MICRO 303 Exam 3 - Regulation of Metabolism

environment

a microorganism has to respond to the ________, by changing enzymes, membranes, etc.

steps of regulation

1. signal that things have changed

2. signal recognized

3. changes transcriptional, translational, post-translation regulation

specific

Regulation needs to be ______

global

Regulation also has to plug into the _____ state of the cell

rapid

Regulation is typically ______, both on and off and much of it is tuned (never all the way off)

allosteric proteins

- contain allosteric site, which is separate from active site

- binds small molecules and changes conformation of the enzyme, which changes subsequent activity

mechanism of regulation

DNA

RNA

Protein

DNA in regulation

• Change access to genes via DNA structure

• DNA rearrangements

• DNA methylation

RNA in regulation

• Regulate whether mRNA is made (initiation, elongation and termination of transcription)

• Stability of RNA

• Binding of proteins, RNA or small molecules to mRNA to affect its translation

Proteins in regulation

• Affect translation and processing of protein

• Protein stability

• Protein function can be regulated by interactions with other proteins or small molecules and by covalent modification

regulation of transcription

- common: regulation of initiation by changing the activity of RNA polymerase

- regulation of elongation/termination

regulatory protein

has two domains:

- one binds DNA

- the other responds to allosteric signal and modifies activity

negative regulation

when regulator binds, there is a decrease in transcription

repressor

A protein that suppresses the transcription of a gene (negative reg.)

active repressor

- good promoter

- binds near promoter, which blocks it

- RNA pol cannot transcribe operon

inactivation of repressor

- repressor falls off of binding site

- promoter open

- RNA pol can proceed

induction (neg. regulation)

repressor is synthesized in an active state, which prevents transcription from the operon. Inducer binds (which is a small signal molecule), which causes it to fall off of the DNA

repression (neg. regulation)

repressor is synthesized in an inactive state. Co-repressor (small molecule) causes repressor activation. The binding site is located on DNA, and transcription stops

positive regulation

binding of an activator, which increases transcription. These are poor promoters, and the binding near the promoter allows protein-to-protein contact with RNA pol (which transcribes DNA)

MalT operon

activator protein, binds to activator binding site only when maltose is present, transcribes the genes (positive)

antisense RNA (regulation)

- small RNA that binds mRNA and disrupts translation

- cis/trans

cis antisense RNA

transcript directly from complementary strand of DNA

trans antisense RNA

transcript from separate gene

SymE

protein that degrades all mRNA

SymR

antisense RNA represses

SOS response

SymR inactive, SymE degrades mRNAs

riboswitches (regulation)

Small molecule that binds to RNA and disrupts translation/transcription-can turn gene expression OFF OR ON

ydhL gene

adenine efflux pump (too much is toxic to the cell), so if conc. is too high, pump gets turned on by riboswitch (anti-terminator), gene is expressed and excess adenine is pumped out of the cell

frameshift mutation

insertion or deletion of nucleotides where the number of bases added or removed is not divisible by 3

C

A frameshift mutation in the lacZ gene would cause

A. The microbe to grow slowly on lactose

B. The microbe will synthesize the lac enzymes constitutively

C. The microbe will be unable to grow on lactose

D. No effect

lacZ

encodes the enzyme B-galactosidase, which breaks down lactose into glucose and galactose

LacY

encodes lactose permease, and transmembrane symport protein crucial for transportin5g lactose into the cell alongside a proton

LacA

codes for the enzyme galactosidase acetyltransferase, which plays a role in breaking down lactose

C

There are mutations that can be isolated that will cause the

repressor to not bind its allosteric effector molecule, allolactose. The protein can still bind DNA. Such a mutation would cause

A. The microbe to grow slowly on lactose

B. The microbe will synthesize the lac enzymes constitutively

C. The microbe will be unable to grow on lactose

D. No effect

lac I

encodes the lac repressor (A DNA binding protein) that inhibits the transcription of the lac operon

C

The lac operon is an example of

A. Positive Regulation

B. Repression

C. Induction

D. Feedback Inhibition

E. Attenuation

D

A mutation that inactivates the repressor (LacI) would cause...

A. The microbe to grow slowly on lactose

B. The microbe will synthesize the lac enzymes constitutively

C. The microbe will be unable to grow on lactose

D. Very little effect

Trp operon

Anabolic pathway - Synthesizes tryptophan

Negative regulation - Repression (trpr) in inactive state

repressor - low tryptophan

synthesized in inactive state

trp R does not bind to operon

RNA pol binds promoter and begins mRNA synthesis

repressor-high tryptophan

Accumulation of tryptophan

binds to trp.R and activates repressor

blocks polymerase

B

Regulation of the trp operon by TrpR is an example of

A. Positive regulation

B.Repression

C.Induction

D.Feedback Inhibition

E.Attenuation

A

A frameshift mutation in trpA would

A. Make the cells unable to synthesize tryptophan

B. Would increase the synthesis of tryptophan

C. Would inactivate the trp repressor

D. Not enough information to tell

D

A deletion mutation of the trpR gene would

A.Would decrease the expression of the trp genes

B.Would cause constitutive expression of the trp genes

C.Would make the cells unable to grow in minimal medium

D.Not enough information to tell

attenuation

second layer of regulation often seen in amino acid biosynthesis operons

operates at the levels of transcription and translation

leader peptide

short sequence of amino acids

contains tryptophan codons

pauses

If concentration of tryptophan is low, ribosome ______ (at 1) cannot form Rho-independent = transcription

reads through

If concentration of tryptophan high, ribosome ______ _______ and pauses at end

C

If you deleted sequence #2 from the leader sequence in the trp operon....

