MICROM 410 Exam 1

Are DNA and RNA symmetric or asymmetric?

Asymmetric

What is on the 5', 3' and 2' spots in DNA?

5' phosphate, 3' OH, 2' Hydrogen, phosphodiester bonds link 5' and 3' carbons

What is on the 2' spot of RNA?

OH instead of just a hydrogen like DNA. Because it's bulkier, you can't form double stranded structures. OH can also perform chemistry, making RNA a possible catalyst and less stable.

What end do polymerases add onto a nucleus acid primer?

3'. Polymerases translocate along template 3' to 5'. Polymerases add bases to growing strands 5' to 3'.

Why do DNA polymerases need primers?

They can't make dimers! Joining 2 free dNTP bases together is chemically hard, to activate the 3' OH you need a chain of more than one DNA base.

Fill in the blanks: replication is ___________ and ___________.

Bidirectional and semiconservative

What happens in theta replication?

Synthesis on a circular genome!

Where does Thera replication begin?

The origin of replication oriC

What is a feature of the theta replication bubbles?

Replication forks

Where does theta replication end?

The terminus

What is DnaA?

It is in initiator protein that binds to oriC

What is DnaB?

It is a helicase that unwinds the helix to expose template strands

What is DnaG?

A primer that synthesizes RNA primers

What is DNA Pol III?

A major replication enzyme

What is DNA pol I/RNaseH do?

It replaces a RNA primer with DNA

What does DNA ligase do?

Seals the nick in DNA strands after DNA pol I replaces the primer

What does DNA gyrase do?

relieves DNA supercoiling

What are the steps in initiation of replication?

1. DnaA-ATP binds to oriC at 9bp sequences. Multiple copies of DnaA-ATP bings to each 9bp site, DNA bends.

2. 13bp region near DnaA-ATP binding site melts

3. DnaC loads DnaB on a single stranded bubble

4. DnaC proceeds away from oriC

What does the replisome contain?

DnaB helicase, DnaG primase, clamps, DNA pol III leading and lagging strands

What happens in replication and elongation?

Elongation proceeds along leading strand from initial primer. Lagging strand proceeds in okazaki fragments with multiple RNA primers. Each okazaki fragment is about 1000 bases long, with RNA primer initially at each 5' end.

What is important info about the contents of a replication bubble?

Each replication fork has a leading and a lagging strand. Each new piece of DNA has a leading strand on the 3' end and a lagging strand on the 5' end.

What is the process of removing RNA primers?

DNA pol III makes most of a new strand DNA. RNAseH or DNA pol I removes primers from RNA-DNA hybrid at beginning of okazaki fragments. DNA pol I then replaces RNA primer with DNA. Final bond made by DNA ligase between 3' OH and 5' Pi.

What is important about the efficiency of DNA polymerases?

Replication is fast 750-1000 bases/sec. Replication is accurate, partly due to fidelity of dNTP binding and 3' -> 5' exonuclease (proofreading exonuclease, found in DNA pol I & III). Approximately 1 mistake in 10^9 nucleotides.

What do tripoisomerases do in bacteria?

Introduce supercoils to condense DNA. Reduce strain during replication (Gyrase). These 2 processes are held in balance. Quinilones are antibiotics that block function of DNA gyrase.

What is the process of terminating replication?

Replicating ends on the opposite side of chromosome from oriC at terminus. Multiple short terminator sequences (ter) are clustered on the chromosome. A protein called Tus binds to these sequences and acts as an orientation specific counter helicase.

How does Tus create a one way gate?

Tus binds more strongly to one DNA backbone. When traveling in direction of arrow DnaB (helicase) can pass Tus. When travelling in opposite direction of arrow DnaB cannot pas Tus.

How can loops get knotted?

Linked catenanes are formed during replication of circular DNAs. Separated by topoisomerase IV and the proteins XerC and XerD acting on sequence in ter region.

What is important about the timing of DNA replication?

Origin binds to molecule in the cell membrane. Can restart replication before cell division complete.

What is the process of coordinating growth with DNA replication?

DNA replication takes ~40 min for many bacteria. In slowly growing cells, chromosomes will segregate to progeny, with a delay in re-initiation of replication until completion of cell division. In rapidly growing cells, 2nd round of DNA replication will initiate before the previous one has finished. This enables generation times that are shorter than time required for chromosome replication.

How do concentrations of DnaA and ATP regulate replication frequency?

DnaA is only active when bound to ATP -> must have ATP available, once oriC is replicated, there are 2x as many binding sites for the same # of DnaA, so need to make more DnaA before we will be able to replicate again. In rapidly growing cell -> lots of ATP, lots of protein production, lots of active DnaA-ATP, replication starts again quickly. In a slow growing cell -> fewer ATP, less protein production, fewer active DnaA-ATP available, replication start is slow.

