Assignment 1:Identify from two independent sources (ex. research paper, news, blog, etc.) two articles discussing the role for genetic transfer in bacteria in a topic of your interest (ex. human health, or industry). For each, in a couple sentences summarize the findings and implications of the study.
Be sure to include proper citation/referencing. Good luck.
Assignment 2:Identify a single regulation factor (could be a protein regulator like LacI, or a small regulatory RNA). In a few sentences describe what it regulates, how it regulates and the biological significance of this regulation. Do not include any regulators we have discussed in class.
Be sure to include proper citation/referencing. Good luck.
Assignment 3:The metabolic abilities of prokaryotes are vastly more diverse than those of eukaryotes. Identify 3 metabolic pathways that are unique to prokaryotes (i.e. are NOT present in eukaryotes). In one or two sentences, describe the reactants and products of each metabolic pathway.
Please include a citation (reference) for each.
(G. Hoffmann, 2012)
Prokaryotic gene regulation
MCB 3020
Ch. 10
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Rationale for regulation
WHY REGULATE THE PRODUCTION OF GENES AND PROTEINS?
Ex. 1: Damage
Ex. 2: Catalysis
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Types of gene expression
(Armache et al 2010)
Ex. housekeeping genes; some regulatory genes
CONSTITUTIVE
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Ex. Structural, regulatory, accessory
Types of expression, cont’d
INDUCIBLE v. REPRESSIBLE
(Bacterial Infections and Immunity © 2016)
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Types of expression, cont’d
Expression
Time
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Regulating gene expression
Bacteria use regulatory proteins to “sense” and respond to conditions within (ENDOGENOUS) and outside (EXOGENOUS) of the cell
These protein REGULATORS then alter gene expression accordingly
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IN
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Regulating gene expression, cont’d
TRANSDUCTION of exogenous signals
Ex. Two-component signal transduction (TCS)
(Modified from Fig. 10.3)
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Regulating gene expression, cont’d
Regulators bind regulatory sequences, and can be REPRESSORS or ACTIVATORS (or both!)
(Modified from Fig. 10.1)
Regulatory sequences used for repression are referred to as OPERATORS; ACTIVATION SEQUENCES are for activators
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Basics of transcription regulation
Ex. 1 Transcription REPRESSION
(Modified from Fig. 10.1)
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Types of expression, cont’d
Expression
Time
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lac
Jacques Monod
ca. 1965
Andre Lwoff
Francois
Jacob
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(biochemistry.es/)
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Basics of lactose metabolism
(Modified from Fig. 10.6)
Products of the lac operon are required for lactose uptake and catabolism
lacY (lactose permease) & lacZ (B-galactosidase)
“B-gal”
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The lac operon
lacZYA encompass the lac operon (tricistron)
OPERONS are groups of genes COTRANSCRIBED from a common promoter (i.e. PlacYZA)
(Modified from Fig. 10.5)
lacZYA constitute a single TRANSCRIPIONAL UNIT
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The lac operon, cont’d
lacI encodes the repressor of PlacZYA, LacI
lacI is transcribed from PlacI
(Modified from Fig. 10.5)
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The lac operon, cont’d
There are two lac operators that LacI binds for repression
lacO and lacOI
(Modified from Fig. 10.5)
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lac repression
When lactose is absent, lac operon expression is not needed:
PlacZYA must therefore be repressed!
