Bio 2EE3 Lectures 16-34

how do we know dna is a hereditary molecule?

dna replication, transcription (rna to produce enzymes), translation (rna molecules coded for enzymes), dna repair

griffith experiment

non pathogenic r strain, pathogenic s strain… anything that contained s (dead or alive) killed the mouse while r did not

avery macleod mccarty experiment

attempted to determine if dna, rna or protein responsible for “transformation” effect observed in griffiths experiment, indicated dna was responsible

hershey chase experiment

did not trust amm, used radioactive labelling of proteins or dna in bacteriophages and only labeled phage dna went into bacterial cells proving that dna was hereditary molecule

structure of dna

\-double stranded double helix

\-five carbon sugar 2-deoxyribose

\-phosphate group on 5’ carbon sugar

\- nitrogenous base attached to 1’ carbon of sugar

\-a pairs with t, 2 hydrogen bonds

\-g pairs with c, 3 hydrogen bonds

\-phosphodiester covalent bonds for the backbone

\

structure of dna: bacteria, archaea, eukarya

bac: single circular chromosome

arc: single circular chromosome packaged around histone proteins

euka: multiple linear chromosome packaged around histone proteins

structure of dna: eukaryal histones

wrapping of dsDNA around histones helps compact very large chromosome structures of eukaryotic cells, 8 core histones that form the nucleosome, additional histone= H1 stabilizes the nucleosome structure

how does dna replicate

semiconservative process, each time dsDNA is copied, each copy carries one strand of the original and one newly made

origins of replication: bacteria

jacob brenner and cuzin discovered how dna replication was initiated in e coli cells

replication begins at origin of replication= oriC, dnaA protein binds

dnaB= helicase and dnaC= helicase loader, dnaG= primase that lay down rna primers so dna polymerases work

single stranded dna binding proteins SSBs recruited to help keep dna unwound

origins of replication: eukarya

multiple origins of replication on each chromosome due to large size

studied in yeast, similar to bacteria just different proteins

dna replication: initiation and elongation

virtually identical in bacteria and eukarya

at replication fork forms, dna polymerase iii adds nucleotides to initial rna primers \\

continuous leading and discontinuous lagging strands (forming okazaki fragments) are formed

dna replication: initiation and elongation → lagging strand

dna polymerase i removes the primers and fills gaps with new nucleotides

dna ligase seals the sugar/phosphate backbone

termination of dna replication of circular chromosome

oriC and ter site on opposite sides

tus protein stops elongation

topoisomerase ii disentangles them

termination of dna replication in linear chromosome

once rna primer removed at 5’ end telomerase binds causing repeated translocation and extension of 3’ end

telomerase is released and completion of complementary dna strand occurs

dna ligase links adjacent nucleotides

what is gene

segment of dna that gets transcribed(copied) into ssRNA

differnt forms of rna and each serve a different purpose in transcription/ translation/ regulation processes of gene expression

transcription processes:

initiation, elongation, termination

mRNA

messenger rna, coding molecules translated into proteins

tRNA

transfer rna, involved in translation, charged with amino acids

rRNA

ribosomal rna structural components of ribosomes

miRNA

microrna, various regulatory functions

transcription: initiation and elongation

starts at promoter

rna polymerase separates dna and lays down complementary strand of rna (does this by binding to promoter region of dna and reading template strand)

transcription: initiation and elongation → bacteria

sigma factors bound to rna polymerase core enzyme direct the combined holoenzyme to a promoter (core enzyme interacts with the sigma factor, forming the functional holoenzyme)

once rna polymerase is situated, sigma factor dissociates

transcription proceeds

different sigma factors can direct core rna polymerase enzyme to different genes as needed

transcription: initiation and elongation → eukarya

individual transcription factor proteins associate with promoter region

rna polymerase (3 possible versions) recruited to transcription factor/ dna complex

binding initiates unwinding of dna and start of actual transcription process

transcription: initiation and elongation → archaea

not as well understood, resembles eukaryal

rna polymerase does not directly bind to dna

transcription factors direct rna polymerase to promoter regions

transcription: termination → bacteria

rho- dependent= rho protein follows rna pol and pops it off dna when reaches termination sequence

rho- independent= dna sequence is transcribed forms rna hairpin loop that causes rna pol to dissociate from dna

