell accomplishes this by: activating the trp operon Bacterial Gene Regulation

29. Tryptophan +/- The cell ‘wants’: some tryptophan but not too much  fine tune production The cell accomplishes this by: attenuating (taper off) the trp operon As tryptophan increases in the cell  production decreases As tryptophan decreases in the cell  production increases The result is a steady-state, or homeostasis Attenuation involves the leader strand (trpL) segment of the trp operon mRNA Somehow, increased tryptophan availability results in the premature termination of trp operon transcription Bacterial Gene Regulation

30. Tryptophan +/- As trp increases rate of trp operon transcription decreases Of the transcripts that are produced, more and more consist only of the first 140 nt from the 5’ end of trpL Bacterial Gene Regulation trp = Full length transcripts Partial (inviable) transcripts

31. Tryptophan +/- trpL contains Four repeated DNA sequences Can form stem-loop structures A region that codes for a 14 AA polypeptide Two back-to-back codons code for tryptophan Bacterial Gene Regulation

32. Tryptophan +/- trpL contains Four repeated DNA sequences Can form stem-loop structures A region that codes for a 14 AA polypeptide Two back-to-back codons code for tryptophan Bacterial Gene Regulation

33. Tryptophan +/- The four repeats can form three, mutually exclusive structures 2-3 loop = antitermination loop 3-4 loop = termination loop Bacterial Gene Regulation

34. Tryptophan +/- 3-4 loop = termination loop Remember WAY back in chapter 8? Intrinsic termination If 3-4 loop forms, transcription of trp is stopped Bacterial Gene Regulation

35. Tryptophan +/- 2-3 loop = antitermination loop 3-4 loop cannot form  transcription continues How does the cell control this in such a way to encourage or discourage trp expression? As tryptophan increases in the cell  transcription decreases As tryptophan decreases in the cell  transcription increases Bacterial Gene Regulation

36. Tryptophan +/- How does the cell control this in such a way to encourage or discourage trp expression? Depends on whether or not the ribosome is stalled in region 1 If tryptophan is readily available, ribosome has no trouble filling the need for two sequential tryptophans during translation Ribosome moves rapidly through 1 and covers 2, preventing 2 from interacting with 3 3-4 termination loop forms, halting transcription of the full operon Bacterial Gene Regulation

37. Tryptophan +/- If tryptophan is readily available, ribosome has no trouble filling the need for two sequential tryptophans during translation Ribosome moves rapidly through 1 and covers 2, preventing 2 from interacting with 3 3-4 termination loop forms, halting transcription of the full operon Bacterial Gene Regulation

38. Tryptophan +/- How does the cell control this in such a way to encourage or discourage trp expression? Depends on whether or not the ribosome is stalled in region 1 If tryptophan is in short supply, ribosome has difficulty filling the need for two sequential tryptophans during translation Ribosome stalls at 1, allowing 2 to interact with 3 2-3 antitermination loop forms, allowing transcription of the full operon Bacterial Gene Regulation

39. Tryptophan +/- If tryptophan is in short supply, ribosome has difficulty filling the need for two sequential tryptophans during translation Ribosome stalls at 1, allowing 2 to interact with 3 2-3 antitermination loop forms, allowing transcription of the full operon Bacterial Gene Regulation

40. Tryptophan +/- http://highered.mheducation.com/sites/dl/free/0072835125/126997 /animation28.html Bacterial Gene Regulation