Cell Molecular Biology

DNA model

semi-conservative, template strand, 5-3 always

DNA polymerase

nucelotide polymerizing enzyme. DNA primer strand polymerizes in the direction of 5 -3, new nucleotide added to 3’ end of OH group (triphosphate, hydrolysis occurs to make it a monophosphate —> this energy is what drives the reaction)

DNA creation Bonds

Covalent bond in backbone, Hydrogen Bonds between base pairs

Processive Polymerase

Particular Enzyme can facilitate numerous reaction before letting go

high fidelity

fidelity —> the degree of exactness with which something is copied or reproduced.

Correcting Mistakes

1) DNA Polymerase —> Already favors correct nucleotides (wraps tighter, more favorable)

2) Exonucleolytic proofreading —> happens if there is a wrong template, or nucleotide wasn’t attached properly, exonuclease will chop off the wrong nucleotide —> on an editing site in DNA polymerase (OCCURS IN 3’-5’ DIRECTION)

3) Strand Directed Mismatch Repair —> removes replication errors made by DNA polymerase that were missed by exo

Replication Forks

Multienzyme complex that facilitates replication —> creation of okazaki fragments and lagging strand that isn’t in the proper direction of 5’-3’

Primers

RNA primer synthesized by DNA primase, DNA polymerase lengthens it to create okazaki fragment, DNA polymerase finishes the fragment, and removes primer. DNA ligase then comes in and fix the gap. Mistakes in RNA Primer region usually, so DNA poly must fix

DNA Helicase + SSB proteins

prevent self base pair binding/hairpin helices

cooperative binding

once one binds, the rest come and bind quicker

Replication Origins

Local Opening of DNA helix by initiator proteins (typically AT rich —> weaker pairing (2 h bonds, so easier to pull apart)

2 for eukayrotes

Telomerase

replicate chromosome ends. extends ends and serves as template so lagging strand can be filled out by. dna poly —> because the final RNA primer cannot be replaced with DNA, a single-stranded overhang remains, causing chromosomes to shorten each cycle.

Telomerase extends the template strand's 3' end, allowing primase and DNA polymerase to complete the lagging strand.

RNA molecules

single stranded that can fold into specific structures like polypeptides, can leave nucleus, ribose sugar, uracil instead of thymine

Transcription vs Replication

Similarities:

• begins with unwinding of DNA

• nucleotide is determined by complementary base pairing

• proceeds 5’-3’

Differences:

• RNA strand does not remain hydrogen bonded to the DNA template strand

• RNA chain is displaced and the DNA helix retwists

• RNA polymerase catalyst not DNA poly

RNA polymerase

catalyzes formation of phosphodiester bond, linking nucleotides together. Growing RNA chain extended by one nucleotide at a time in the 5’-3’ direction.

RNA poly DNA poly

substrates used to give energy to drive the reaction —? ATP GTP UTP CTP etc

RNA poly vs DNA poly

RNA poly- can start rna chain w/o primer, because transcription doesn’t need to be as accurate as DNA replication- it only has 1 site and NO correction site in the exonucletic editing activity on DNA poly

NOT STRUCTURALLY SIMILAR (convergent evolution lolololol)

mRNA

code for proteins

ribosomal RNA

form ribosomes and catalyze protein synthesis

transfer RNAs

serve as adaptors between mRNA and amino acids

microRNA

regulate gene expression

other NON-CODING RNA

used in RNA splicing, gene regulation, telomere maintenance, and other processes

Steps for transcription: (in prokaryotes)

1) rna poly binds to a SIGMAAAAAA factor on the dna chain RNA polymerase binds to, which locates a promoter DNA on dna sequence.

2) rna poly unwinds and transcription bubble created

3) polymerase breaks interactions with promoter DNA after 10 nuces

4) sigma factor releases and polymerase tightens around DNA and moves 50 nuc/sec

5) polymerase encounters the terminator DNA sequence that dissociates polymerase

6) transcription terminated, and transcript and polymerase released from DNA

Start and Stop sequences in DNA (PROKAYROTES)

Start: the -35 sequence or the -10 sequence, specific sequences determine promoter strength and attraction factor for sigma.

PROMOTER DETERMINES DIRECTION OF TRANSCRIPTION.

