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Transcription regulation in prokaryotes. Background: major and minor grooves of DNA.

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Presentation on theme: "Transcription regulation in prokaryotes. Background: major and minor grooves of DNA."— Presentation transcript:

1 Transcription regulation in prokaryotes

2 Background: major and minor grooves of DNA

3 Available interactions in minor and major grooves

4 Schematic view of available chemical groups in minor and major grooves In major groove all 4 nucleotides can be differentiated by a binding protein In minor groove only AT or GC base pairs can be differentiated The minor groove is also much narrower, which makes it less accessible for binding proteins As a consequence, most sequence specific DNA binding proteins bind to major groove

5 Helix-turn-helix DNA binding motif Helix-turn-helix motif is the most common DNA-binding motif in prokaryotes, present in many transcription repressors and activators One of the helices, DNA recognition helix, gets inserted in the major groove of DNA Helix-turn-helix proteins are often dimeric, with two recognition helices recognizing two adjacent DNA sequences Why dimeric? 1)Dimer binds to DNA stronger than monomer 2)By changing the relative positions of monomers, the dimer activity can be easily turned on and off

6 A common principle to activate or inactivate dimeric helix-turn-helix proteins Ligand changes the position of DNA binding a helices, so they do not bind DNA any more Or the opposite – ligand changes the position of helices, so they do bind to DNA (ligand)

7 The main features of interactions between DNA and the helix-turn-helix motif in DNA-binding proteins 1. Sequence unspecific interactions contribute to overall stability of complex, but do not differentiate the ound DNA sequence 2. Sequence specific interactions determine, with which regions of DNA the interaction will occur

8 Bacterial promoters 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) UP element pre –10 element +1 Transcription start +1

9 The  factors  factors are required for promoter recognition and transcription initiation in prokaryotes  factors have analogous function as general transcription factors in eukaryotes A variety of  factors exist in E.coli For expression from most promoters  70 is required For expression from some bacterial promoters one of other  subunits is needed instead  70 is essential for cell growth in all conditions, while other sigmas are required for special events, like nitrogen regulation (  54 ), response to heat shock (  32 ), sporulation, etc

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

11 The promoter specificity of some  factors in E.coli  70 TTGACA – 17 bp – TATAATN 3-6 -A  -35 -10 +1  32 CTTGAAA – 16 bp – CCCCATNTN 3-10 -T/A -35 -10 +1  54 GG – N 12 – GC/T – 12bp – A -24 -12 +1

12 Some  70 promoters in E.coli The –35 and –10 sequences of individual  factors are conserved (yellow boxes) The spacer sequence between –35 and –10 is not conserved, but the spacer length is 17  1 bp

13 The 3D structure of bacterial RNA polymerase holoenzyme N-term  1 Inhibition  2 -10 binding  3 -10 binding  4 -35 binding  factor domains : 33

14 The UP element 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  subunits -35-10UP +1 44  2-3  RNAP  NTD  CTD RNAP

15 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

16 Regulation at promoters Promoters can be regulated by repression and/or activation Many  70 promoters are controlled both by repression and activation, whereas, for example  54 promoters are controled solely by activation

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

18 a) Repression by steric hindrance (most cases) Examples: Trp repressor, lac repressor

19 b) Inhibition of transition to open complex RNA Polymerase –  complex (RNAP-  can bind to promoter, but transition to open complex is blocked

20 c) Inhibition of promoter clearance The transcription bubble can be formed, but further RNAP-  movement is blocked

21 e) Anti-sigma factors An anti-  factor is defined by the ability to prevent its cognate  factor to compete for core RNA polymerase Mostly used for  factors, other than  70, for example in life cycle regulation (sporulation, etc) Some bacteriophages use their own anti-  factors to prevent expression of cellular proteins -10 -35 RNAP  -10 -35 RNAP  anti- 

22 d) Anti-activation Repressor molecule removes the activator weak promoter +1 weak promoter ABS +1 ABS RNA pol -  Activator RNA pol -  Activator Repressor Activator binding sequence

23 Two examples of steric hindrance Trp repressor Lac repressor

24 The tryptophan repressor The trp repressor controls the operon for the synthesis of L-tryptophan in E.coli by a simple negative feedback loop When enough tryptophane (blue dots) is made, it binds to repressor, which now is able to bind to promoter and block RNA polymerase binding 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

25 The 3-D structure of trp repressor

26 The conformational change upon binding tryptophan molecules induces a conformational change in trp repressor

27 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-  -thiogalactoside) instead of native lactose

28 The structure of lac repressor monomer DNA binding domain Tetramerization helix Core N subdomain Core C subdomain Inducer binding pocket Hinge helix

29 Functional lac repressor is a homotetramer Each dimer binds to a distinct DNA sequence at –82 and +11 respective to transcription start site This results in DNA looping, preventing the DNA polymerase from binding to –35 and –10 sequences -82 +11 lac repressor -35 -10 -82+11

30 The lac repressor binds both to major and minor grooves of DNA

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

32 Mechanisms of activation a) Regulated recruitment b) Polymerase activation c) Promoter activation

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

34 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-  to bind as well All CAP–dependent promoters have weak –35 sequence, so that RNAP-  is unable to bind the promoter without CAP assistance


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

37 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  CTD subunit is making different interactions

38 3-D structure of CAP-cAMP-DNA complex Cyclic AMP binds to the N-terminal domain and causes the two long “C” helices to reorient and move the DNA binding domains apart, so that CAP can bind to DNA 80º Binding of CAP causes DNA bending by 80º C C

39 CAP activates lac operon

40 AraC – repressor and activator of arabinose promoter RNAP-  Transcription + arabinose ( ) AraC promoter DNA binding domain of AraC

41 Structure of AraC dimers in presence (A) and absence (B) of arabinose Without arabinose, the monomers interact with the  barrel domains. An important interaction is stacking of Tyr31 of one subunit on Trp 95 of the other Arabinose binds in close proximity to Trp95, making the stacking interaction impossible Monomers associate in a different way – 4 helix bundle interactions by the helical domains

42 b) Polymerase activation This works for  54 promoters RNAP-  54 forms a stable complex with DNA, but needs to be activated to form an open complex

43 RNAP-  54 activation RNAP-  54 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-  54 resulting in the open bubble formation upon ATP hydrolysis  54 ATP ATP+P i

44 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

45 The BmrR-DNA-drug complex BmrR binds to a variety of toxic compounds, including tetraphenylphosphine (TPP) Both TPP bound and unbound forms of BmrR bind to promoter, but only TPP bound form induces transcription

46 In 19-bp spacer variant, -35 and –10 binding regions not only are too far from each other, but also on the opposite sides of double helix In BmrR-TPP bound vairiant, DNA double helix is underwound, so that –10 and –35 regions are at the same distance as in regular 17-bp spacer

47 Transcription termination In prokaryotes two types of transcription termination occur – rho indepedent termination and rho dependent termination In rho independent case, the termination is achieved by a secondary structure of mRNA – RNA stem-loop, followed by an AU rich region A rho protein is required for rho-dependent termination

48 Rho independent termination

49 Attenuation Regulation of transcription by the behavior of ribosomes Observed in bacteria, where transcription and translation are tightly coupled Translation of a mRNA can occur as the mRNA is being synthesized

50 Attenuation in trp operon

51 Rho dependent termination As polymerase transcribes away from the promoter, rho factor binds to RNA and follows the polymerase When polymerase reaches some sort of pause site, rho factor catches up with polymerase and unwinds the DNA-RNA hybrid, resulting in release of polymerase

52 Anti-termination

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