Cell and Development Prelim 1

Resolving power

Ability to distinguish two close objects. The maximum resolution is determined by the wavelength of illumination.

Light microscopy

Microscopy method that uses photons of visible light to resolve objects up to 200 nm apart

Electron microscopy

Microscopy method that uses electrons with wave properties to illuminate structures as small as atoms, with a resolution of 0.2 nm.

2 specific methods:

1) Indirect immunofluorescence

2) Green fluorescent protein (GFP)

Advantages:

a) Much better resolution than light microscopy

Disadvantages:

i) Much more effort to get a 3D view of the cell

ii) Much more difficult to label specific proteins

iii) Cannot be used in live cells

Indirect immunofluorescence

Mechanism:

1) Fix cells by using a detergent to damage plasma membrane (kills the cells) but everything else stays intact

2) Now primary antibody can be added - serves as a marker of the protein

3) Then a secondary antibody can be added that recognizes the primary antibody. This will have a fluorescent marker on that will allow us to visualize it under the microscope.

Characteristics:

a) Specificity: Uses an antibody to your protein of interest "antigen"

b) Sensitivity: Utilization of fluorescence - emitting light of a different wavelength showcases a target protein

Green fluorescent protein (GFP)

Protein tagging that utilizes the fluorescent protein with same name to watch the movement of a protein and see its mechanisms in action.

Characteristics:

a) Specificity: GFP fluorescence

b) Sensitivity: GFP protein "fusion"

Central dogma

Describes how information is passed through different systems in the cell in molecular biology. It begins with the DNA sequences, before becoming RNA through RNA transcription, which then is translated into proteins.

Macromolecules

Signature of life. They provide function to the cell in every way. There are 4 types of macromolecules:

1) Carbohydrates

2) Proteins

3) Lipids

4) Nucleic acids

In order to create all of these polymers, each monomer pair goes through a condensation reaction, which creates a bond through the removal of water.

Carbohydrates

Made up of sugar monomers (monosaccharides) to produce polysaccharides.

Proteins

Amino acids bind to create polypeptide chains, which bind to create these.

Lipids

Fats and oils made from fatty acid chains.

Nucleic acids

Nucleotides are the building blocks for all genetic information - there are 4 types for DNA and RNA. Bound by phosphodiester linkages.

1) DNA's monomers are known as deoxynucleotides

a) Made up of a deoxynucleoside (base and deoxyribose sugar) and phosphate group in the 5' end

b) Contains a hydrogen on 2nd carbon of sugar rather than hydroxyl, which is what ribose contains, which is what makes DNA more stable than RNA and evolutionarily might be why DNA become the main form of storing genetic information

c) 4 different bases that can be divided into 2 subcategories

i) Pyrimidines (singular ring) : cytosine, thymine

ii) Purines (contain double rings): adenine, guanine

d) Adenine pairs with Thymine, Cytosine pairs with Guanine

2) RNA's monomers are known as nucleotides: made up of a ribonucleoside (base and ribose sugar) and phosphate group

DNA

Characteristics:

1) strands will always be antiparallel to one another within their double helix

2) Complementary base pairs create hydrogen bonds between the two strands. Hydrogen bonds define the genetic code because their presence indicates a genetic code is available to be replicated - indicates that there are templates to be replicated.

3) Replication is a semi-conservative process. One old strand and one new strand is included in each daughter cell. Per parent cell there are 2 daughter cells, meaning a total of 4 strands are seen per replication round, with one old strand and one new strand per daughter cell. The molecules acts as a template for its own replication.

DNA replication

Process:

1) Synthesis begins at the replication origins, where the double helix is opened up to create replication forks

2) Many replication origins form for eukaryotic cells

3) Primase synthesizes short RNA primers that provide the 3' end needed for DNA polymerase to start adding nucleotides.

4) DNA polymerase adds nucleotides to the 3' end, meaning that synthesis occurs in the 5'-3' direction. Synthesize very quickly. DNA polymerase has its own checking method and can go back if it makes a mistake when adding nucleotides.

5) The leading strand goes through this process uninterrupted. The lagging strand must allow for primase to add multiple RNA primers, creating short fragments of copied DNA known as Okazaki fragments along the template lagging strand.