Stop: sequences that form RNA hairpins, many variable sequences

Transcription in Eukayrotes:

Multiple types of RNA poly. Rna 2 transcribes most protein coding genes, Rna 1 transcribes rrna, Rna 3 transcribes trna

After rna is transcribed —> poly a tail at 3’ end, 5’ cap at 5’ end, allows assessment of whether both ends of RNA are present

—> this processing occurs at the same time as transcription

Transcription Initiation in Eukayrotes

transcription initiation: needs general transcription factors (not just sigma factors). transcription must occur in nucleosomes and higher forms of chromatin structure

GTF’s —> position RNA poly, aid in pulling apart DNA, release rna poly frm promoter

1) DNA promoter sequence has a TATA box, located 25 nucleotides from start of transcription

2) TFID and TBP recognizes and binds to the TATA box, and facilitates binding of TF2B (transcription factor 2b), which causes a kink in DNA that facilitates association of other factors

3) TF2H uses ATP hydrolysis to melt helix and expose template strand

4) TF2H also phosphorlyates RNA poly 2 in c terminal domain releasing general transcription factors to begin elongation phase of transcription

THEN

1) due to the chromatin, ‘upstream’ of TATA box, there is an enhancer region, where activator proteins bind, bring mediator to promoter.

Activator Proteins

1) at enhancer, they recruit ATP dependent chromatin remodeling complexes and histone modifying enzymes, this exposes segments of DNA for transcription

2) proteolysis of activator proteins

chromatin remodeling complexes

removes dna that is wrapped around histones, so they can be expressed

histone modifying enzymes

changes ability of chromosomes to be opened or closed to allow for expression of DNA into proteins

processing rna

Exons and Introns

exons- expressed sequences

introns - intervening sequences (pre-rna) —> must be spliced

splicesome —> recognizes splicing signals on pre mRNA molecule, catalyes rxn

Introns

Splicing out different introns allows the same gene to produce multiple different protein isoforms

over time, the introns may allow for evolutionary advantage or just emergence of new proteins through genetic recombination

mRNA selective nuclear export

5 cap and poly a tail marked by proteins to recognize these modifications, deeped export ready

Translation

An mRNA sequence is decoded in set of three nucelotides (codons)

polypeptides synthesized stepwise from N to C terminal end

Codons

ALWAYS written 5’-3’, multiple per one amino acid, AUG = tranlsation initiation codon

tRNA

80 long nucleotide adapter RNA molecules that recognize and bind both the codon and to the appropriate amino acid. has an ANTICODON that pairs with correct codon on mRNA

Aminoacyl tRNA synthetase

20 different synthetases link the proper amino acid to the proper tRNA a process called charging. Amino acid is bonded thru energy to correct anticodon/tRNA

Ribosome

small subunit —> framework where tRNA are accurately matched to mRNA codon.

Large subunit —> catalyzes formation of peptide bonds. 2 amino per sec in eukayrote, 20 / sec in pro

4 binding sites —> one for mRNA, A site, P site, E(xit) site

Translation Termination

Stop codons —> UAA, UAG, UGA

release factors bind to any ribosome with a stop codon position at the A site

Forces peptidyl transferse (in ribsome) to catalyze addition of water molecule, instead of amino acid

Amino Acid R groups

Some are hydrophobic or hydrophillic, and this affects the folding of the proteins (by forming hydrogen bonds located outside the surface of the protein)

electrostatic attractions between r groups to change the structure of protein

peptide bond

by hydrolysis and dehydrationsynthesis, strong covalent bond

primary structure

sequence of amino acids, determines shape and function, covalent peptide bonds

secondary structure

a helix —> where each carbonyl group in the backbone forms a hydrogen bond with an amide group four groups away. Rt handed coil, outward facing

b sheet —> hydrogen bonds between the strand that runs parallel across from it R-groups project alternatively above and below the plane (Parallel/antiparallel sheets). Hydrogen bonds stabilize interactions of local amino acids along the polypeptide backbone.

tertiary structure

determined by Interactions Amino Acid Side Chains

-hydrophillic bs hydrophobic

- Ionic bonds

-Hydrogen bonds

-Van der waals forces

Quarternary Structure

2 or more polypeptide chains or subunits, Each has its own tertiary structure, ex: DNA polymerase

Diffusion-limited reactions

• rate determining step

so they need: Multienzyme complexes or Feedback Inhibition or Intracellular membrane systems ( the cell with mitochondria, golgi, ribsomes, etc)

Multienzyme complexes

evolved binding sites that concentrate them with other proteins of related function in

particular regions of the cell, thereby increasing the rate of efficiency of the reactions

that they catalyze

Enzyme domains are near each other in the folded protein

single ‘scaffold protein’ that can catalyze many reactions at once

Intracellular membrane systems

• Confining enzymes to particular cellular compartments

• Cells control how many molecules of each enzyme it makes

• phosphorylation

• Rate of protein destruction by targeted proteolysis

Feedback Inhibition

Direct, reversible change in the activity of an enzyme in response

to the specific small molecules that it binds

• stops the pathway when a large amount of the final product accumuluates

• will reverse when product level falls

Three major classes of cytoskeletal polymers

microtubules, actin filaments, intermediate filaments

microtubules

about 25 nm deep, a protofilament of a microtubule is made out of alphabeta - tubulin heterodimers (this is their subunit)

consists of 13 protofils, has + and - ends, hollow tube,

functions: cytoplasmic: organization and maintenence of cell shape and polarity, chromosome movements, intracellular transport

actin filaments

about 8 nm deep, the subunit is Globular-actin monomers

Structure: two intertwined chains of filamentous actin, 7 nm diameter, barbed pointed ends

