like anchoring proteins keep these loops relatively independent

50 loops

Topoisomerases

T1

T2

Topoisomerasesregulate the supercoilingof DNA

•Type I nick the DNA and relieve supercoils

•Type II cleave both strands and introduce supercoils–requires ATP

origin of replication(oriC) and all the DNA that is replicated as a unit from that origin

Most prokaryotic chromosomes are single replicons

–A plasmid is a replicon

reads 3' -> 5'

oh -> phos

Writes 5' -> 3'

Helicase recruits primase(RNA polymerase) to begin replication

Primasessynthesize a small RNA molecule (10-12 nucleotides) that acts as a primer for the start of DNA replication

DNA polymerase III synthesizes new DNA strand .must have a primer.

Energy for polymerization comes from phosphate groups on added base..

a new base is added to 3'OH. New nucleic acids grow to extend 3'end.

DNA polymerase III has proof-reading activity. Exonucleaseactivity to cut off mispairedbases. Effectively corrects errors

Helicase – unwinds the two strands of DNA

•SSBs(single-stranded DNA binding protein) – keep the single strands apart during unwinding and copying

•Primase – RNA primer

•DNA polymerase III(two per replisome) – synthesizes the complementary strand of DNA according to the base-pairing rules, from 5’to 3’

•RNaseHremoves the RNA primers
–One primer for each leading strand
–Many primers on the lagging strands –one per Okazaki fragment

•DNA polymerase I fills the gaps in the DNA strand

•DNA ligase seals and completes the lagging strand

Lagging strand growth

strand lags after the fork

1kb piece

Rolling Circle

Unidirectional replication

-starts at nick

-Makes new "+" strand removing the old + strand

- New + and old - make new plasmid

-Old + and new - make new plasmid

Areas of Microbial Genomics

-functional

comparative

metagenomics

Functional Genomics: Study how genes in a genome operate

•Comparative Genomics: Compare different genomes to understand their function and evolution

•Metagenomics(Environmental genomics): the study of genetic material recovered directly from environmental samples

•Shot-gun cloning of DNA directly from environmental samples

•Including genetic material from uncultivableorganisms

•Discovery of significant, abundant, and novel genes

•mRNA –messenger RNA

–carry genetic information from DNA toprotein

•rRNA–ribosomal RNA

–rRNA is the major structural component of ribosomes

•tRNA–transfer RNA

–rRNAand tRNAare non-coding RNA

•Other non-coding RNAs

–sRNA–small RNA: Regulate gene expression through transcription and translation

 Can base pair with target mRNA and block or activate translation

 –Catalytic RNA –enzymatic activity

Core Enzyme: αωßß’–RNA synthesis

•Holoenzyme: core enzyme plus Sigma factor

•Sigma factor (σ):–Recognizes promoters by binding to -10(PribnowBox=TATAAT) and -35regions of genes

–Guide the core enzyme to initiate transcription

once DNA is open sigma factor is released

Stem loop forms at the stop codon, physically interferes with movement of rna polymerase

 The RNA ploymerase binds with the loop but also continues translation of the U. The weak U-A bonds break because the loop bind is stronger, stopping transcription

5->3

Ribosomesbind mRNA while mRNA is still being synthesized

 Multiple ribosomesbind to each mRNA, proteins are made rapidly

Longest peptides are on ribosomesfurthest from the start codonAUG

Very large rRNA-protein complexes

–2 subunits (30s and 50s), 52 proteins, 3 rRNAs
•30s subunit –16s rRNA
•50s subunit –5s and 23s rRNA

•rRNAforms the catalytic center of the ribosome
–Can bind 1 mRNA + 3 tRNAs

tRNAs bind individual amino acids

–tRNAs have clover leaf structure

tRNAshave 3-base anticodon

•Base pair to codons in mRNA

–Aminoacyl-

tRNA transferases charge tRNA

Initiation
–Ribosome-binding site (RBS codon) on mRNA allows binding to 30s subunit
–Formylmethionine tRNA binds AUG (start) codon the Formyl group will be removed later
–50s subunit (shell) docks to 30s subunit –fMet-tRNA occupies the “P”site

•Elongation
–Next charged tRNAbinds to “A”site
–Peptide bond forms

•Translocation
–Ribosome moves from one codonto the next and the empty tRNAis released from the “E”site
–Next charged tRNAbinds to empty “A”site

•Process continues until stop codon

trna binds to codon -> ribosome binds at trna to p site and is added to chain -> trna binds to codon at a site-> ribosome move so p->e and a->p -> E codon ejected and new trna binds a site.  read p site to chain

genetic islands- Large insertions of sequence

 horizontal gene transfer - Transferred from other species

vertical gene transfer - from mother cell to daughter cells through cell division

 Mutations - slow, but accumulated through time

- Natural transformation: free (naked) DNA taken up from the environment and then integrated into genome, Plasmid transformation

–Conjugation: cell-cell contact (sex pilus)