6) RNA primers are removed and replaced with DNA to seal gaps. For the lagging strand, however, there is a gap at the end of the strand where the 3' end of the parent strand is still left uncopied. If no mechanism is present to fix this, the strands of DNA in daughter cells would grow shorter over time with every replication. This is known as the 'end-replication problem'.

7) Telomerase synthesizes telomeres, a short RNA molecule. This telomere can bind to the end of the chromosome and serve as a template for that empty portion of the lagging strand's new copy.

DNA genome organization

1) Nucleosomes: DNA is compacted into the nucleus by being wrapped around histones to form a protein complex

2) Chromatin fiber: an array of DNA-histone molecules strung together, like beads on a string.

3) Chromosome: Further condensation of chromatin fiber by scaffolding proteins that occurs during cell division to eventually be segregated to daughter cells.

Transcription

Occurs in the nucleus of the cell, and one DNA strand serves as the template strand for this RNA molecule, known as messenger RNA (mRNA). Allows for amplification and regulation. Depending on the transcription factors and the promoters in play, which sequences get transcribed can be controlled and regulated.

Regulation of prokaryotic transcription

1) A promoter sequence within the DNA strand is the indicator for transcription to begin.

2) RNA polymerase binds to the promoter region with the assistance of transcription factors. In prokaryotes, the transcription factor is known as a sigma factor.

3) RNA polymerase reads the template strand, and synthesizes the molecule in the 5'-3' direction. It tends to have more errors than DNA, but this is tolerable because many molecules of RNA transcript are being constructed so if one is incorrect it doesn't affect the entire system of synthesizing proteins.

4) RNA polymerase stops at the terminator sequence on the DNA strand, causing it to fall off from the DNA strand and releases the RNA transcript.

Regulation of mRNA

1) 5' capping and 3' polyadenylation: co-transcriptional regulation; within the nucleus

2) Alternative splicing: post-transcriptional regulation; within the cytoplasm prior to translation.

Alternative splicing

1) Small nuclear RNAs (snRNAs) known as spliceosomes come in and take out portions of the mRNA known as introns when mRNA arrives in cytosol.

2) Introns are sequences of the mRNA that can code for proteins, but have been chosen not to be expressed for this specific protein synthesis

3) Exons are sequences that remain in mRNA after splicing and directly play a role in the synthesis of the protein

4) Allows for one transcript to code for many different proteins, and thus can serve as an amplification tool, or a regulation tool by allowing certain proteins to eventually be synthesized by choosing which introns to take out each time the same transcript arrives for splicing.

5' capping & 3' polyadenylation

Within the nucleus, specialized nucleotide attaches to the 5' end of the mRNA, while the 3' end gets poly-A-tails to attach (long stretch of adenine nucleotides). The addition of these two nucleotides during transcription affects how stable the mRNA molecule is, and influences the efficiency by which the mRNA will be translated.

Types of RNA

1) mRNA: protein coding

2) tRNA: protein synthesis (adaptors to mRNA)

3) rRNA: protein synthesis (machinery)

tRNAs

What pairs codons to their corresponding amino acids through the use of an anticodon.

Mechanism

1) The anticodon is located on the 5' end of this molecule.

2) In order to ensure that each molecule has the correct amino acid before the anticodon finds its codon, a protein called amino-acyl-tRNA synthetase attaches the two together via ATP hydrolysis.

3) There is at least one amino-acyl-tRNA for each amino acid.

Ribosome subunits

1) Small ribosomal subunit: Binds mRNA and matches tRNA to mRNA codons

2) Large ribosomal subunit: Catalyzes peptide bond formation. Also known as a ribozyme.

Amino acids

Characteristics:

1) Amino group: NH2 - N-terminus end

-> At pH 7, carries a positive charge

2) Carboxyl group: COOH - C-terminus end

-> At pH 7, carries a negative charge

3) Side chain: R

-> Identifies what amino acid we are looking at

-> Can be either hydrophobic and hydrophilic, and this determines how protein folding and function occurs.

4) alpha-Carbon: center C

5) Bound by peptide (covalent) bonds, formed through condensation reactions.

Translation

Begins when the mRNA arrives in the ribosome, containing rRNA and proteins ready to assist with peptide synthesis.

