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| Growth Conditions of E. coli Control the Stability of cII Protein and thus the Lytic/Lysogenic Choice |
Infection to host growing in rich media: tends to propagate lytically, releasing progeny into an environment rich in fresh host cells.
Infection to host growing in poor media: form lysogens and sit tight, there will likely be few host cells in the vicinity for any progeny phage to infect.
High multiplicity of infection.
More templates produce more of the CII protein, which stimulates PRE.
Phage sense that it is too crowded.
Poor nutrient conditions for host.
Low [glucose] leads to increase in [cAMP].
Increased [cAMP] will repress the host gene hfl (protease FtsH).
Less FtsH protease leads to less degradation of the CII protein |
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mcs5109 Wed, 19 Nov 2008 06:55:59 GMT |
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| negative feedback for lamba repressor |
lamba repressor activates PRM trx and produces more repressors. Once [repressor] reaches high, repressor start binding at OR3 to repress PRM trx. |
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mcs5109 Wed, 19 Nov 2008 06:40:31 GMT |
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| Lysogenic Induction |
-UV or other damage cleaves lamba repressor
-cannot bind DNA (OR1 and OR2) cooperatively anymore
-triggers PR and PL trxs (goes to lytic growth).
For induction to work efficiently, the level of repressor in a lysogen condition must be tightly regulated
Negative feedback: lamba repressor activates PRM trx and produces more repressors. Once [repressor] reaches high, repressor start binding at OR3 to repress PRM trx. |
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mcs5109 Wed, 19 Nov 2008 06:40:31 GMT |
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| bacteriophage gamma |
-repressor= cI gene product
-two domains joined by flexible linker
1. N-ter: DNA binding (HTH motif) and activating region
2. C-ter: dimerization and tetramerization.
-can both activate and repress
Binding affinity: OR1 > OR2/OR3 (10-fold)
Another transcription factor in l phage gene regulation system, Cro (control of repressor and other things) has a single domain, binds 17 bp DNA w/o cooperatively and only repress transcription.
Binding affinity: OR3 > OR1/OR2 (10-fold) |
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mcs5109 Wed, 19 Nov 2008 06:08:15 GMT |
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| Lysogenic induction |
switch from lysogenic to lytic growth |
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mcs5109 Wed, 19 Nov 2008 05:46:16 GMT |
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| Bacteriophage gamma |
-both lysis and lysogeny |
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mcs5109 Wed, 19 Nov 2008 05:46:16 GMT |
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| Lysogeny (prophage) |
After infection, the phage DNA integrates into the host genome and resides there passively |
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mcs5109 Wed, 19 Nov 2008 05:46:16 GMT |
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| Lysis |
Infection by phage produces many progeny and breaks open (lyses) the host bacterium |
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mcs5109 Wed, 19 Nov 2008 05:46:16 GMT |
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| araBAD Operon |
-same regulatory proteins can be both activators and repressors
-w/ Arabinose, AraC dimer activates transcription.
-w/o Arabinose, AraC represses
-depends on [arabinose] |
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mcs5109 Wed, 19 Nov 2008 05:44:54 GMT |
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NtrC Works from DNA Sites Far from the Gene
??????????? |
-NtrC is an activator of s54-dependent transcription
-s54 holoenzyme itself not able to form open complex
-Under low nitrogen condition, NtrC is phosphorylated by NtrB, and binds upstream of DNA (150 bp). Binding site can be moved further w/o loss of RNAP activation ability.
After direct interaction w/ RNAP, using ATP hydrolysis energy, NtrC activates RNAP to make an open complex.
Some NtrC-dependent promoter need DNA architectural protein (e.g., IHF) between NtrC and RNAP binding sites. DNA looping enables NtrC-RNAP direct interaction. |
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mcs5109 Wed, 19 Nov 2008 05:44:54 GMT |
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| How MerR activates transcription |
-MerR activates merT gene expression
-effector: Hg2+
-merT promoter has an unusual 19 bp distance between -35 and -10 elements
-not optimally separated or aligned
-MerR protein binding merT promoter locks the promoter in this conformation
When Hg2+ binds MerR, the protein undergoes a conformational change that twists DNA to show the -35 and -10 elements on the same DNA surface |
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mcs5109 Wed, 19 Nov 2008 05:44:54 GMT |
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| bacteriophage SPO1 infects B. subtilis |
-3 sigmas cascade enables ordered gene expressions
-genes expressed in the order they're needed |
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mcs5109 Wed, 19 Nov 2008 05:31:55 GMT |
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| alternative sigmas |
-Expression of alternative s factor displaces housekeeping sigma (s70 in E. coli)
-ex: heat shock increases translation of s32--> displaces s70 and transcribes genes for thermostability
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mcs5109 Wed, 19 Nov 2008 05:31:55 GMT |
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| CAP and Lac repressor structural motif |
-helix-turn-helix
-recognition helix fits into major groove of DNA
-proteins bind as a homodimer to their DNA binding sites that contain inverted repeat sequences |
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mcs5109 Wed, 19 Nov 2008 04:46:54 GMT |
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| CAP |
-Catabolite activator protein
-activates the lac operon by recruiting RNAP to the promoter
-positive control mutation: binds DNA in one region, interacts w/ RNAP in another region of mutation
-binding controlled by cAMP (increases if no glucose) |
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mcs5109 Wed, 19 Nov 2008 04:46:54 GMT |
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| Lac operator |
-21 bp sequence is 2-fold symmetric
-overlaps with the promoter (RNAP binding site)
-Lac repressor binding is able to exclude RNAP binding to the promoter |
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mcs5109 Wed, 19 Nov 2008 04:37:19 GMT |
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| Lac repressor |
-binds lac operator (21 bp sequence is 2-fold symmetric)
-two subunits of Lac repressor binds on each half-site |
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mcs5109 Wed, 19 Nov 2008 04:37:19 GMT |
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| Allolactose |
-releases Lac repressor from DNA
-binds to Lac repressor and triggers conformational change
Expression of lac genes is leaky: even when they are repressed, an occasional transcript gets made.