Where are the sites and what is the timing of DNA replication?

In rod shaped bacterial cells, DNA replication occurs at mid cell, where DNA pol/replisomes are located, replicated DNA moves away from cell center. -In cells that grow with generation time faster than 40 mins, rapid regeneration of DnaA-ATP from DnaA-ADP will allow initiation of another round of synthesis before the previous round has completed, daughter cells are born pregnant.

What are similarities in eukaryotes? (DNA and RNA related)

Bidirectional, semi-conservative replication from origin. 5' to 3' synthesis with leading and lagging strands. Basic types of enzymes needed are the same -> helicase, replicative polymerase, primase, primer replacing polymerase, topoisomerase, clamps.

What are differences in eukaryotes? (DNA and RNA related)

Usually have linear chromosomes so we need to have a special way to deal with ends (Telomers). Chromosomes have many origins. Most enzymes have evolved more domains that increase way to regulate replication. Often tightly regulated -> especially in multicellular organisms. Cannot begin replication before cell division is complete.

What are some features of archaea? (DNA and RNA related)

All known archaea have circular chromosomes -> most have multiple origins, like eukaryotic chromosomes, some have to come from horizontal gene transfer. Dna PolB is similar to bacterial pol III. Most other replication enzymes are most closely related to eukaryotic enzymes than bacterial ones. Regulation more similar to bacteria as not multicellular.

What do plasmids do?

Contain origin of replication. Usually code for something useful, not essential: Antibiotic resistant genes, Pathways to use unusual carbon sources, Conjugation machinery, Toxins and their antidote, Many other cool things. Plasmids used to insert genes of interest into cells.

What does generation mean?

One cell becoming 2 cells.

What are the various ways that bacteria grow?

Continuous culture, biofilms, batch culture

What is continuous culture?

A constant influx of new media

Where does a batch culture occur?

a lab

What are the different phases of bacteria growth?

lag, log, stationary, death

What happens in the lag phase of bacteria growth?

preparing to grow, length of phase determined by environmental conditions and previous condition

What happens in the log phase of bacterial growth?

doubling

What happens in stationary phase of bacterial growth?

growth arrest, ran out of nutrient, buildup of toxic byproducts, turn on stress responses to increase viability

What happens in the death phase of bacterial growth?

bacteria die with a half-life similar to radioactive decay, negative exponential curve

When are bacteria the most resistant to osmotic stress?

stationary phase

What is specific to batch culture?

Stationary phase and death phase

What is a benefit of continuous culture?

No toxic buildup of metabolic byproducts

What are biofilms?

Present in 65-85% of all bacterial infections (breast implant, catheter, eye contact lenses, burn, sinusitis, cystic fibrosis, etc). Form when nutrients are plentiful. Can be single species or multi-species.

How do cells communicate with each other to coordinate in a biofilm?

1. Attachment to monolayer by flagella (use flagella to stick to the surface) if using pilli they can move on surface

2. Become non-motile and form microcolonies (more and more cells build up, cells can grow, cells can start communicating to each other to coordinate EPS production)

3. Exopolysaccharide (EPS) production (different for different bacteria): generally composed of DNA, polysaccharides, etc. Increases resistance to antibiotics and other stressors

4. Mature biofilm

5. Once nutrients become scarce, individuals detach: dissolution and dispersal (dispersed cells have increased expression of adhesion factors as well as virulence production)

What are physical parameters a microbe can be classified based on?

temp, pH, osmolarity, oxygen, pressure. Only prokaryotes grow above 65 degrees, only archaea above 95 degrees C (extremophiles). Thermus aquaticus = thermophile, identified in 1960s in a hot spring at yellowstone (Taq polymerase).

How can microbes be classified based on their carbon and energy acquisition?

Autotroph: CO2 is fixed and assembled into organic molecules. Heterotroph: preformed organic molecules

What is a Chemolithoautotroph?

Something which produces energy from oxidizing inorganic molecules, this energy is sued to fix CO2 into biomass.

What is the different media that E.coli can grow on?

LB broth (complex mixture), M9 media defined (slower growth)

What are the different media growth times?

Complex 20 min, Defined + glucose (60 min), Defined + glycerol (120 min)

What is important info about uncultured microbes?

99% of bacteria will not form colonies on an agar plate. In addition to the 6 macronutrients (C, O, N, H, P, S) cations (Mg 2+, Fe, Ca, K, Na) & 6 Macronutrients (Mn, Zn, Cu, Co, Mb, Ni) many bacteria require specific growth factors or signaling molecules.

How do bacteria obtain nutrients?

Passive diffusion (small uncharged molecules). Osmosis (water diffuses into cell). Membrane-permeant molecules (weak acids and bases). Transport (passive/facilitated diffusion or active)

P goes from high to low. Active:

Coupled transport.

What is teichoic acid and lipopolysaccharides?