Process:

1) Met-initiator tRNA binds to the start codon Methionine (AUG). Only one that can initiate translation.

2) Met-initiator tRNA, translation initiation factors, and small ribosomal subunit binds to 5' end of mRNA, marked by the 5' cap. Starts to slide down towards 3' end.

3) Translation initiation factors disassociate to allow the large ribosomal subunit to bind.

4) The Met-initiator tRNA is now in the P-site of the ribosome. It is the only tRNA molecule that will not bind with the A site first.

5) The next amino acid-loaded tRNA enters the A site of the ribosome at the second codon.

6) The peptide bond forms between the second amino acid, in the A site, and the methionine in the P site. The large ribosomal unit catalyzes this peptide bond formation through the assistance of a ribozyme. The amino acid releases from the tRNA in the P site.

7) The tRNA slides into the E site of the ribosome, which allows for the molecule to be ejected from the ribosome.

8) Because no tRNA binds to a stop codon, this is how the ribosome knows that process has ended. Stop codons bind in the A-site along with release factors, and this interaction causes a hydrolysis reaction, where the ribosome adds a water molecule to the peptide, allowing for the ribosome, mRNA, and release factor to disassociate from the peptide sequence.

Noncovalent interactions involved in protein structure

1) Electrostatic interactions

2) van der Waals interactions

3) Hydrogen bonds

4) Hydrophobic interactions

Protein structure interactions: Electrostatic interactions

Between the positively charged amino group and the negatively charged carboxyl group

Protein structure interactions: van der Waals interactions

Due to electron clouds surrounding the side chains, natural, weak interactions occur between neighbouring amino acid side chains, regardless of the charge they present.

Protein structure interactions: Hydrogen bonds

Formed between the backbones of two amino acids

Protein structure interactions: Hydrophobic interactions

Depending on whether a side chain is polar or nonpolar, will determine if the side chain wants to be on the outside of the protein structure or towards the inside. If nonpolar, it is hydrophobic, and wants to stay away from the side of the protein that faces surrounding fluid. The hydrophobic side chain wants to minimize the surface area that is exposed to water, and thus the protein will fold inward.

Protein structure levels

1) Primary: the peptide sequence of amino acids that forms after translation

2) Secondary: alpha-helices and beta-sheets

-> Alpha-helix: hydrogen bonds forming between C-O of one peptide bond to N-H of another peptide bond. Creates a backbone of C-O and N-H where side chains stick out of the helix. The hydrogen bond forms between every 4th amino acid.

-> Beta-sheet: Hydrogen bonds between adjacent strands where the side chains alternate which way they stick out. C-O and N-H backbone forms in the middle of the sheet rather than a spiraling staircase.

-> Can be parallel or antiparallel

3) Tertiary: 3D structure of a single polypeptide chain

-> Protein domain: a segment of a polypeptide chain that can fold independently into a compact, stable structure. Each domain within a polypeptide chain has different functions, and bind to different things to carry out these different functions.

4) Quaternary: 3D structure of multiple polypeptide chains

Lock and Key Mechanism

The selective binding of a ligand to a protein is dependent on how the ligand can form noncovalent interactions with the binding site of said protein. The ligand must fit into the binding site in order for these noncovalent interactions to occur. The side chains of the protein form the binding site, and are what interact with the ligand. Thus, the side chains are the key to how proteins bind with ligands and thus function.

Enzyme

Characteristics:

1) Catalyze chemical reactions while remaining unchanged themselves

2) Lower the activation barrier in order to make favourable and unfavourable reactions occur. It does not change the energy difference between a reactant and its product, but rather speeds up a reaction that may or may not have occurred.

3) Change the energy of the transition state, which is the amount of energy needed for the reaction to occur.

4) Can be used to also drive unfavourable reactions, through a mechanism known as reaction "coupling": unfavourable reaction will be paired with a favourable reaction so that the entire reaction is made to be favourable

-> Many unfavourable reactions are driven by ATP hydrolysis, since it is the largest chemical energy source in cells.