transcription: termination → eukarya

more complex, depends on which type of rna pol (i, ii, iii) that does transcription

rna modified AFTER transcription: 5’ cao added, poly a tail added, introns spliced out and exons joined together

how are proteins made

during translation the info contained in the mrna molecules is decoded to form proteins

intitation, elongation and termination events

most energy intensive process, very tightly controlled/ regulated

translation

ribosomes interact with mrna and trnas (each trna that brings an amino acid to the ribosome needs to be charged with the amino acid, aminoacyl-tRNA synthetase charges)

each nucleotide triplet (codon) matches to complementary on trna molecule

peptide bonds formed between amino acids

translation: wobble

codon on mrna does not need to be an exact match to the anticodon, called wobble and allows for 61 codons to code for insertion of 20 amino acids into proteins

stop codons= uaa, uag, uga

translation: initiation and elongation → bacteria

depends on interaction between small ribosome subunits and the shine- dalgarno sequence on mrna molecule =, helps align machinery

translation: initiation and elongation → shine- dalgarno

multiple shine- dalgarno sequences allow for bacterial mrna to be polycistronic= coding for more than one protein

euk mrna= usually monocistronic

translation: termination

ribosomes reach a stop codon, release factors cause the complex to come apart, releasing the new protein for folding and modification

translation: protein folding, processing and transport

must fold into secondary/ tertiary forms

some proteins need multiple subunits to form quaternary structure

foldings depend on number of interactions between amino acids in primary polypeptide sequence

translation: protein folding, processing and transport → molecular chaperones

proteins identified in all three domains of life

originally referred to heat- shock proteins because they appear after exposure of cells to heat

assist in correct folding/refolding of polypeptide sequences

translation: protein folding, processing and transport → protein processing

many proteins further modified after initial translation= post- translational modifications, can be phosphorylation, glycosylation

translation: protein folding, processing and transport → protein transport

fully formed and functional proteins need to be directed to proper location (cytoplasm, membrane, etc)

basic steps same for each domain

signal peptides, short amino acid sequences near N-terminus act as zip code to direct

once in right location, signal peptide is often cleaved

translation: role of the nuclear membrane

lack of nuclear membrane in bacteria, transcription and translation can be coupled

eukarya transcription occurs in nucleus, translation in cytoplasm

how are gene expression and enzyme activity controlled?

cells do not require gene products at all time

\-constitutive genes= need to be constantly on

\-inducible genes= required at particular times

gene expression can be controlled at several levels (transcription, translation, post translation)

differential gene expression: activity of enzymes

inhibition of enzymes may result in binding of inhibitor molecule (alters conformation so no longer binds= allosteric inhibition)

covalent modification may alter enzyme conformations, altering activity

differential gene expression: production of enzymes

translation takes tons of energy, enzymes is a better use of energy

lactose

disaccharide of galactose and glucose joined by beta-1→ glycosidic linkage, lactose intolerance is deficiency of lactase (beta- galactosidase family)

how do regulatory proteins control transcription

operon= transcriptional unit with series of structural genes and their transcriptional regulatory elements

ex is lac operon

why is the lac operon important

grow e coli in media with glucose and lactose as sources of carbon

glucose consumed first and when all gone THEN lactose is consumed

creates a small moment of lag in curve= diauxic growth, this is beta- galactosidase and permease produced

lac operon= 3 structural genes

lacZ, Y, A, along with a single promoter and an operator

Z= beta- galactosidase, y= permease, a= beta galactosidase transacetylase

regulatory gene = lacI, located upstream of operon and encodes lacI repressor protein which controls the operator

lac operon

lactose metabolized by e coli, multiple control elements on both the dna and accessory proteins

inducible expression, system only turned on when needed

permease and lactose into the cell

lactose cleaved into glucose and galactose that can be further metabolized by cell

lactose can be converted to allolactose which can turn on the lac operon

key components of operon (8)

promoter= site on dna bound by rna polymerase, promoter directs the initiation of transcription

activator= protein that binds to site on dna, activator assists binding of rna polymerase to the promoter, resulting in increased transcription

activator binding site= site on the dna bound by the activator

repressor= protein that binds to the operator on the dna, inhibiting transcription

operator= site on the dna bound by the repressor

effector= small molecule that binds to activator or repressor proteins, modifying their gene regulation activity