Functions: muscle contraction, cell locomotion, cytokenesis, cytoplasmic streaming

intermediate filaments

8-12 nm deep, the subunit is an IF dimer

monomers: several different proteins

Functions: structural support, maintenence of animal cell shape, formation of nuclear lamina, strengthening of nerve cell axons, Desmin is a key intermediate filament protein that provides structural integrity to muscle cells by linking Z-discs, mitochondria, and the nucleus, particularly in cardiac and skeletal muscles.

Actin

Muscle: Actin + myosin = 60% of total protein (discovered 1940s)

Non-muscle cells: Actin = 15% of total protein (100 μM) (discovered 1960s)

Actin binds ATP or ADP. The affinity for ATP is higher, so

given the higher concentration of ATP in cells, G-actin is

saturated with ATP.

Globular Actin

Globular actin (G-actin) is folded into two domains that are stabilized by an adenine nucleotide lying in between

Filamentous Actin

has 13 actin molecules arranged on six left-handed turns repeating every 36 nm. The rise per subunit is 2.75 nm. The morphology of the actin helix is two intertwined, long- pitch, right-handed helices. Along each of the morphological helices, the actin monomers are spaced by 5.5 nm.

Actin assembly

nucleation, elongation, steady state

nucleation

lag phase, energetically unfavorable, it doesn’t want to create new phase, but once it does it will favor being in the new phase than ‘dividing’ again

elongation

Barbed ends elongate 100-fold faster than pointed ends

internal molecular clock

ATP hydrolysis provides an IMC,

Actin filament severing proteins

ADF/Cofilin —> they exclusively target OLD ADP actin filament, chopping them up to disassemble them

Actin filament bundling/crosslinking proteins

proteins with one-two+ binding sites, with filaments inbetween it

Molecular ‘motors’

For actin filaments, they are called Myosins. To bond actin filaments, they motor will pull the minus (-)/ pointed end towards each other to intertwin

Myosin II

dimers. they have a head thats a motor, where it will bind to actin. about 150 nm long (filaments intertwined), the tail binds cargo (to other myosin II’s).

Mechanism:

ATP hydrolysis drives the whole action.

1) ATP binds to head/motor domain

2) The myosin lets go of actin filament (due to confirmational change)

3) ATP hydrolysis occurs, phosphate released

4) ATP releases, changing confirmational shape of myosin, allowing the motor to bind and PULL the actin

processive motor - non processive

can take multiple steps along an actin filament before letting go, will let go after one

Myosin 5

PROCESSIVE MOTOR, walks towards the barbed end

Walks in a ‘hand over hand’ motion. One head is bound, the other swings over it and binds, then the cycle repeats.

Processive motor allows for long distance transport of filaments. allows cell to get bigger. LONG RANGE DIRECTED TRANSPORT

Microtubules

GTP. Exchangable GTP in periphery/Beta tubulin, NON EXCHANGABLE GTP in the center of tubulin (important for structure does NOT hydrolyze)

Also have lag elongation and steady state

POLAR POLYMER —> + end grows quickly

Microtubules (are the traintracks, that motors ‘walk’ on)

Nucleation of Microtubule: gamma tubulin is put in this ring, and templates the addition of alpha beta tubulin assembly

Nucleation occurs at the minus end (?) —> ASK

Dynamic Instability

Polymerizes and Depolymerizes (grows and shrinks)

A PROMINENT BEHAVIOR OF MICROTUBULES

Mechanism:

1) GTP in Beta tubulin at + end = GTP cap (rapidly elongates)

2) GDP after phosphorylation, changes to shrinking (GROWING TO SHRINKING CALLED CATASTROPHE)

3) Rapid shrinkage

4) If cap is reformed with new GTP, it will grow again

GTP presence has a straight configuration of protofilaments. Straight protofilaments can bond with each other very easily,

GDP tubulin has a bent confirmation - Bent cannot bond easily

ALLOWS A MICROTUBULE TO BE ABLE TO TAKE MULTIPLE TRIES TO CAPTURE CHROMOSOME IN MITOSIS, INSTEAD OF JS MISSING IT

Microtubule Associated Proteins

similarities to the actin ones (ask TA, do we need to memorize all of these), freaking out a little

KINESIN (microtubule motor protein)

processively walk ‘hand over hand’ , uses ATP hydrolysis, walks towards + end.

processive motor proteins

Dyneins (microtubule motor protein)

ODD ONE OUT —> walks to negative end (pointed end)

The motor domains ATP hyrdolysis occurs and has to travel some distance to get to stalk (isn’t this how all of them work?)

limit of resolution

seperation of where two objects sit, distinct

Fourescence Microscopy

you will have a molecule that is tagged with a flourescent protein.