–Transduction: DNA transfer is mediated by bacteriophage

Competent cells - Uptake of DNA directly from the environment

Gram Positive -  Bacteria secrete competent factor & Accumulation of competent factors induces the assembly of translocasome located in the cell membrane –quorum sensing

 Gram Negative - Stress induced competence via CaCl2 and low temperature(4°C) or driven in through heat shcok @42C best done early in log phase

Type IV secretion system = sex pilus

 Pilusproteins encoded on fertility plasmid or F factor and is single stranded DNA

F+ = male or donor

F- = Female or recipiant

Female turns into male after mating

Uses rolling circle repilcation to old + into F- the F- minus makes new -  and become F+

Hfr- when DNA is integrated into F-'s chromosome instead of become F+

-Binds IS3, B gene first then C, then A so i the host chromosome goes A, IS3 F plasmid, IS3, B,C

-occurs during interupted mating

Gene Transfer: Transduction

Lytic Life Cycle

Viruses (phage) inject viral DNA into host cell

Bacteria then replicated virus until the burst from the cell with 

-normal phages - DNA that tricks bact. into making more

-Transducing phages - contains combination of bact and virus dna made via lysogeny

Trans ducing phage are mistakenly picked up due to excision error by Bact. This causes recombination to form new bact. DNA in transduced cell with cells DNA, virus DNA, and old bact DNA(Transduction)

Transformation - Fragments of DNA or sent out of Bact cell and picked up by another cell and is "recombination" into new dna in host cell

Transduction - Virus with some host bact dna and some virus dna inject the combination into new bact cell and "recombination" into new dna in host cell

Conjugation - old + DNA from F+ injected into F- and old+ and new - form new DNA

restriction exonucleases - degrade injected most DNA

Restriction endonucleases -  cut foreign DNA to pieces at palindromic sequence, host prevent this happening to own dna by adding methyl group at sequences

,

- Coexsisting as Extrachormosomal DNA

Recombination - Merge into chromosome

Mutation

Transition

Transversion

Transition: purine -> purine via base pair that encodes for purine

Transversion: purine -> pyridimine or vice versa

UV and ionizing radiation cause the formation of toxic oxygen radicals, Radicals cause two adjacent pyrimidinebases to dimerize-Thymine dimers, prevents DNA transcription

Photoreactivation-split thymine dimersapart in the presence of light

Nucleotide excision repair - Operates in dark

–UvrABC protein complex removes the dimeralong with flanking nucleotides

–DNA polymerase I fills the gap and DNA ligaseseals the strand

Chemical Mutagens

-base analogs

-DNA Modifing Agents

-Intercalating Agents

-base analogs: similar but not exact to base pair but leads to point mutation

  -5-Bromouracil

-DNA Modifing Agents: Changes structure of DNA so instead of A-T in G-T

Uses bacterial mutant that can not synthesize histidine

–has a mutation in hisG gene

 -can't grow without Histidine

-Reverse Mutation - hisG change to normal histimine gene so bacteria can make own histidine and grow

DNA Repair

DNA polymerase III proofreads and corrects errors during replication

-Methyl mismath indicated error

 - DNA polymerase III can't replicate DNA due to thymine dimmer so it leaves a gap in the newer strand

-The undamaged strand is copied and placed into gap leaving space at error, the error (thymine dimmer) is removed

 Base excision repair - DNA glycosylaseclips the damaged basesfrom the phosphodiesterbackbone

DNA Polymerase I fills gap and DNA ligasw seals strand

LexA represses SOS genes

RecA is activated by DNA Damage which deactivates LexA

DNA mutases repair DNA rapidly but produce errors

Segments of DNA that can hop from one place in DNA to another –transposition

transposases: enzymes promote transposition

- in all living organisms

simplest transposable element; encodes transposase flanked by inverted repeats

Composite transposon - carry functional genes between two IS elements: e.g. antibiotic resistance genes

Transposable element (Tn) jumps from one site to another

EX: A-Tn-B + CD => AB + C-Tn-D

Transposable element is copied and the copy is inserted into target

A-Tn-B + CD => A-Tn-B + C-Tn-D

Posttranscriptional control –No enzymatic activity

Translational control –No protein synthesis

Transcriptional control –No mRNA synthesis

expressed continuously

housekeeping genes

Highly expressed only when needed

•Basal level of transcription: occurs in the absence of any specific activation

•Protein level rises (~3 vs. 3,000 molecules) in the presence of inducer–

Repressible gene: transcription level decreases in the presence of corepressor

corepressor - a protein that decreases gene expression by binding to a transcription factor

Sensor kinase - membrane spanning protein

•Binds to signal

-phosorylates Histidine

which then phosphorylates aspartic acid on Response regulator

- RR Binds DNA to regulate gene transcription

Biosynthetic Enzymes - If end product not present enzymes get made

Catabolic Enzymes - if substrate is present and prefered energy source is not enzymes are made

biphasic growth curve

    _/

/

grows fast until glucose is exhausted, lag during gene expression switch, slow growth using lactose instead of glucose

