Transcription in Prokaryotes

1. Transcription in Prokaryotes

2. Environmental change DNA RNA Turn gene(s) on/off protein Proteins to deal with new environment Transcriptional Control • Very important to: • express genes when needed • repress genes when not needed • Conserve energy resources; avoid expressing unnecessary/detrimental genes

3. Transcriptional Control DNA RNA protein Many places for control Transcription Initiation Elongation Termination Processing Capping Splicing Polyadenylation Turnover Translation Protein processing

4. Prokaryotic Transcription • Operons Groups of related genes transcribed by the same promoter • Polycistronic RNA • Multiple genes transcribed as ONE TRANSCRIPT • No nucleus, so transcription and translation can occur simultaneously

5. RNA Structure • Contain ribose instead of deoxyribose • Bases are A,G,C,U, • Uracil pairs with adenine • Small chemical difference from DNA, but large structural differences • Single stranded helix • Ability to fold into 3D shapes - can be functional

6. RNA Structures Vary • RNA more like proteins than DNA: structured domains connected by more flexible domains, leading to different functions • e.g. ribozymes – catalytic RNA

7. RNA synthesis • RNAP binds, melts DNA • Nucleosides added 5’  3’

8. Types of RNA • Messenger RNA (mRNA) – genes that encode proteins • Ribosomal RNA (rRNA) – form the core of ribosomes • Transfer RNA (tRNA) – adaptors that link amino acids to mRNA during translation • Small regulatory RNA – also called non-coding RNA

9. Transcriptional Control Transcription Initiation Elongation Termination Processing Capping Splicing Polyadenylation Turnover Translation Protein processing Control of initiation usually most important.

10. Initiation • RNA polymerase • Transcription factors • Promoter DNA • RNAP binding sites • Operator – repressor binding • Other TF binding sites Start site of txn is +1 α α ββ’σ

11. Initiation • RNA polymerase • 4 core subunits • Sigma factor (σ)– determines promoter specificity • Core + σ = holoenzyme • Binds promoter sequence • Catalyzes “open complex” and transcription of DNA to RNA

12. RNAP binds specific promoter sequences • Sigma factors recognize consensus -10 and -35 sequences

13. RNA polymerase promoters TTGACA TATAAT Deviation from consensus -10 , -35 sequence leads to weaker gene expression

14. Bacterial sigma factors • Sigma factors are “transcription factors” • Different sigma factors bind RNAP and recognize specific -10 ,-35 sequences • Helps melt DNA to expose transcriptional start site • Most bacteria have major and alternate sigma factors • Promote broad changes in gene expression • E. coli 7 sigma factors • B. subtilis 18 sigma factors • Generally, bacteria that live in more varied environments have more sigma factors

15. Sigma factors s70 s54 sS sS sF s32 Extreme heat shock, unfolded proteins E. coli can choose between 7 sigma factors and about 350 transcription factors to fine tune its transcriptional output An Rev Micro Vol. 57: 441-466T. M. Gruber

16. What regulates sigma factors • Number of copies per cell (σ70 more than alternate) • Anti-sigma factors (bind/sequester sigma factors) • Levels of effector molecules • Transcription factors

17. Bacterial RNAP numbers • In log-phase E. coli: • ~4000 genes • ~2000 core RNA polymerase molecules • ~2/3 (1300) are active at a time • ~1/3 (650) can bind σ subunits. • Competition of σ for core determines much of a cell’s protein content.

18. Lac operon control • Repressor binding prevents RNAP binding promoter • An activating transcription factor found to be • required for full lac operon expression: CAP (or Crp)

19. lac operon – activator and repressor CAP = catabolite activator protein CRP = cAMP receptor protein

20. Activating transcription factors Crp dimer w/ DNA • Helix-turn-helix (HTH) bind major groove of DNA • HTH one of many TF motifs

21. glucose cAMP Crp lac operon no mRNA Cofactor binding alters conformation • Crp binds cAMP, induces allosteric changes glucose cAMP Crp mRNA

22. Cooperative binding of Crp and RNAP Binds more stably than either protein alone

23. Enhancers • activating regions not • necessarily close to RNAP • binding site NtrC example: • NtrC required for RNAP to • form open complex • NtrC activated by P • P NtrC binds DNA, forms loop • that folds back onto RNAP, • initiating transcription • signature of sigma 54

24. Bacterial promoters Transcription start +1 UP element • Most bacterial promoters have –35 and –10 elements • Some have UP element • Some lack –35 element, but have extended –10 region -35 element -10 element (Pribnow box) +1 pre –10 element

25. E. coliRNA polymerase composed of 5 subunits: • Subunits: b, b’, a(2), , and s • Core enzyme: b, b’, a(2),  • Holoenzyme : b, b’, ,a(2) and s • The s subunits give specificity for site of initiation- promoter

26. NTD NTD  ’ CTD CTD  s Subunit structure of bacterial RNA polymerase 160 kDa 150 kDa 40 kDa DNA Holoenzyme-b’ba2s. Functions in initiation. Core enzyme-b’ba2. Functions in elongation.