Negative receptors for bacteriophages.

What organism would be completely resistant to lysozyme?

Sulfolobus (archaea).

Has different linkage that lysozyme can't cleave.

What organism is most sensitive to penicillin?

Gram pos because peptidoglycan is most accessible.

What is the S-layer?

Protein/glycoprotein component of cell enveloped. Some bacteria have one

G + linked to PG

G - linked to LPS. Porous and can move, provides some structure. Bacteria lose genes that encode S layer in the lab (reductive evolution). Many archaea have one but not all, linked to pseudoPG or membrane.

What are capsules and slime layers?

Most prokaryotes have polysaccharide layer outside the cell wall called glycocalyx.

Capsule is discrete later (E.coli can make 50 different types).

Slime layer is less discrete layer associated with the cell wall. Helpful for Attachment/Adhesion. Protection from dessication & phagocytosis. Capsules contribute to pathogenesis

What is important about rotary flagella?

Rotate together in a bundle behind the cell. Archaea have flagellum. 10-15 microns long. Flagellin = FliC = ~20,000 subunits. Movement is generated by the rotation of the flagellum (either clockwise or counterclockwise). Energy that is required is generated by proton motive force

What different types of cells have different flagella?

Monotrichous cells have a singular flagellum. Lophotrichous cells have flagella at the ends. Peritrichious flagella are randomly distributed around the cell.

What is important about flagella in spirochetes?

Long tight spirals. Periplasmic flagella. Rotation forces entire cell to twist like a corkscrew.

What is chemotaxis?

Moving towards a nutrient or away from a toxin. Biased random walk- ends far away from where they started. Flagella moving counterclockwise is moving towards attractant. Flagella moving clockwise (tumbling) is stopping forward motion so the cell tumbles and changes direction. Each run is about the same length.

What is important about chemoreceptor arrays?

If signaling molecule present, chemoreceptor is on. Attractant decreases- CheA is phosphorylates, transfers phosphoryl group to CheY, CheY-P binds flagellar motor to switch its direction, CheZ dephosphorylates CheY-P to reset.

How do bacteria grow?

Increase in length and divide into 2 daughter cells- binary fission. Some bacteria divide asymmetrically.

What does bacterial growth mean?

Population growth and an increase in cell numbers or biomass

What happens during bacterial elongation?

Chromosomes are replicated so one daughter cell gets a copy.

What is a microbe?

a living organism that requires a microscope to be seen

What are prokaryotes?

Bacteria and Archaea

What are eukaryotes?

algae, fungi, protozoa

What are the 3 domains of life?

Bacteria, Archaea, Eukarya

What is the chromosomal material for all cells on earth?

Double stranded DNA

What is bacteria and archaea cell volume?

1-100 um^3

What is eukarya cell volume?

1-10^6 um^3

What is the type of DNA chromosome for archaea and bacteria?

Circular

What is the type of DNA chromosome for eukarya?

Linear

What is the DNA organization for bacteria and eukarya?

Nucleoid

What is the DNA organization for eukarya?

Nucleus with a membrane

What's the gene organization for bacteria and archaea?

Multigene operons

What's the gene organization for eukarya?

Single genes

What's the metabolism for bacteria and archaea?

Dentrification, N2 fixation, lithotropy, respiration, and fermentation

What's the metabolism like of eukarya?

Respiration and fermentation

What's the different taxonomic hierarchy of classification sections?

Domain, phylum, class, subclass, order, family, genus, species

What is the endosymbiotic theory?

Bacteria diverged from archaea and eukarya before archaea and eukarya diverged from each other

What is endosymbiosis?

one organism living inside another

What are some examples of endosymbiosis?

Chlorelle growing with paramecium, drosophila carrying wolbachia

What are the different categories for classifying bacteria?

Cell shape, cell arrangements, motility, staining properties

What are some differential stains?

Gram stain, malachite green, acid fast

What percent of total weight of a bacteria is water?

70%

How thick is the cell membrane?

8 nm thick

What are some components of the cell membrane?

Hopanoid, transporter protein, phospholipid, proton driven atp synthase

What is the function of the cell membrane?

Site of Energy generation, key location of about 200 proteins, permeability layer & location of transport proteins

What type of molecules is the cell membrane an effective barrier for?

Polar and charged molecules

What are phospholipids?

Fatty acids attached to glycerol backbone via ester linkages.

What is triclosan?

An antibiotic that targets fatty acid synthesis

What are some things that can vary membrane phospholipid composition?

Temperature, acid stress, starvation stress

What do hopanoids do?

Stabilize the membrane by filling gaps between hydrocarbon chains

What are the variation in phospholipid side chain structures for archaea?

L-Glycerol, ether links between glycerol and fatty acids, isoprenoid chains, cross-linked lipids, cyclopentane rings, lipid monolayer