micro exam ch 7-8

Gene expression

Gene expression

How genetic information stored in a gene is transcribed and translated into a protein

Gene regulation

The cell conserves energy by only making proteins that are needed at a particular time

Constitutive genes

(also called housekeeping) are expressed all the time and 75% of bacterial genes are constitutive genes

Regulated genes

(also called facultative or inducible) are only transcribed and translated when the cell needs them. This allows the cell to conserve energy

Gene expression controls

controls the amount of an enzyme synthesized

Regulate level of transcription

Regulate translation - Slower process (minutes)

Protein regulators regulate gene expression

Enzyme regulation controls

The activity of preexisting enzymes

feedback inhibition

Allosteric activation or allosteric inhibition

rapid process (seconds)

Negative transcription control

(prevents transcription) uses repressor proteins to repress transcription

Positive transcriptional control

uses activator proteins to induce expression

Typically anabolic reactions are

repressed

Typically catabolic reactions are

induced

Repressible system

can be turned off

the end protein can go back and stop transcription (anabolic)

Ex. arginine synthesis

the repressor protein is inactive until the co-repressor turns it on and inhibits the biosynthesis of the enzyme.

inducible system

catabolic

ex sugar pathway (lac operon)

repressor protein is activated and shuts the inhibitor off, activating the pathway

Co-repressors and inducers

“effector” molecules are often the products (anabolic) or substrates (catabolic) of a particular pathway

Transcription in bacteria

Sigma recognizes the promoter and initiation site

transcription begins; sigma released, RNA chain grows

Termination site is reached; chain growth stops

Polymerase and RNA released

Inverted repeats

specific binding sites for regulatory proteins

Homodimeric proteins

proteins composed of two identical polypeptides

Structure of DNA-Binding proteins

Helix-turn-helix two alpha helices connected by a short “turn” sequence

First helix is the recognition helix (interacts specifically with DNA)

Second helix is the stabilizing helix, (interacts with the first helix through hydrophobic interactions)

many different DNA-binding proteins from bacteria contain

helix-turn-helix

lac and trp repressors of E. coli

outcomes after DNA binding

DNA-binding protein may catalyze a specific reaction on the DNA molecule

The binding event can activate transcription (positive regulation)

The binding event can block transcription (negative regulation)

Inducer

substance that induces enzyme synthesis

corepressor

substance that represses enzyme synthesis

Effectors

collective term for inducers and co-repressors

(typically small molecules that can be structural analogs of substrates(inducer)/products(repressor))

lac operon model

first proposed by Francois jacob and jacques monod

based on enzymes needed for lactose catabolism in E. Coli

became known as the lac operon model of gene expression

(has both positive and negative control)

operon

cluster of consecutive genes whose expression is under control of a single promoter/operator

Negative control

a regulatory mechanism that stops transcription

repression

preventing the synthesis of an enzyme in response to sufficient amounts of a product-specific effect

• widespread as control for amino acid and nucleotide precursors

• usually final product of a biosynthetic pathway represses enzymes

• usually affects biosynthetic/anabolic enzymes

repressors role is inhibitory, so it is called negative control

When RNA polymerase is bound to the promotor region in the arginine operon and the repressor is alone

transcription proceeds

when RNA polymerase is bound to the promotor region in the arginine operon and the repressor has a co-repressor

transcription is blocked

induction

production of an enzyme in response to presence of substrate

(typically affects catabolic enzymes

ensures enzymes are synthesized only when needed)

When RNA polymerase is bound to the promotor region in the lactose operon and the repressor is alone

transcription is blocked

when RNA polymerase is bound to the promotor region in the lactose operon and the repressor has a co-repressor

transcription proceeds

positive control

regulator protein activates the binding of RNA polymerase to DNA

(binds to the activator-binding site not operator)

RNA polymerase cannot bind to the promotor region in the maltose operon without first binding to what

the maltose activator protein with an inducer (maltose)

Maltose activator protein can not bind to DNA unless it first binds to

maltose (inducer)

Activator protein helps RNA polymerase recognize promoter in the maltose operon to initiate

transcription

activator proteins interact with DNA and/or RNA polymerase to influence

transcription

regulon

multiple operons are controlled by the same regulatory protein

global control systems

regulate expression of many different genes simultaneously

catabolite repression is an example of

global control

(controls use of carbon sources if more than one present)

(synthesis of unrelated catabolic enzymes is repressed if glucose is present in growth medium)

(also called the glucose affect)

(ensures that the best carbon and energy source is used first)

Cyclic AMP and cyclic AMP receptor protein

in catabolite repression, transcription is controlled by

cyclic AMP receptor protein, an activator protein, and is a form of positive control

CRP binds to DNA only if it has bound cAMP first

cyclic AMP formation is inhibited by

glucose

For lac genes to be transcribed

cyclic AMP level must be high enough for CRP protein to bind to CRP-binding site (no glucose) positive control

Lactose or another inducer must be present to prevent lactose repressor (Lacl) binding. negative control