inducer= effector that increases transcription by enabling an activator or disabling a repressor

corepressor= effector that decreases transcription by enabling a repressor

operon: negative control of transcription → lac operon

repressor protein binds to the operator, blocking rna polymerase and therefore transcription

when inducer (allolactose) binds to repressor (laci) and prevents repressor from binding to operator, transcription proceeds

operon: negative control of transcription → trp operon

encodes for proteins in the tryptophane synthesis pathway

when tryptophan binds to the repressor, it can bind to the operator and prevent transcription

transcription proceeds in the absence of the corepressor tryptophan, when the repressor cannot bind to the operator

operon: positive control of transcription → lac operon

regulatory molecules bind and increase transcription rates

usually an activator molecule increases affinity of rna polymerase for promoters

effector molecule binds the activator, alters shape to increase binding affinity for regulatory site on dna

low glucose, cAMP increases which binds to crp which increases affinity of rna polymerase for the promoter resulting in transcription

operon: positive control of transcription

lac operon represents a composite of control methods

lecture 18, slide 15

operon: constitutive laci repressor mutants of lac operon

mutant class i: laci mutants, constantly expressed the lac operon even with no lactose

mutant class ii: lacO mutants, other mutant strains couldn’t shut down lac expression even when good laci was indroduced

how does a single enviro stimulus control multiple operons?

regulons are genes that are coordinated to respond to the same regulatory systems

catabolite repression= shutdown of several systems that utilize various nutrients when glucose is present

sos response= multigene system for wide- scale dna repair

pho regulon= gene whose expression is regulated via the concentration of phosphate in the media

sos response: uv exposure

allows a cell to recognize and respond to serious dna damage

uv exposure induces nonspecific repair, bacteria pre exposed then infected repaired the phage but induced more mutations

sos response: promoter probe

promoter probe reporter lacz gene transposon, when inserted next to gene expressed dna damage and itself (blue colonies) so showed which genes were required for repair

sos response: 2 important regulators

recA= recombination and regulation of sos response, binds to single stranded dna

lexA= dna binding transcriptional repressor of sos regulon genes

alternative sigma factors (4)

use different sigma factors directs rna polymerases to certain genes

most ecoli recognized by sigma 70

70= houskeeping

54= regulating nitrogen utilization genes

32= heat shock protein gene regulator

38= general stress response gene regulator (Stress= starving, low temp, etc)”

post initiation control of gene expression: how can mRNA be controlled

regulatory rna, all genomes carry regions of dna coding for non- translated rna

small noncoding rna (sRNA) that can control gene expression at transcription or translation points

ex, antisense rna works by binding to complementary mrna to shut sequence down

post initiation control of gene expression: control of transcription by mRNA secondary structure (attenuation)

attenuation= interaction between translation and transcription processes

if ribosome follows rna polymerase, terminator hairpin rna loops are formed in the leader sequence and the polymerase detaches, stalling out of ribosome in mrna leader sequence allows transcription to continue

can’t occur in eukaryotes

post initiation control of gene expression: attenuation

control of the trp operon by attenuation

regulation by attenuation occurs after transcription initiation

trpL gene is 1st gene in operon, encodes a leader peptide containing attenuator sequence

high levels region 3 binds to 4, stopping transcription

low levels region 3 binds to 2, allowing transcription

post initiation control of gene expression: control of transcription by mrna secondary structure (riboswitches)

riboswitches= regulatory molecules that bind rna and alter its shape

changed shape in leader areas of mrna can prevent continuation of transcription, this around start codons in mrna can prevent ribosomes from translating mrna

post initiation control of gene expression: control of transcription by mrna secondary structure → riboswitches

mrna can bind to effector molecules such as vitamins or amino acids that regulate gene expression

some riboswitches affect transcription

some riboswitches regulate translation by inhibiting ribosome binding

how do bacteria communicate with neighbors?