27. The 3D structure of bacterial RNA polymerase holoenzyme s3 s factor domains : N-term s1 Inhibition s2 -10 binding s3 -10 binding s4 -35 binding

28. The s factors • s factors are required for promoter recognition and transcription initiation in prokaryotes • s factors have analogous function as general transcription factors in eukaryotes • A variety of s factors exist in E.coli • For expression from most promoters s70 is required • For expression from some bacterial promoters one of other s subunits is needed instead • s70 is essential for cell growth in all conditions, while other sigmas are required for special events, like nitrogen regulation (s54), response to heat shock (s32), sporulation, etc

29. RNA pol s Holoenzyme Promoter region -35 -10 Closed complex Open complex Promoter escape Elongation mRNA s release The overview of s factor function

30. The promoter specificity of some s factors in E.coli s70 TTGACA – 17 bp – TATAATN3-6-A -35 -10 +1 s32 CTTGAAA – 16 bp – CCCCATNTN3-10-T/A -35 -10 +1 s54 GG – N12 – GC/T – 12bp – A -24 -12 +1

31. The UP element RNAP RNAP a NTD • UP element is an AT rich motif present in some strong (e.g. rRNA) promoters • UP element interacts directly with C-terminal domain of RNA polymerase a subunits s a CTD s4 s2-3 UP -35 -10 +1

32. Constitutive and inducible promoters • Certain genes are transcribed at all times and circumstances -Examples – tRNAs, rRNAs, ribosomal proteins, RNA polymerase -Promoters of those genes are called constitutive • Most genes, however, need to be transcribed only under certain circumstances or periods in cell life cycles -The promoters of those genes are called inducible and they are subject to up- and down- regulation

33. Regulation at promoters • Promoters can be regulated by repression and/or activation • Many s70 promoters are controlled both by repression and activation, whereas, for example s54 promoters are controled solely by activation

34. Cartoon of the transcription cycle

35. Mechanisms of repression • Repression by steric hindrance • Inhibition of transition to open complex • Inhibition of promoter clearance • Anti-activation • Anti-sigma factors

36. e) Anti-sigma factors • An anti-s factor is defined by the ability toprevent its cognate s factor to compete for core RNA polymerase • Mostly used for s factors, other than s70, for example in life cycle regulation (sporulation, etc) • Some bacteriophages use their own anti-s factors to prevent expression of cellular proteins RNAP RNAP anti-s s s -10 -35 -10 -35

37. d) Anti-activation • Repressor molecule removes the activator RNA pol - s Activator ABS weak promoter +1 Activator binding sequence Activator RNA pol - s Repressor ABS weak promoter +1

38. Two examples of steric hindrance • Trp repressor • Lac repressor

39. In the absence of tryptophane the trp repressor (red blob) shows no affinity to promoter (black box) and the RNA polymerase (yellow blob) transcribes the operon When enough tryptophane (blue dots) is made, it binds to repressor, which now is able to bind to promoter and block RNA polymerase binding The tryptophan repressor • The trp repressor controls the operon for the synthesis of L-tryptophan in E.coli by a simple negative feedback loop

40. The lac promoter Lac promoter is widely used in artifical plasmids, designed for protein production For practical purposes it is easier to use non-hydrolyzable lactose analog – IPTG (isopropyl-b-thiogalactoside) instead of native lactose

41. A cartoon, ilustrating events upon IPTG binding to lac repressor (IPTG) As IPTG binds, the DNA binding domains scissor apart

42. Mechanisms of activation • a) Regulated recruitment • b) Polymerase activation • c) Promoter activation

43. a) Regulated recruitment • Activator “extends” the binding site for RNA polymerase strong or weak affinity RNA pol - s Activator ABS weak promoter +1 strong affinity weak affinity

44. Catabolite Activator Protein: CAP • Activates transcription from more than 150 promoters in E.coli • Upon activation by cAMP (cyclic Adenosine MonoPhosphate), CAP binds to promoter and helps RNAP-s to bind as well • All CAP–dependent promoters have weak –35 sequence, so that RNAP-s is unable to bind the promoter without CAP assistance

46. Models for Class I and Class II promoter activation Class I CAP binding sites can be from –62 to –103. CAP interacts with the carboxy terminal domain of the RNAP a-subunit (aCTD) Class II CAP binding sites usually overlap the –35. CAP interacts with the aCTD, aNTD (N-terminal domain), and the s factor Busby and Ebright, 2000, J. Mol. Biol. 293:199-213

47. Model for Class III promoter activation • Activation of Class III promoters requires binding of at least two CAP dimers or at least one CAP dimer and one regulation-specific activator • Interactions can be similar to those of ClassI and/or ClassII promoters, except that each aCTD subunit is making different interactions

48. DNA binding domain of AraC AraC RNAP-s promoter + arabinose ( ) Transcription RNAP-s AraC – repressor and activator of arabinose promoter

49. RNAP-s54 activation • RNAP-s54 open complex formation requires ATP hydrolysis • Activator protein with ATP-ase activity binds to “enhancer” site about 160 bp upstream from –24 sequence. DNA then gets looped and activator interacts with RNAP-s54 resulting in the open bubble formation upon ATP hydrolysis ATP+Pi ATP s54 s54

50. c) Example of promoter activation: MerR activator family • MerR is an activator that controls genes involved in the response to mercury poisoning • Other MerR family activators (CueR, BmrR, etc) respond to a variety of different toxic compounds such as other heavy metal atoms or drugs • In MerR activated promoters, -10 and –35 regions are separated by 19bp instead of optimal 17bp