Protein regulation

2 branches

1) Control protein amount

a) Rate of mRNA transcription

b) Rate of mRNA degradation

c) Rate of mRNA translation into protein

d) Rate of protein degradation

2) Control protein activity

i) Cellular localization (targeting to nucleus)

ii) Inhibition or activation by ligands (feedback inhibition): the last product in a chain reaction of enzymatic activity and products can inhibit the activity of the first enzyme that acted in this chain-reaction.

-> Allostery: conformational change of an enzyme by a product that was created in a chain reaction that the said enzyme initially catalyzed. This conformational change inhibits the enzyme's activity

iii) Inhibition or activation by another protein

iv) Protein modification (phosphorylation): occurs for proteins that contain residues that have an -OH group. A phosphate is added by a protein kinase to the residue holding the hydroxyl group, in the process hydrolyzing ATP. This changes the activity of the enzyme. Can either increase or decrease enzyme activity.

Cytoskeleton components

Consistis of 3 filament systems that provide shape and structure to cells:

1) Microfilaments - actin: Contractile machinery and giving the cell shape/movement

2) Microtubules - tubulin dimer: Organize the cells in terms of organelle distribution and important for organelle transport

3) Intermediate filaments: Cell and tissue integrity

Microtubules

Characteristics:

1) Organized by centrosomes. Centrosomes serve as the microtubule organizing center (MTOC).

2) They form hollow tubes of alpha and beta tubulin dimers. These tubes are formed by 13 protofilaments. Each protofilament is alternating alpha and beta tubulin dimers. Both alpha and beta tubulins are GTP-binding proteins - when GTP is bound to the dimers, it stabilizes the dimer and allows the protofilaments to assemble and for this polymer to grow.

a) Alpha-tubulin: serve as the minus end of the microtubule. This is the end that favours disassembly. Towards the cell body of an axon, embedded in the centrosome.

b) Beta-tubulin: serves as the plus end of the microtubule. This is the end that favours assembly. They hydrolyze GTP to GDP over time. Is attached to a GTP cap, and allows for the constant polymer growth. Grows towards the periphery edges of the axon.

3) Transport cargo along a nerve cell axon with the assistance of motor proteins. They serve as highways for the transport of organelles between the cell body and axon terminal. There are two motor proteins working for inward (toward cell body) and outward (axon terminal) - kinesin and dynein. The globular heads of these motor proteins allow for the movement along the microtubules. Their tails are the portions that hold cargo along these pathways. The globular heads are ATP-binding.

Kinesin

Motor protein moving towards plus end of microtubule in hand-over-hand motion.

Characteristics:

1) Moves ER towards the edge of the cell (axon terminal)

2) Couples ATP hydrolysis to conformational change

a) Leading head has ADP, lagging head has ATP.

b) ATP hydrolysis, particularly release of phosphate, loosens the attachment of lagging head

c) ADP is released from leading head and replaced with ATP, causing a conformational change that pulls "lagging" head with ADP forward by 16nm, resetting the process.

Dynein

Motor protein moving towards minus end of microtubule.

Characteristics:

1) Moves Golgi towards the center of the cell (cell body)

Myosin

Actin-binding protein facilitating muscle contraction.

Associate to form filaments. Contain head domains that bind to ATP. Moves in a non-processive motion.

Mechanism:

1) ATP binds to myosin head, causing the release of myosin from actin filament.

2) ATP is hydrolyzed, causing the myosin head, now tightly bound to an ADP and a phosphate, to become cocked, a conformational change of the head.

3) The new cocked state allows for the myosin head to weakly bind to the actin filament, causing the release of the phosphate.

4) This phosphate release causes another conformation change, called a power stroke, which moves the actin filament relative to the myosin filament towards the minus end

5) ADP is released, myosin remains attached, allowing for the cycle to repeat.

Actin Filaments

Characteristics:

1) ATP-binding filaments.

2) They are thin, flexible protein threads.

3) They contain a plus end and a minus end, but grow out only from the plus end.

4) Hydrolyzes ATP as it grows, and dissociates when bound to ADP.

Sarcomeres

Contractile units of muscle, and are made up of actin filaments and myosin filaments.

Sliding Filament Mechanism

The power stroke interaction creates this sliding motion of the myosin and actin filaments, where the actin filaments move towards the center of the sarcomere as a result of the myosin interactions.