The first filter only lets light through that will excite the particular color of flourescence. the beam splitting mirror will reflect the light onto a sample. the sample will be excited by the blue light, and then will emit light (based on color of protein) (get’s rid of background flouresence)

multi-color fluorescence microscopy

• can find different parts of a cell, by labeling each part with a different flourescent protein, and then repeat microscopy (just switch the filter sets)

Antibodies - labeling with flourescence

1) Have something you want to label (like a microtubule)

2) PRIMARY ANTIBODY —> recognizes the object

3) SECONDARY ANTIBODY —> flourescent molecule that recognizes the primary

flourescent protein

• using translational fusion, to code this protein into the genome of the organism ur studying, so when the organism ur studying expresses this particular protein, you can see it thru flourescence

flourescent dye

• very bright and don’t bleach, but they aren’t encoded

• to get them to label the protein of interest (USE THE ANTIBODIES), add this dye onto a secondary antibody

TIRF microscopy

SINGULAR molecule thru fluoresence

• the laser comes in at a angle, so it only catches a few molecules that are in the top layer, so it removes any background molecules

electron microscopy

• lowest limit of resolution very small

Allosteric Regulation Linkage - POSITIVE

Positive when = regulatory molecule is bound to it for it to be active (TURNS SOMETHING ONNN)

Allosteric Regulation Linkage - Negative

Negative when = regulatory molecule is bound to it for it to be inactive (turns it off and stops it from producing more) (TURNS SOMETHING OFF)

Protein Phosphorylation

• addition of a phosphate group to one or more of its amino acid side chains

• it can —> cause major confirmation change, and attract positive charges (since P is negative)

• an attached p group can change the structure / binding sites for another protein to recognize

• addition of a phosphate group can mask minding site that holds proteins together (proteins become detached)

phosphorylation

Can change confirmation of protein, changing what binds to it, changing what it attracts

Kinase

catalyzes the phosphorylation reaction (ATP —> ADP, P to a serine, threonine, or tyrosine amino acid)

phosphatase

catalyzes dephosphorylation

GTP-binding proteins

• self hydrolyzes to GDP to turn something off.

• Then a new GTP can come in and turn in back on to turn protein on

• in the on phase, it can bind to another molecule to ‘power it’ (molecules called effectors)

GAP

regulatory protein to control activity of GTP binding protein to determine whether GTP or GDP is bound

• turns GTP back to GDP, to turn protein off (INHIBITOR)

GEF

regulatory protein to control activity of GTP binding protein to determine whether GTP or GDP is bound

• turns GDP to GTP, to bind to protein (ACTIVATOR)

Ubiquitylation

Ubiquitin, a protein that can be covalently attaches to target proteins.

small regulatory protein that tags other proteins for degradation, trafficking, or functional modification within eukaryotic cell

1) ATP hydrolyzes to ubiquiting from E2 to E1 molecule

2) E2 complex targets a certain protein, and tags it with a polyubiquitin chain (notifying cell of what to do to it)

Nucelosomes (structure/function)

basic units of chromosome structure (packed bit of DNA)

—> 8 histones, 147 nucleotide pair long of DNA

—> its bound so tight bc since histones are positive, and phosphate backbone of DNA is negative —> tight binding

nucleosome to chromatin

• nucleosomes are like beads on a string

• The linker histone H1 binds to the outside of the nucleosome, sealing the DNA and aiding in further compaction.

• allows for further bonding/tightening

linker histone H1

5th histone, binds to the outside of the nucleosome, sealing the DNA and aiding in further compaction.

• allows for further bonding/tightening

Regulation of chromosome structure

chromatin remodeling complex

histone modifying enzyme

chromatin remodeling complex

use ATP hydrolysis to loosed nucleosomal DNA

histone modifying enzyme

tails of all core histones are subject to modifications (addition or removal of groups), changes structure

• Acetylation

• Methylation

• Phosphorylation

DIFFERENT FOR DIFFERENT HISTONES

histone modifying enzyme

can change configuration to create Heterochromatin, or to create euchromatin

Heterochromatin

tightens dna to stop expression

euchromatin

loosens dna to increase expression

For DNA creation

triphosphate, hydrolysis occurs to make it a monophosphate —> this energy is what drives the reaction

exonuclyetic proofreading

happens if there is a wrong template, or nucleotide wasn’t attached properly, exonuclease will chop off the wrong nucleotide —> on an editing site in DNA polymerase (OCCURS IN 3’-5’ DIRECTION)