Microbiology (Lectures 10-18)

DNA binding proteins

homodimeric; bind at inverted repeats on DNA to control gene activity, repair chromosomes, or copy/fix genetic material; interact with nucleic acids (specificity due to R-groups)

domains

functional region of protein, divisions of polypeptide subunit

Helix-turn helix

two polypeptide chains in alpha helix, connected by short seq (turn —> 3 amino acids)

first helix

recognition helix, binds to DNA

second helix

stabilizing helix, stabilizes other helix via hydrophobic interactions

transcription factors

proteins that bind to specific sequences of DNA and control the rate of gene transcription

activator proteins

transcription factors that bind to the promoter region of DNA and enhances transcription

repressor proteins

transcription factors that bind to the operon region of the DNA to prevent RNA polymerase from binding and decreases transcription

co-repressor

molecule that binds to a repressor, changes conformation, and makes it stick to the operator

inducer

induces transcription by removing repressor or activating activator

Effector molecules

control ability of activator and repressor proteins to bind to DNA

operon

group of genes that are transcribed as a single mRNA and controlled by one promoter; all genes required for metabolic processes found here

regulon

more than one operon is under the control of a single regulatory protein (sigma or transcription factor)

sRNA

non-coding RNA molecules that can base pair to target mRNA and block transcription (open RBS to facilitate translation); can also increase or decrease mRNA stability

trans-sRNA

encoded in DNA; target mRNA with limited complimentarity

Hfq

protein that facilitates interaction between trans-sRNAs and target mRNA (binds to both)

Two component regulatory system

comprised of a sensor kinase protein and response regulator protein

sensor kinase

integral membrane protein with the ability to transfer a phosphoryl group onto another protein

response regulator protein

DNA-binding protein in the cytoplasm, activated by transfer of phosphoryl group from sensor kinase (enhance)

feedback loop

phosphotase removes phosphate —> dephosphorylated state causes system to reset

Catabolite repression

controls the use of carbon sources (E. coli prefers glucose over other carbon sources, grows faster when consuming glucose then lag phase follows)

heat shock response

protect cells from protein denaturation

heat shock proteins

counter protein damage and help cell recover from stress, 5 major classes

Hsp70 = DnaK

prevents aggregation if newly synthesized proteins and stabilizes unfolded proteins

Hsp60 & Hsp10

molecular chaperones that correctly fold mis-folded proteins

Hsp100

proteases that degrade denatured or irreversibly aggregated proteins

Hsp90

molecular chaperone that works to refold denatured proteins

RpoH

modulates the expression of genes encoding heat shock proteins; degraded quickly at lower temperatures

chemotaxis

sense/respond to nutrient limitations and toxin accumulation by moving away from repellants or towards attractants

MCP bound to attractant

unmethylated

MCP bound to repellant

methylated

CheR

responsible for methylation

CheB

response regulator that can demethylate MCPs when phosphorylated; how system resets after responding to stimulus

Quorum sensing

allows bacteria to detect each other vis autoinducers, decide when to engage in group behaviors

Acyl-homoserine lactone

lipid soluble molecule that can diffuse across membrane and binds to activator protein; also responds to QS from other bacteria

High stress & high AI-3 levels

sensor kinases bind and autophosphorylate —> transcriptional regulators become phosphorylated —> activate expression of toxin genes (recognizes both bacterial and eukaryotic cells)

nutritional auxotroph

mutation occurs in essential gene for biosynthesis enzyme, mutant can no longer synthesize and requires a growth medium

exonuclease

DNA Pol III uses this to remove the last base and add in the correct one

fidelity

mistake rates for different DNA Polymerases

Reversion

mutant phenotype can go back to its original phenotype

same-site revertant

A → G → A (true revertant), A → C → G (amino acid change but phenoype restored)

second-site revertant

mutation in a different place in genome compensates for original defect

error-free repair

deals with mismatched bases or chemically modified bases, complementary DNA strand is there to direct perfect repair

error-prone repair

coping with massive DNA damage, some repair is better than no repair

MutHSL

recognizes mismatched base pair, clips backbone of non-methylated strand, removes a piece of one strand, DNA Pol I repairs and ligase closes

Excision repair

recognizes modified bases and removes them [UvrAB cuts out strand of DNA with thymine dimers (sun damage)]

