For each, in a couple sentences summarize the findings and implications of the study.

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

Riordan 2013

*

*

Rationale for regulation

WHY REGULATE THE PRODUCTION OF GENES AND PROTEINS?

Ex. 1: Damage

Ex. 2: Catalysis

Riordan 2013

*

*

Types of gene expression

(Armache et al 2010)

Ex. housekeeping genes; some regulatory genes

CONSTITUTIVE

Riordan 2013

*

*

Ex. Structural, regulatory, accessory

Types of expression, cont’d

INDUCIBLE v. REPRESSIBLE

(Bacterial Infections and Immunity © 2016)

Riordan 2013

*

*

Types of expression, cont’d

Expression

Time

Riordan 2013

*

*

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

Riordan 2013

OUT

IN

*

*

Regulating gene expression, cont’d

TRANSDUCTION of exogenous signals

Ex. Two-component signal transduction (TCS)

(Modified from Fig. 10.3)

#1

#2

*

*

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

Riordan 2013

*

*

Basics of transcription regulation

Ex. 1 Transcription REPRESSION

(Modified from Fig. 10.1)

Riordan 2013

*

*

Types of expression, cont’d

Expression

Time

Riordan 2013

*

*

lac

Jacques Monod

ca. 1965

Andre Lwoff

Francois

Jacob

Riordan 2013

*

*

(biochemistry.es/)

Riordan 2013

*

*

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”

#1

#2

#2

Riordan 2013

*

*

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

Riordan 2013

*

*

The lac operon, cont’d

lacI encodes the repressor of PlacZYA, LacI

lacI is transcribed from PlacI

(Modified from Fig. 10.5)

Riordan 2013

*

*

The lac operon, cont’d

There are two lac operators that LacI binds for repression

lacO and lacOI

(Modified from Fig. 10.5)

Riordan 2013

*

*

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)

Riordan 2013

LacI dimers interact—bending the promoter region between lacO and lacOI

*

*

Riordan 2013

*

*

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

Riordan 2013

*

*

Metabolic regulator

cAMP-CRP

*

*

cAMP-CRP

Cyclic AMP (cAMP) is cyclized adenosine monophosphate

cAMP accumulates endogenously when nutrients (i.e. glucose) are sparse

AMP

cAMP

Riordan 2013

*

*

cAMP-CRP, cont’d

cAMP interacts with cAMP RECEPTOR PROTEIN (CRP)

Together they regulate hundreds of genes, including lacZYA

(Modified from Fig. 10.7)

Riordan 2013

*

*

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

Riordan 2013

X

*

*

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)

Riordan 2013

*

*

Reality check

Blue-white screening by lacZ α-COMPLEMENTATION

Riordan 2013

X-GAL

aka (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)

(© 2016 Thermo-Fisher)

*

*

Reality check, cont’d

Blue-white screening by lacZ α-COMPLEMENTATION

Riordan 2013

X-GAL

aka (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside)

(© 2016 Thermo-Fisher)

*

*

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)

*

*

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)

*

*

Reality check, cont’d

Step 3: Select “transformants” defective in X-gal hydrolysis

X

(© 2016 Thermo-Fisher)

*

*

Blue-white

screen

*

Basics of transcription regulation

Ex. 2 Transcription REPRESSION

(Modified from Fig. 10.1)

Riordan 2013

*

*

Types of expression, cont’d

Expression

Time

Riordan 2013

*

*

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

Riordan 2013

*

Riordan 2013

*

*

TrpR

trpEDCBA

In contrast, genes for anabolic pathways are regulated by repressors that bind pathway end-products

Repressible metabolic pathways

Riordan 2013

*

Tryptophan biosynthesis

Tryptophan is an aromatic amino acid used primarily in protein biosynthesis

It is synthesized from chorismate and glutamine

Riordan 2013

*

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)

*

Riordan 2013

*

*

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)

Riordan 2013

*

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

*

(Modified from Fig. 10.14)

Riordan 2013

*

*

Basics of transcription regulation

Ex. Transcription ACTIVATION

(Modified from Fig. 10.1)

Riordan 2013

*

*

the

ara operon

dual regulation of transcription

Riordan 2013

*

*

Arabinose metabolism

Arabinose is catabolized to D-xylulose-P, an intermediate in the pentose-phosphate shunt

Riordan 2013

(R. Schleif 2000)

*

*

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

Riordan 2013

*

*

The ara operon

Non-coding/regulatory

Riordan 2013

Structural genes (araC-araBAD)

Regulatory elements (PC; PBAD; araO1, araO2, araI1, araI2, CAP (aka cAMP-CRP box)

*

*

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)

Riordan 2013

*

*

ara regulation, cont’d

This shuts down transcription from PC and PBAD

(Modified from Fig. 10.13)

Riordan 2013

X

X

*

*

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)

*

*

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

Riordan 2013

*

Levels of regulation

(J. Lee)

Riordan 2013

*

*

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

Riordan 2013

*

*

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

Riordan 2013

*

*

Levels of regulation, cont’d

Regulation at the level of translation (initiation and rate): controlled by TIR, and sRNA

Riordan 2013

*

*

Levels of regulation, cont’d

Post-translational regulation is fast, but least efficient energetically: alterations in protein activity via cleavage, phosphorylation, methylation, acetylation &co

Riordan 2013

*

Levels of regulation, cont’d

Alterations in DNA sequence? (Ex. promoter inversion)

(van der Woude et al 2004)

Riordan 2013

*

*

(Hengge-Aronis 2002)

Levels of regulation, cont’d

A single gene can be regulated at multiple levels (Ex. rpoS)

Riordan 2013

*

*

Sigma factors

Riordan 2013

*

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

Riordan 2013

*

*

Sigma factors, cont’d

Riordan 2013

*

*

Text

Text

Sigma factors, cont’d

(F. Fang 2005)

Riordan 2013

*

*

Sigma factors, cont’d

The σS REGULON—a set of genes/operons that are unlinked, but functionally related

Riordan 2013

*

*

Regulatory RNA

Riordan 2013

*

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)

Riordan 2013

*

*

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)

Riordan 2013

*

*

Quorum sensing

Riordan 2013

*

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

Riordan 2013

*

*

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

Riordan 2013

*

*

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

Riordan 2013

*

*

Recap

Levels of regulation; trade-offs

Sigma factors; regulons

Regulatory RNA; sRNA mechanism

Quorum sensing; autoinducers

Riordan 2013

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

*

“Get 15% discount on your first 3 orders with us”
Use the following coupon
FIRST15

Order Now