gene expression can be means of communication between microbes

chemical signaling known as quorum sensing

cells release autoinducer molecules into enviro as population density increases, detecting changes in autoinducer levels causes regulation of gene expression

lux, prototypical quorum sensing system

found in vibrio fischeri, freely/ in symbiosis with squid

cells only emit light via enzyme luciferase when in light organ of squid when in high density

at low desnity cells do not produce enough AHL to emit light

luxR regulator interacts with ahl to bind to lux box dna regulatory site

leads to transcription of luxa/luxb= luciferase protein and luxi= positive feedback loop forming more ahl

quorum sensing: cell density

low= transcriptional regulation by quorum sensing involves the production of small amounts of ahl molecules by enzyme luxi encoded by the luxi gene

high= concentration of these ahl molecules increases and they can bind the luxr transcriptional activator protein encoded by luxr

results in increased affinity of transcriptional activator protein for the lux box and increased transcription of the lux operon

quorum sensing: widespread occurrence

broad range of microbes possess quorum- sensing systems

mechanisms controlled include: motility, conjugation, biofilm formation, pathogenesis

autoinducers may even play a role in competition, interrupting or inhibiting a control pathway in other organisms in the enviro

two- component regulatory systems: how do enviro conditions affect gene expression?

systems can use one protein as a sensor and another to control transcription

allows for response to changes in enviro

signal transduction induced inside the cell alters it to respond appropriately

ex, sensor= virA, regulator= virG, function= tumor formation in agrobacterium tumefaciens

two- component regulatory systems: involve…

sensor kinase (histidine protein kinase= hpk) to detect the enviro stimulus

response regulator (Rr) to regulate transcription

two- component regulatory systems: virulence of agrobacterium tumefaciens

vir genes found on ti plasmid

vira= transmembrane hpk protein

virg= transcriptional activator rr protein

two- component regulatory systems

allow microbe to respond differently to enviro stimuli

pairing particular hpk and rr cells can better control whch genes are expressed in response to cues from the enviro

how can bacterial cells regulate their behaviour

mechanism of chemotaxis!

ex, complex bacterial behaviour modulated by shifts in protein activity

controlled at level of protein activity, rather than via changes in gene expression

chemotactic bacteria sense changes in chemical gradients over time

changes induce altered direction and duration of flagellar rotation, leading to directed movement over time

chemotaxis

chemotaxis mutants isolated using a capillary tube filled with nutrients

microbes with normal chemotaxis move into tube

those with mutations in chemotactic proteins will remain outside the tube

multiple che gene mutant strains have been isolated and studied in this manner

chemotaxis: study of chemotaxis using mutants → che

che proteins= two- component regulatory systems

cheA works as the sensor kinase, becoming phosphorylated

cheA then phosphorylates cheY= rr protein, controls direction of flagella rotation

chemotaxis: study of chemotaxis using mutants → mcp

methyl- accepting chemotaxis protens (mcp) also work in chemotaxis systems

by interacting with cheW proteins, autophosphorylation cheA is modulated

attractants decrease phosphorylation

repellants increase phosphorylation

chemotaxis: absence of attractant

cheW promotes phosphorylation of cheA, which then phosphorylates cheY

this interacts with flagellum and signals cw rotation causing tumble

removal of phosphate from chey-p by cheZ resulting in ccw rotation so cell runs

chemotaxis: presence of attractant

phosphorylation of cheA is inhibited so cheY is not phosphorylated either

absence of cheY-P results in ccw rotation causing cell to run

chemotaxis: study of chemotaxis using mutants → mcp, adaptation

regulates attraction during periods of very high attractant levels

if high levels arent maintained phosphorylation of cheA/B will lead to eventual demethylation of mcp

food spoilage

perishable= easily support microbe growth, meat and fruit

semi- perishable= do not spoil as quick, nuts and potatoes

non- perish= remain edible for long periods, flour and sugar

food spoilage: intrinsic and extrinsic factors

intrinsic

* water availability
* osmolarity
* nutrient content
* ph and buffering capacity
* antimicrobial constituents
* biological structures such as rinds or shells

extrinsic

* temp
* humidity
* presence and concentration of gases

desirable food storage

dairy

raw milk= suitable for growth of natural microbiota contaminating microorgs

processing of milk= various products aims to change physical and chemical form of milk to make less favorable for growth of spoilage microorgs

food preservation, what techniques?