To achieve this, LacI binds to both lacO & lacOI operators
(Modified from Fig. 10.5)
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LacI dimers interact—bending the promoter region between lacO and lacOI
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lac induction
When lactose is present, it is co-transported (Lac + H+) by LacY permease into the cell
B-gal catalyzes transgalactosylation of lactose to ALLOLACTOSE ligand
(Modified from Fig. 10.5)
LacI is displaced, and RNAP gains access to PlacZYA “inducing” lac
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Metabolic regulator
cAMP-CRP
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cAMP-CRP
Cyclic AMP (cAMP) is cyclized adenosine monophosphate
cAMP accumulates endogenously when nutrients (i.e. glucose) are sparse
AMP
cAMP
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cAMP-CRP, cont’d
cAMP interacts with cAMP RECEPTOR PROTEIN (CRP)
Together they regulate hundreds of genes, including lacZYA
(Modified from Fig. 10.7)
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cAMP-CRP, cont’d
At cAMP-CRP regulated promoters, RNAP cannot easily initiate transcription
The aCTD domain of RNAP at these promoters tethers the complex
(Modified from Fig. 10.7)
Binding of cAMP-CRP complex releases the tether, allowing for transcription
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Full induction of lac requires (I) LacI binding to allolactose; and (II) loading of cAMP-CRP complex; and (III) interaction of cAMP-CRP with aCTD of RNAP
cAMP-CRP & lac
(Modified from Fig. 10.8)
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Reality check
Blue-white screening by lacZ α-COMPLEMENTATION
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X-GAL
aka (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)
(© 2016 Thermo-Fisher)
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Reality check, cont’d
Blue-white screening by lacZ α-COMPLEMENTATION
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X-GAL
aka (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)
(© 2016 Thermo-Fisher)
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Reality check, cont’d
Blue-white screening by lacZ α-COMPLEMENTATION
Step 1: Ligate foreign DNA (“insert”) into plasmid vector lacZ locus at multiple-cloning site (MCS)
(© 2016 Thermo-Fisher)
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Reality check, cont’d
Blue-white screening by lacZ α-COMPLEMENTATION
Step 2: Transform recombinant vector with disrupted lacZ gene into lacZ null E. coli
(© 2016 Thermo-Fisher)
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Reality check, cont’d
Step 3: Select “transformants” defective in X-gal hydrolysis
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(© 2016 Thermo-Fisher)
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Blue-white
screen
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Basics of transcription regulation
Ex. 2 Transcription REPRESSION
(Modified from Fig. 10.1)
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Types of expression, cont’d
Expression
Time
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LacI
lacZYA
Repressor proteins that control catabolic pathways typically bind the initial substrate/analog of the pathway
This leads to “induction” of the pathway for its utilization
Repressible metabolic pathways
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TrpR
trpEDCBA
In contrast, genes for anabolic pathways are regulated by repressors that bind pathway end-products
Repressible metabolic pathways
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Tryptophan biosynthesis
Tryptophan is an aromatic amino acid used primarily in protein biosynthesis
It is synthesized from chorismate and glutamine
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trp operon regulation
trp is a five gene operon (trpEDCBA)
trpR and trpEDCBA are divergently transcribed
In the absence of tryptophan, the trp operon is expressed from PtrpEDCBA and PtrpR
(Modified from Fig. 10.14)
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TrpR
The TrpR repressor protein exist as an inactive APOREPRESSOR
Conversion to an active repressor requires binding of its cognate COREPRESSOR, tryptophan (Trp)—yielding a holorepressor
Aporepressor
Co-repressor(Trp)
Holorepressor
(Modified from Fig. 10.14)
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trp operon regulation, cont’d
As endogenous tryptophan accumulates, it binds TrpR as a co-repressor to yield the TrpR holorepressor
Binding of TrpR to trpO (operator) interferes with RNAP binding at PtrpEDCBA, thus reducing expression
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(Modified from Fig. 10.14)
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Basics of transcription regulation
Ex. Transcription ACTIVATION
(Modified from Fig. 10.1)
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the
ara operon
dual regulation of transcription
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Arabinose metabolism
Arabinose is catabolized to D-xylulose-P, an intermediate in the pentose-phosphate shunt
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(R. Schleif 2000)
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AraC dual regulator
AraC is the prototype for the family of AraC/XylS regulators
Thousands of members in bacteria; regulate many discrete biological phenomena (ex. Table 10.1)
(Modified from Fig. 10.13)
The AraC dimer can form two conformations based on ligand (arabinose) binding
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The ara operon
Non-coding/regulatory
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Structural genes (araC-araBAD)
Regulatory elements (PC; PBAD; araO1, araO2, araI1, araI2, CAP (aka cAMP-CRP box)
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ara regulation
In the absence of arabinose, AraC represses expression of the genes for arabinose utilization (araC-araBAD)
The AraC dimer w/o arabinose is rigid and elongated; C-terminal ends bind araO2 and araII
?