Transcription of lac operon occurs only when glucose levels are low AND lactose is present

Repressor: LacI

Activator: cAMP Receptor Protein (CRP)-cAMP

lactose permease(LacY) - Lactose needs transporter to pass through cell membrane, driven by PMF

Induces

 CRP-cAMPis the activator protein–Binds to activator, promotes transcription of lac and other operons

CRP acts as activator only when bound to cAMP–inducer and if LacI is not present

LacI turns off operon

Phosphotransferase system - Glucose is transported though the membrane

Glucose is supposed to become Glucose-6-P but if it doesn't it inhibits Adenylate Cyclase  which is the catayse for cAMP

High expression - low glucose, available lactose

Low Expression - high gluctose, lactose avail.

No Exp: High (no cAMP) or Low glucose, Lactose unavailable (no lacI)

 lacL-CAP-P-O-lacZ-lacY-lacA

lacY brings lactose into cell and derepresses lac operon, CRP binds to Camp both bind to CAP, If no lacI, RNA polymerase binds and translates, lactose converted to glucose by lacZ, 

–Responsible for arabinose catabolism

–Activated by cAMP-CRP

-Repressed by AraC

–With arabinose: activator
– Without arabinose: repressor

w/o= folded at araC binding araO2 and araL1 via araC unit (two tables onto top of on another)

with- AraC is bound with Arabinose causing conformation change to straight genome, AraC comound now binds AraL1/2

araO2-araC-araO1-cAMP-CRP binding site-araL1-araL2-araB

- only in prokaryotes

- Mechanism to terminate transcription

 –Attenuation is important when end product is low, not a repressor

- Trp in E.coli when attenuated produced 15% of  capability, cut off early

Concentration of tryptophan is:

high: TrpR a repressor is activated preventing tryp from being made 3-4 loop

Low: TrpR not Active but TrpL is, attenuation, only 15% is made 2-3 loop

Starvation: TrpR and TrpL deactivated, Tryp is made at max rate

TrpL is nonfunctional leader peptide upstream of the structural genes

–The mRNA of TrpL has 2 adjacent tryptophan codons

Ribosome binds to mRNA after transcription starts

•Translates leader peptide

•Ribosome stalls if level of tryptophan is VERY low because of the two adjacent trpcodons–Stalled ribosome prevents mRNA from forming a transcription terminator

Small effector molecules may bind to leader mRNA and change its folding pattern to regulate translation

In the presence of the ligand, ribosome binding site forms a stem-loop with the leader mRNA and ribosome binding is blocked

Under low iron conditions, iron acquisition gene (ent) and rhyBare expressed.

 rhyB produces a sRNA, which suppresses the translation of sucCDAB, which is involved in iron storage

 Under high iron conditions, Fur acts as a repressor protein for ent and rhyB, sucCDAB is transcribed and translated to promote iron storage

Individual cells assess population density

Cells work together at high cell density

Diffusible signaling molecule: acetyl homoserinelactone(AHL) aka AUTOINDUCER

 -species specfic

-accumulates in high cell densities

lux = lumensense operons

luxL- encodes AHL synthase

luxR activates AHL which activates lux->light

Restriction enzymes - Cut DNA at the specific restriction sites

 DNA ligase: only matching ends can be ligated

5'---G AATTC---3'

3'---CTTAA G---5'

Sticky ends

5'---CCC GGG---3'

3'---GGG CCC---5'

Blunt ends

put DNA in new host

1.Origin of Replication

2.Antibiotic Resistance gene

3.Multiple Cloning Site (MCS)

-blue cells = recombinants

Cells with transposons grow with antibiotics present

 Create insertion mutations that knocks out gene function

-identify

negatively charged dna migrates from anode to cathode

migration rate is determined by size and conformationof the DNA

DNA-DNA hybridization

For identifying a particular sequence of DNA in a complex mixture of DNA fragments

Labeled DNA probe will form a “hybrid”with its complementary sequencesand be detected

Amplificationof millions of copies of a specific DNA fragment

DNA polymerase: Taqpolymerase

•No proofreading activity

all of the cell’s mRNA molecules under a given growth condition

-unique to each condition

all of the cell’s proteins under a given growth condition

unique to each condition

•Complementary DNA (cDNA) is produced by reverse transcription(RT) from mRNA and the hybridized

Green: Expression only in control

Red: Expression only in expirimental

Yellow: Expressed in both

PolyU downstream of pause site

•DNA-mRNA U-A base pairs are instable

–Even less stable when RNA polymerase is stalled at the pause site

•mRNA breaks off from DNA, polymerase is released

Rhoprotein binds to the mRNA at GC rich sequences

•Physically interacts with RNA polymerase when it stalls at the pause site

•ATP-dependent helicase activity of Rho releases the mRNA from DNA

•RNA polymerase is released