SOS response

stop cell division and metabolism as a whole; enhanced excision repair (uvrAB)

Translesion repair

repair with no DNA template as a guide using error-prone DNA polymerase (Pol V)

transposons

DNA that can hop around the genome

transposase

enzyme that mediates excision and re-insertion (coded for between inverted repeats)

screen

look at phenotype of every bacterium and pick up those with a phenotype of interest

Cofactors

micronutrient-trace elements found in these enzymes, include Fe

Coenzymes

non-protein molecule that contributes to enzyme function

Growth factors

vitamins, amino acids

defined media

precise chemical composition known

complex media

composed of digests of chemically undefined substances (yeast, meat extract)

MacConkey agar

selective for gram - bacteria and differential for lactose fermenters (pH indicator)

Great plate anomaly

direct microscopic counts of samples are much higher than viable counts (~10-1000X)

turbidity

optical density, absorbance; light scattering by cells

spectrophotometry

lower sensitivity and higher wavelength = more accurate; cell shape affects light scattering so make a standard curve to estimate cell number

Horizontal gene transfer

movement of DNA between cells, not linked to reproduction

Mobile DNA

DNA that has evolved to move between cells, can evolve independently of their cellular hosts

Core genome

found in all strains, 20% of genome

pan genome

specific genes, not conserved; 80% of genome

homologous recombination

due to recA mediated DNA repair

non-homologous recombination

due to diverse mechanisms, doesn’t require similar DNA sequences, but often requires target sequence and specific enzymes

plasmids

small DNA elements w/non-essential but usually beneficial genes; often encode for their own transfer from conjugation; usually contain antibiotic resistant ‘marker genes’

viruses

can accidentally transfer host DNA along with viral DNA upon infection

transposons and integrons

DNA elements with enzyme that catalyzes excision of element followed by insertion into new site

cassette

small, mobile segment of DNA that contain several other genes and can replace genes via restriction sites/homologous recombineering resulting in gene disruptions and knockout mutations

genome islands

region of genome that appears to have inherited through a single large recombination event

conjugation

donor cell gives plasmid to recipient from direct physical contact; need pilus

transduction

transducing particle (from bacteriophage-lytic cycle) mediated gene transfer

transformation

Lysis of donor cell → DNA released into environment → Donor DNA integrated into recipient cell genome

naturally competent

bacteria that can take up extracellular DNA

transposition genes

in F-plasmid, allows for reintegration into host chromosome

specialized transduction

selective transfer of small region of bacterial chromosome; can lead to production of defective phages (become gene transfer agents)

transposable elements

stretches of DNA that can move from one site to another

insertion sequences & transposons

has transposase and DNA segment for transposition; has short inverted terminal repeats

transposition

transposase recognizes, cuts, and ligates DNA, inserts transposable element into chromosome; can result in mutations where genes in a chromosome become disrupted

conservative transposition

only recipient DNA has copy of the transposon

replicative transposition

both donor and recipient DNA have copy of the transposon

heterologous expression

expression of a gene from one organism in a different host organism

qPCR (qualitative)

make DNA from RNA template, measures amount of DNA

nucleic acid hybridization

determines if a gene is expressed and how much expression by identifying specific RNA/DNA sequences in cells

Southern hybridization

detects the presence of specific genes in DNA sequences

Northern hybridization

detects the presence of specific genes in RNA sequences w/gene probe

in situ hybridization

DNA probe used to find a cell that contains a specific sequence

restriction enzymes

cut phosphodiester backbone of restriction sites and form sticky ends or blunt ends in DNA

recombineering

generate recombinant vector with gene of interest; dependent on the expression of a recombinase; occurs at homologous regions

Parameters for expressing foreign genes in bacteria

• promoter

• introns spliced

• codon usage modified

• if gene is eukaryotic in origin, modifications must occur after translation to yield active protein

expression vector

facilitate control of expression of cloned genes

artificial synthesis

modifications to mRNA → converted to cDNA → inserted into plasmid for expression

site directed mutagenesis

use of synthetic DNA to introduce mutants at a specific site; dependent on DNA oligonucleotides (primers or probes)

inclusion bodies

insoluble protein form aggregates

reporter gene fusion

target + reporter = gene fusion; reporter expressed under target gene promoter (ex. green fluorescent protein)

His-tag protein

binds to metal protein for selective purification of a protein