(Modified from Fig. 10.13)
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ara regulation, cont’d
This shuts down transcription from PC and PBAD
(Modified from Fig. 10.13)
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ara regulation, cont’d
In the presence of arabinose, AraC activates expression of the genes for arabinose utilization (araC-araBAD)
The AraC dimer w/ arabinose assumes a compact form; C-terminal ends bind araI1 and araI2
Transcription from PC and PBAD ensues
(Modified from Fig. 10.13)
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Recap
Regulatory protein repressors and activators
Paradigms of LacI, TrpR, and AraC
Operons
Paradigms of lac, trp and ara; structural and regulatory features; function
cAMP-CRP
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Levels of regulation
(J. Lee)
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Levels of regulation
Expression of genes/operons can be regulated at multiple levels
Different levels of control confer different advantages
Levels include:
DNA sequence
Transcription
RNA/transcript stability
Translation
Post-translational control
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Levels of regulation, cont’d
Regulation at the level of transcription/stability is the most efficient, but response is slow: controlled by protein regulators, sigma factors, RNase, and sRNA
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Levels of regulation, cont’d
Regulation at the level of translation (initiation and rate): controlled by TIR, and sRNA
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Levels of regulation, cont’d
Post-translational regulation is fast, but least efficient energetically: alterations in protein activity via cleavage, phosphorylation, methylation, acetylation &co
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Levels of regulation, cont’d
Alterations in DNA sequence? (Ex. promoter inversion)
(van der Woude et al 2004)
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(Hengge-Aronis 2002)
Levels of regulation, cont’d
A single gene can be regulated at multiple levels (Ex. rpoS)
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Sigma factors
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Sigma factor regulation
Bacteria often need to upregulate large sets of genes/operons under specific conditions (i.e. stress, host response &co)
One way to achieve this is to control the expression or activity of RNAP sigma factors
Sigma factors are the dissociable subunits of RNAP that specify transcription from promoters
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Sigma factors, cont’d
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Text
Text
Sigma factors, cont’d
(F. Fang 2005)
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Sigma factors, cont’d
The σS REGULON—a set of genes/operons that are unlinked, but functionally related
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Regulatory RNA
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Regulatory RNA, cont’d
Some untranslated RNA have regulatory functions
Some tend to be small (100-200 nucleotides)
aka (small regulatory RNA/sRNA) (Table 10.2)
Have direct complementarity with endogenous mRNA target
mRNA
sRNA
Many are encoded intergenic; some are cis-antisense RNA (asRNA)
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sRNA regulates the stability of target mRNA by binding to them; forming an sRNA:mRNA duplex
This either increases or decreases target mRNA stability
Regulatory RNA, cont’d
sRNA
mRNA
sRNA
mRNA
(Modified from Fig. 10.21)
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Quorum sensing
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Quorum sensing
Quorum sensing refers to a process where bacteria act in a cooperative manner by communicating using small secreted molecules at high cell density
It was first discovered in V. fischeri, a bioluminescent bacterium that colonizes the light organ of bobtail squid
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QS requires pheromones (aka autoinducers) produced endogenously
Autoinducers can interact with regulatory proteins as a ligand
Acyl homoserine lactone (AHL) is a common QS autoinducer
Quorum sensing, cont’d
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For V. fischeri, AHL is the autoinducer and LuxR is the protein regulator
LuxR-AHL activates transcription of genes conferring bioluminescence
Quorum sensing, cont’d
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Recap
Levels of regulation; trade-offs
Sigma factors; regulons
Regulatory RNA; sRNA mechanism
Quorum sensing; autoinducers
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