* reduction of water activity
* control of temp
* increase in acidity of food
* addition of chemical preservatives
* irradiation of food
* used of modified atmosphere packaging
* hurdle technology

food preservation: water activity

reduction of water can be achieved by dryng out food or adding solutes (sugar/ salt)

for growth most bacteria require an Aw > 0.9 (water = 1.0)

microorgs require usually above 0.99

bacterial pathogens cannot grow at less than 0.86 and yeasts/ molds cant grow at less than 0.65

food preservation: temp

cooling= refrigeration or freezing slows rate of chemical reactions

heating= pasteurization, usually heating liquids to 63 for at least 30 mins

canning= combined with pressure to eliminate endospores, heating under pressure beforehand

food preservation: acidity

increasing acidity prevents spoilage

pickling foods in vinegar allows fermentation to naturally drop ph over time, so microbes cant grow

food preservation: chemical preservatives

often by reducing ph

natural antimicrobial preservatives: bacteriocins, lactic acid, acetic acid

artificial: sodium benzoate, nitrites

food preservation: irradiation

strength of microbe elimination depends on type of radiation, but all cause damage to microbe dna

non- ionizing radiation (uv)= surface only not strong

ionizing radiation (Gamma/ x rays)= stronger, more penetrating

food preservation: map

vacuum packing takes oxygen out of package, some are flooded with co2 to acheve same effect

red meat packed in high oxygen enviro to reduce growth of harmful anaerobe

food preservation: what is hurdle tech

multiple levels of antimicrobial control in food

employ multiple constraints each of which an organism must overcome to proliferate, can allow less destructive preservation methods when combined

food/ water bourne illness

illness due to microbial toxins= foodbourne intoxiction

actual microbes= foodbourne infection

salmonellosis= food poison

illness like cholera spread through either food or water

food/ water bourne illness: clostridium botulinum

gram pos, spore forming, obligate anaerobe, neurotoxing

causes botulism through production of botulism toxin

associated with improper canning, controlled by ph, Ae, hgh o2 and temp

food/ water bourne illness: ecoli O157:H7

gram neg, facultative anaerobe, severe/ acute diarrhea

consuming raw milk and beef

prodcues shiga toxin which inhibits proten synthesis in target cells

acquired by horizontal gene transfer from integration of a prophage encoding it in genome

food/ water bourne illness: listeria monocytogenes

gram pos, facultative anaerobe

unpasterized dairy, raw veg and meet, processed meet

severe illness

listeria killed in cooking process

food/ water bourne illness: campylobacter jejuni

gram neg, non spore forming, microaerobic

food poisoning from contaminated chicken/ food and water

diarrhea and other serious complications

food/ water bourne illness: norovirus

non enveloped, (+) ssRNA

infects intestinal epithelium, most common cause of gastroenteritis

non bloody diarrhea, vomitng, stomach pain

fecal oral route (Contaminated food water or surfaces)

fermentation

produce a number of different types of food

starter cultures of microbes are added to food to begin the fermentation process

used by microbes to regenerate NAD+

used by facultative anaerobes like e coli or s cerevisiae under anaerobic conditions

waste products= fermented food

fermentation: milk products

lactic acid bacteria= lab, used to produce cheese, yogurt, salam, sauerkraut

starter culture= prep of microorgs to help control fermenting

lab ferments lactose to produce lactic acid which lowers ph and play role in flavour

holes in swiss cheese by propionibacterium

fermentation: milk products → cheese

starter culture induce acid (also important for flavor aroma texture)

acid production coagulates milk protein and forms curds

loose cheese= curds and liquid whey mixed together

creamy/ hard cheese= curds are separated from whey and pressed

ripened= aged by storage

unripened creamy= not aged

fermentation: with acid

extensively in asia for miso, tempeh, soy sauce, sake

koji= starter of various molds for aerobic fermentation to break down proteins and polysaccharides

maromi= secondary anaerobic fermentation where bacteria and yeasts further ferment

fermentation: vinegar manufacture

small amounts used to flavor food

larger amounts used as a preservative

produced by fermentation of ethanol by acetic acid bacteria into acetic acid, source of ethanol can imbue different flavors

trickle/ quixk method uses acetic acid bacteria on inert support material for fermentation

fermentation: gone wrong

wrong microbes can get in the mix and produce the wrong fermentation product (like lactic acid instead of ethanol in wine)

infection of microbes by lytic bacteriophages can stop a desired fermentation from occurring

starter culture microbes may also lose plasmids with desirable genes for fermentation

fermentation: kombucha

starter culture= scoby

rich source of probiotics

brew tea, add sugar, ferment with scoby, bottle