w/o Allolactose: repressor binds operator tightly
w/ Allolactose: repressor can no longer bind DNA |
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mcs5109 Wed, 19 Nov 2008 04:48:52 GMT |
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| Lac Operon |
LacZ: b-galactosidase, cleaves lactose into galactose and glucose.
LacY: lactose permiase, transport lactose into the cell.
LacA: thiogalactoside transacetylase, rids the cell of toxic thiogalactoside which is transported by LacY.
These genes are expressed at high level in E. coli cells only when lactose is available and glucose is not in their growth media. |
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mcs5109 Wed, 19 Nov 2008 04:37:19 GMT |
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| Allostery Gene regulation |
-effector molecules induce conformational change of regulatory proteins that switch their DNA binding affinities
-ex: allolactose in Lac Repressor or cAMP in CAP |
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mcs5109 Wed, 19 Nov 2008 04:37:19 GMT |
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| Cooperative binding gene regulation |
-Group of regulators often bind DNA cooperatively
-2 or more activators and/or repressors interact with each other and with DNA.
Cooperative binding can produce sensitive switches (fully active or inactive)
Cooperative binding can also serve to integrate signals |
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mcs5109 Wed, 19 Nov 2008 04:37:19 GMT |
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| DNA looping |
-enables action of regulator binding far from the RNAP binding site
-b/w activator binding site and promoter
-"architectural proteins" |
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mcs5109 Wed, 19 Nov 2008 04:37:19 GMT |
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| MerR |
-activator that induces conformational change in promoter DNA |
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mcs5109 Wed, 19 Nov 2008 04:14:07 GMT |
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| NtrC |
-activator that interacts w/ RNAP bound in closed complex at the promoter |
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mcs5109 Wed, 19 Nov 2008 04:14:07 GMT |
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| Stable closed complex #s |
high KB but low kf, RNAP is not able to form open complex spontaneously |
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mcs5109 Wed, 19 Nov 2008 04:14:07 GMT |
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| Allosteric activators |
-Some activators interact w/ RNAP
-increase rate of open complex formation
-NtrC/MerR |
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mcs5109 Wed, 19 Nov 2008 04:14:07 GMT |
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| activation of transcription |
-Activator helps RNAP for binding to the promoter (KB) and for opening DNA (kf)
-binds to activator binding site |
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mcs5109 Wed, 19 Nov 2008 04:07:32 GMT |
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| operator |
-where repressor binds |
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mcs5109 Wed, 19 Nov 2008 04:07:32 GMT |
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| repression of transcription |
-Repressor blocks RNAP from binding to the promoter
-binds to operator
-Blocking is the most common repression mechanism in prokaryotes |
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mcs5109 Wed, 19 Nov 2008 04:07:32 GMT |
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| Basal level of transcription |
-transcription by RNAP itself
-in absence of regulatory proteins, RNAP binds only weakly
-constitutive level called basal level |
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mcs5109 Wed, 19 Nov 2008 04:07:32 GMT |
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| Promoter Regulators |
-basal level
-activators
-repressors |
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mcs5109 Wed, 19 Nov 2008 04:07:32 GMT |
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| Regulate the amount of protein |
Transcription (RNA synthesis)
RNA processing (capping, polyA addition, splicing)
RNA turnover
Translation (from RNA to protein)
Protein processing, assembly, turnover |
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mcs5109 Wed, 19 Nov 2008 04:07:32 GMT |
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| Regulate protein activity |
Allostery
Covalent modification
Localization |
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mcs5109 Wed, 19 Nov 2008 04:07:32 GMT |
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| regulate protein activity vs. amt |
Regulate protein activity:
Allostery
Covalent modification
Localization
Regulate Protein amt:
Transcription (RNA synthesis)
RNA processing (capping, polyA addition, splicing)
RNA turnover
Translation (from RNA to protein)
Protein processing, assembly, turnover |
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mcs5109 Wed, 19 Nov 2008 04:07:32 GMT |
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| To transcribe gene to RNA, RNAP has to: |
1) binds to promoter (KB)
2) forms an open complex (kf)
3) initiates transcription
4) escapes from the promoter
5) elongates
6) terminates transcription
All are potential targets for regulation |
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mcs5109 Wed, 19 Nov 2008 03:45:03 GMT |
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| What are genes controlled by? |
-extracellular signals
-these signals are communicated to genes by regulatory proteins |
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mcs5109 Wed, 19 Nov 2008 03:45:03 GMT |
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| Repressor |
Negative regulators, decrease or eliminate transcription of regulated genes. |
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mcs5109 Wed, 19 Nov 2008 03:45:03 GMT |
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| Activators |
Positive regulators, increase transcription of regulated genes. |
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mcs5109 Wed, 19 Nov 2008 03:45:03 GMT |
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