Study BMB 400 Chap 16 Flash Cards

 
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BMB 400 Chap 16

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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
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.
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.
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)
Lysogenic induction
switch from lysogenic to lytic growth
Bacteriophage gamma
-both lysis and lysogeny
Lysogeny (prophage)
After infection, the phage DNA integrates into the host genome and resides there passively
Lysis
Infection by phage produces many progeny and breaks open (lyses) the host bacterium
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]
NtrC Works from DNA Sites Far from the Gene

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-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.
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
bacteriophage SPO1 infects B. subtilis
-3 sigmas cascade enables ordered gene expressions
-genes expressed in the order they're needed
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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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
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
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)
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
Lac repressor
-binds lac operator (21 bp sequence is 2-fold symmetric)
-two subunits of Lac repressor binds on each half-site
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.
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
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
DNA looping
-enables action of regulator binding far from the RNAP binding site
-b/w activator binding site and promoter
-"architectural proteins"
MerR
-activator that induces conformational change in promoter DNA
NtrC
-activator that interacts w/ RNAP bound in closed complex at the promoter
Stable closed complex #s
high KB but low kf, RNAP is not able to form open complex spontaneously
Allosteric activators
-Some activators interact w/ RNAP
-increase rate of open complex formation
-NtrC/MerR
activation of transcription
-Activator helps RNAP for binding to the promoter (KB) and for opening DNA (kf)
-binds to activator binding site
operator
-where repressor binds
repression of transcription
-Repressor blocks RNAP from binding to the promoter
-binds to operator
-Blocking is the most common repression mechanism in prokaryotes
Basal level of transcription
-transcription by RNAP itself
-in absence of regulatory proteins, RNAP binds only weakly
-constitutive level called basal level
Promoter Regulators
-basal level
-activators
-repressors
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
Regulate protein activity
Allostery
Covalent modification
Localization
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
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
What are genes controlled by?
-extracellular signals
-these signals are communicated to genes by regulatory proteins
Repressor
Negative regulators, decrease or eliminate transcription of regulated genes.
Activators
Positive regulators, increase transcription of regulated genes.
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