Binding of CRP recruits RNA polymerase which initiates transcription followed by translation. The Lacl (active repressor/deactivates negative control) does what

active repressor binds to operator and blocks transcription or binds to an inducer which inactivates the repressor

Prokaryotes regulate cellular metabolism in response to

environmental fluctuations

(external signal may be transmitted directly to the target or

external signal may be detected by sensor and transmitted to regulatory machinery (signal transduction))

most signal transduction systems are

two-component regulatory systems

(especially in prokaryotes, phosphate can be a good transfer group)

Two-component regulatory systems are made up of which two proteins

sensor kinase (in cytoplasmic membrane) and response regulator (in cytoplasm)

sensor kinase (in cytoplasmic membrane)

detects environmental signal and autophosphorylates (found in cytoplasmic membrane whether gram - or + bacteria)

Response regulator (in cytoplasm)

DNA binding protein that regulates transcription (may be repressor or an activator)

What do two-component regulatory systems contain besides the two different proteins

a feedback loop

feedback loop in two-component regulatory systems

terminates signal and uses phosphatase that removes phosphate from response regulator

OmpF and OmpC are regulated by a two-component system

OmpF and OmpC are porins

A sensor kinase (EnvZ) detects changes in osmotic pressure

Osmotic pressure low = high OmpF - most concentrated

Osmotic pressure high = high OmpC and low OmpC

Osmolarity shift causes EnvZ autophosphorylation by histamine which phosphorylates what

ompR is phosphorylated by EnvZ which regulates transcription of OmpF and OmpC

sensor kinase

(EnvZ) detects changes in osmotic pressure

EnvZ is a sensor kinase that detects osmosis and is autophosphorylated on the Histidine residue which phosphorylates

OmpR which is a response regulator that influences OmpF/OmpC

Osmolarity low = OmpF turned up

Osmolarity high = OpC activated

Chemotaxis

moving towards stimulate or away from harm

modified two-component system used in chemotaxis to

sense temporal changes in attractants or repellents

regulate flagellar rotation (spin, powered by proton motive force)

regulate activity of preexisting proteins (flagellum proteins) instead of modifying transcription of genes

(if nutrients decrease, tumble then run)

Regulation of chemotaxis, response to signal

sensory proteins (not kinases) in cytoplasmic membrane sense attractants and repellents, and interact with cytoplasmic sensory kinases

methyl-accepting chemotaxis proteins (MCPs) - in membrane, kinase in cytoplasm

bind attractant or repellent and initiate flagellar rotation (tumble or run)

interact with CheA (sensor kinase) and CheW - works with MCPs

Regulation of chemotaxis - controlling flagellar rotation

controlled by CheY protein (response regulator)

CheY results in counterclockwise rotation (CheY phosphorylated) and runs (not phosphorylated)

CheY-P results in clockwise rotation and tumbling

Regulation of chemotaxis decrease attractant

Decreased attractant binding to MCP triggers phosphorylation of the CheA-CheW complex

CheA-CheW phosphorylate CheY and CheB

MCP is both methylated and demethylated

CheZ dephosphorylates CheY-P

CheY results in counterclockwise rotation and runs

CheY-P results in clockwise rotation and tumbing.

MCP senses a decrease in attractant triggers

CheW which helps stimulate CheA

CheA can either autophorsphorylate (kinase) as a response regulator or phosphorylate CheB

CheW-CheA help phosphorylate CheY

CheY can either bind to the flagellar switch to stimulate flagellum rotation clockwise causing a tumble, or be dephosphorylated by CheZ

When CheA is not present, CheY runs

Adaptation

stop responding and reset

Example of adaptation (feedback loop)

allows the system to reset itself to continue to sense the presence of a signal

relies on response regulator CheB

involves modification of MCPs: methylation stops response to attractants and increases response to repellants

phototaxis

movement toward light - light sensor replaces MCPs

Aerotaxis

movement toward oxygen - redox protein monitors oxygen level

Quorum sensing

mechanism by which bacteria and some archaea assess their population density

ensures that a sufficient number of cells are present before initiating a response that, to be effective, requires a certain cell density

Each species of bacterium produces a specific autoinducer signaling molecule that does what

Diffuses freely across the cell envelops

reaches high concentrations inside cell only if many cells are nearby and making the same autoinducer

binds to specific activator protein or sensor kinase, triggering transcription of specific genes

Virulence factors

secrete small peptides that damage host cells or alter hosts immune system

under control of autoinducing peptide

autoinducing peptide (AIP)

activates several proteins that lead to production of virulence proteins

Quorum-sensing disruptors are potential drugs for what

dispersing biofilms and preventing virulence gene expression

Stringent response

used to survive nutrient deprivation, environmental stress, and antibiotics

Stringent response does what

shuts down macromolecule synthesis and activates stress survival pathways

Stringent response in Escherichia coli

if shifted down from amino acid excess to limitation, rRNA and tRNA synthesis stopped and no new ribosomes are produced.