A.Transcription of the trp operon would increase

B.Regulation would not be affected

C.Transcription of the trp operon would decrease

feedback inhibition

Signal molecule is again tryptophan

• Negative effector (tryptophan) stops anthranilate synthase activity

• First committed enzyme

post translational regulation (at level of ENZYMES)

C

The allosteric site of anthranilate synthase is altered, such that the

enzyme always behaves as if tryptophan is bound. This would

A.cause an excess of tryptophan synthesis

B.have no effect

C.cause a decrease in tryptophan synthesis

D.inactivate the enzyme

chorismate

used in all of the essential amino acids (see picture for feedback loops)

example of global regulation (bassd on three different amino acids)

global regulation

regulators affect activity of many genes all at once

genes not adjacent on chromosome

regulons (instead of operons)

global regulation examples

• Catabolite Repression

• Heat Shock

• Quorum Sensing

catabolite repression

Prioritizes glucose over lactose by suppressing the lac operon when glucose is present.

Low glucose increases cAMP, which activates the Catabolite Activator Protein (CAP) to bind the promoter, enabling high-level transcription only when lactose is present and glucose is absent

B

Under what conditions would expression of the lac operon be highest? A. no glucose, no lactose

B. no glucose, plus lactose

C. plus glucose, no lactose

D. plus glucose, plus lactose

cytoplasm

core RNA polymerase binds to sigma factor in _______, sigma factor directs RNAP to promoter

Alternative sigma factors

recognize different sets of promoters to control expression of specific groups of genes

heat shock response

response to high temperature that includes the synthesis of heat shock proteins together with other changes in gene expression

heat shock proteins

• Regulators

• Molecular chaperones

• Proteases

• Transcription and translation

• DNA repair

• Synthesis of biomolecules (Fe:S centers)

increase

HSP (heat shock proteins) present under all circumstances, but levels _________ at high temps

ethanol

HSP also induced under other stress conditions, such as _______ (solvents)

sigma-H

Master regulator of heat-shock response is _________ (also called rpoH)

translation of rpoH

low temp: secondary structure forms

high temp: secondary structure melts (mRNA translated faster!!!)

regulation by control of TSL initiation via RNA structure

degrade

DnaK/J, GroEL/S, FtsH _______ RpoH at high temps

away

Signal is unfolded or damaged proteins

• This titrates chaperones ______from RpoH

• Regulation by control of protein stability

A

If you removed the secondary structure of the rpoH gene, but did not change the actual codons, expression of heat shock genes would

A. Increase

B. Decrease

C. Stay the same

two-component regulators

- sensor kinase

- not always in membrane

- response regulators not always transcriptional regulators

sensor kinase

if signal is outside and needs to be transduced interiorly, sensor kinase (an enzyme that can phosphorylate something) will activate or repress transcription

quorum sensing

• Bacteria can sense their environment by diffusible signals

• In general, secrete small molecule into environment

Agr Quorum Sensing (Gram-Positive Bacteria)

It detects autoinducing peptides (AIPs) to transition from surface adhesion (biofilm formation) to the secretion of toxins and enzymes, enabling tissue invasion and infection spread

AgrD

prepropeptide, processed to autoinducing peptide (AIP)

gram positive

AgrA/AgrC

two component system - AgrC is the sensor kinase that recognizes AIP, phosphorylates AgrA, which activates transcription

gram positive

SarA

transcription factor

gram positive

III

RNA ______ made binds to other mRNA transcripts

RNA III

- activates/inhibits translation by base-pairing and relieving masks of Shine Dalgarno Sequence

- expressed when high populations/stationary phase

blocks

- RNAIII binds to Shine-Dalgarno region of rot mRNA and _______ translation

- The Rot protein is a negative regulator of many virulence genes

outcome of Agr regulation

-agr system is off in log phase and at lower pH

-increases in stationary phase or elevated pH

-AIP increases, leading to increase in RNAIII

-RNAIII affects expression of many virulence genes

B

If you mutated AgrB so that AgrB could transport, but not degrade AgrD, would the Agr system still work for quorum sensing?

A. Yes

B. No

C. Impossible to tell

Vibrio fischeri-Euprymna scolopes

-Hawaiian squid takes up bacteria into light organ

•Light provides protection for squid

•Bacteria grow and bioluminesce (only at night)

•Gain sugars, amino acids, peptides and proteins

•Light organ is highly selective - pure culture Vf

bioluminescence

luciferase

proteins to make fatty acids

proteins to make aldehyde

luciferase

• Reduces long-chain aldehyde with electrons

• Consumes oxygen

• Produces light

Lux operon

encodes genes for self-regulation and for the production of luminescent proteins

lux c, d, e

fatty acid synthesis

lux a, b

luciferase

lux G

flavin reductase

lux l, r

regulation

Autoinduction

a gene regulatory mechanism involving small, diffusible signal molecules that are produced in larger amounts as population size increases