Protein and DNA synthesis stop, but new amino acids are biosynthesized

Later, rRNa synthesis and new ribosome production begins again at a slower rate

what is the Stringent response triggered by

regulatory nucleotides: the alarmones

Stringent response in Escherichia coli

Growth in rich medium shifts down and flat is the stringent response on a graph

Stringent response activated with uncharged tRNA

• rRNA, tRNA synthesis decreased

• Amino acid biosynthetic operons activated

• Cell division arrested

  • Stress survival pathways activated

Heat shock response with heat shock proteins

counteract damage of denatured proteins and help cell recover from temperature stress

• very ancient proteins

• induced by heat, exposure to ethanol or UV radiation

• Three major classes: Hsp70 (DNAK in E coli), Hsp60 (GroEL in E coli) and Hsp10 (GroES in E coli)

Largely controlled by alternative sigma factor RpoH

The rate of RpoH degradation depends on what

level of free DnaK

Proteins unfold at high temperature

DNAK binds and refolds protein

At low temperatures degradation of RpoH is done by protease and RpoH is released

RpoH is free to transcribe heat shock genes

General stress response

RpoS regulation

• Allows cells to withstand harsh conditions

• controlled by alternative sigma factor RpoS (stationary phase sigma factor)

  • RpoS regulon includes 400+ genes: nutrient limitation, resistance to DNA damage, biofilm formation, responses to osmotic, oxidative, and acid stress

Bacteria cell cycle binary fission

Chromosome replication

Segregation of chromosome

formation of septum

DNAA binding to specific sequences within oriC region leads to unwinding and loading of replisome

most active when linked to ATP (DNAA-ATP)

Regulated by inactivation of DNAA-ATP, competition for oriC binding, and repression of DNAA expression

After replication initiation only the parental strand is methylated, yielding

hemimethylated DNA - facilitates competition for origin binding between DNAA-ATP and SeqA protein

Hemimethylated oriC is strongly bound by SeqA, blocking DNAA-ATP

Newly synthesized daughter strands from binding of oriC by DNAA and SeqA proteins become methylated when

approximately 10 minutes after replication

Genome replication in fast-growing cells

Circular genome replication is bidirectional from origin

E coli’s genome replication takes 40 minutes but is independent of generation time

Multiple DNA replication forks present in each cell

If doubling time is shorter than genome replication time, so a new round begins before previous rounds are completed, some genes are present in multiple copies

The divisome houses what

several essential proteins called fts proteins that form the divisome

FtsZ protein is crucial in binary fission

Related to tubulin (eukaryotic cell-division protein)

Also found in virtually all Archaea

In rod-shaped cells, formation begins with attachment of FtsZ molecules around center of cell in a ring that becomes cell-division plane

Ring attracts other divisiome proteins including FtsA and ZipA

Divisome forms about ¾ the way into cell divison

ZipA

anchor that connects FtsZ ring to cytoplasmic membrane

FtsA

related to actin; recruits FtsZ and other divisome proteins and helps connect FtsZ ring to membrane

The divisome also contains Fts proteins needed for what

peptidoglycan synthesis

FtsL

penicillin-binding protein (activity inhibited by penicillin antibiotic)

The divisome orchestrates synthesis of what

new cytoplasmic membrane and cell wall material and then divides

Min proteins and cell division

Dna replicates before the FtsZ ring forms (ring forms between nucleoids)

• Before nucleoids segregate, they block formation of FtsZ ring (nucleoid occlusion)

MinC, MinD, and MinE proteins guide FtsZ to cell midpoint instead of poles

FtsK and other proteins mediate separation of chromosomes to daughter cells

FtsZ depolymerizes, triggering inward growth of wall materials to form septum

FtsZ also hydrolyzes GTP to provide energy for polymerization and depolymerization of FtsZ ring

MinCD spral oscillates across the long axis of the cell why

to inhibit FtsZ ring formation

MinE also oscillates from pole-to-pole and segregates MinCD to the poles and away from the center why

to allow formation of FtsZ ring in the center of the cell

Prokaryotes contain a cell cytoskeleton that is what

dynamic and multifaceted

Cell shape and MreB

Major shape-determining factor in Bacteria and a few archaea

forms simple cytoskeleton with patch-like filaments around inside of cell just below cytoplasmic membrane

recruits other proteins for cell wall growth to group into a specific pattern

Inactivation causes cells to become cocci

Most coccoid bacteria lack MreB

Filaments move from one side to another, localizing synthesis of peptidoglycan and allowing new cell wall to form at several points

Crescentin

Shape-determining protein found in vibrio-shaped Caulobacter

organizes into filaments ~10 nm wide that localize on concave face of the curved cells

thought to impart curved morphology

similar proteins found in other curved cells

Steps in peptidoglycan biosynthesis

preexisting peptidoglycan needs to be temporarily severed to allow newly synthesized peptidoglycan to form

In cocci, cell walls grow in opposite directions outward from the FtsZ ring

In rod-shaped cells, cell wall growth occurs at several points along length of the cell

Must synthesize new peptidoglycan and export it outside the cytoplasmic membrane

Insertion of new peptidoglycan requires

controlled cutting of existing peptidoglycan and simultaneous insertion of precursors