Bio130

course

Nucleus

The structure that contains the chromosomes (DNA) in the center of the cell

area of transcription

Endoplasmic Reticulum

Irregular maze of spaces interconnected with membrane where cell exports are made(proteins)

very large in excretory cells.

Golgi apparatus

Processes and packages proteins and lipids meant for transport inside or outside of the cell. Stacks or membrane enclosed sacs.

Nucleolus

Produces and assembles ribosomes in the nucleus (rRNA gets transcribed and then assembly occurs)

Prokaryotic Cell

* Cell with no nucleus
* Single Celled
* Bacteria and Archaea
* No membrane-bound organelles
* Smaller
* Less DNA
* Ribosomes in cytosol

Eukaryotic Cell

* Nuclei
* Single Celled OR Multi-cellular
* Plants, Fungi, Animals, Humans
* Several membrane-bound nucleus
* Larger and more Complex

Differences between DNA and RNA

Dna

* Double Stranded
* Has deoxyribose Sugar
* Is transcripted
* Synthesized from dNTPs

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Rna

* Is Translated
* Single Stranded
* Has Ribose Sugar
* Synthesized from NTPs

Parts of Nucleotides

1) Nitrogenous Base

2) Pentose Sugar

3) Phosphate Group

Central Dogma

Genetic information flows in 1 direction; from DNA to RNA to protein

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Origins of Mitochondria

resemble bacteria in many ways, and are thought to derive from bacteria that were engulfed by an anaerobic bacterium that later is an ancestor to present day Eukaryotic cells.

Origins of Chloroplast

Also contain their own DNA, and reproduce by dividing into two and are believed to have evolved from bacteria that were engulfed by an early aerobic eukaryote

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Evolved later in time compared to mitochondria

Nomenclature

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Endosymbiont Hypothesis

1. Mitochondria and chloroplasts still have remnants of their own genomes and their genetic systems resemble that of modern day prokaryotes.
2. Mitochondria and chloroplasts have kept some of their own protein & DNA synthesis components and these resemble prokaryotes too.

Nucleoside

Contains base, and sugar ONLY (can also act as a chemical energy carrier ATP)

Nucleotide

Contains a base (varies), sugar (scaffold for base) AND phosphate group (backbone1,2,3)

Monomer/ subunit of Nucleic acid, order encodes genetic info based on base. Joined with phosphodiester bonds.

Mouse, E-coli, FruitFly, Brewers Yeast, Zebra Fish

Model Organism Examples

* General attributes:
* rapid development with short life cycles
* small adult size
* readily available
* easily manipulated
* understandable genetics

DNA structure

* 2 Chains of complementary nucleotide polymers running antiparallel
* strands held via hydrogen bonds between the bases of nucleotides (inside of helix)
* Double Helix (10 bases per twist)
* phosphodiester bonding between the phosphate group on the 5’ carbon and the hydroxyl on the 3’
* chemical polarity
* information encoded in the sequence of the nitrogen bases
* Major and minor groove (that proteins can interact with)
* deoxyribose sugar
* Bases (Thymine, Adenine (2 hbonds), cytosine, guanine(3hbonds))

Molecular interactions

* Electrostatic attractions
* Hydrogen Bonds
* Van der Waals attractions
* Hydrophobic force

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Purines

* “Al gore stinks PU”
* have two rings
* Adenine guanine

Pyrimidines

* have one ring
* “U C the Pyamids
* bases: U, C, T

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Lysosomes

* Irregular shape
* where intracellular digestion occurs
* releases nutrients into the cytosol
* breaks unwanted molecules for recycling or excreting from cell

Cytoskeleton

* filament often anchored at one end of the membrane or radiating out from a side next to the nucleus
* thinnest filament: actin
* thickest: microtubules
* separates chromosome pairs and pulls them to their respective poles during mitosis
* even found in bacteria for use in division

Mitochondria

* found in all eukaryotes
* double membrane
* inner membrane folded into inward-facing folds
* contain their own DNA and reproduce via division
* generate energy for the cell from the oxidation of food molecules to produce ATP

Transport vesicle

tiny membrane sacs that move materials (such as proteins and molecules) throughout the cell.

transport vesicles move cell proteins to the Golgi apparatus

Peroxisomes

* found in eukaryotic cells
* organelle enclosed by a single membrane
* contains enzymes that degrade lipids and destroy toxins.

Nuclear envelope

* found in eukaryotic cells
* two lipid bilayer membranes: inner nuclear membrane and outer nuclear membrane
* separates contents of the nucleus from the cytoplasm
* structural framework of the nucleus

Cytosol

* intra-cellular fluid (liquid found inside the cell)
* solution of water, proteins, and metabolites
* allows organelles, proteins, and other cell structures to float

Differences between Animal and plant cells

* animal cell:
* no cell wall
* no chloroplast
* contains a centrosome and lysosomes
* plant cell
* contains cell wall
* contains chloroplast
* no centrosome nor lysosomes

endocytosis

* process by which cells absorb external material by engulfing it with the cell membrane
* includes two processes
* pinocytosis (“cell drinking”): soluble materials are ingested by the cells and incorporated into vesicles for digestion
* phagocytosis (“cell eating”): particulate materials are ingested by the cell. plays a role in the elimination of foreign substances.

exocytosis

* process where molecules are transported out of a eukaryotic cell
* involves the fusion of secretory vesicles with the plasma membrane
* releases vesicle content, and incorporates new proteins and lipids into the plasma membrane

RNA structure

* typically single stranded
* contains ribose as its pentose sugar
* contains the pyrimidine uracil instead of thymine
* polynucleotide composed of covalently linked ribonucleotide subunits

proteome

All the proteins in a living cell or organism

Genome

All the DNA sequences in a living cell or organism

Cell Theory

the cell is the basic organizational unit of life

all organisms are composed of one or more cells

cells arise from preexisting cells.

Transcriptome

all the RNA sequences in a living cell or organism

Interactome

all protein-protein interactions in a cell or organism

metabolome

all the small the small molecule metabolites in a cell or organism

Amino Acids

* consist of an alpha carbon that’s attached to an amino group(N-terminus) and a carboxylic group (C-terminus)
* each amino acid has a unique side chair that is attached to the alpha carbon (called the R group)
* They’re the monomers of peptides and polypeptides
* There are 20 unique ones
* there are d an l forms (they’re optical isomers)

Proteins

* long polymers of amino acids linked by peptide bonds and they are always written with the N-terminus on the left side

Peptides

* shorter polymers of amino acids, usually fewer than 50 amino acids long

Amino Acid Families

* acidic
* basic
* uncharged polar
* nonpolar

R group determines type

Amino acids and the Genetic Code

Disulfide bonds

* helps stabilizes protein structure acts almost as “braces”
* there are interchain and intrachain disulfide bonds

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Peptides bonds

* hold together amino acids in their polymers
* forms between the carboxylic group of the amino acid and the amino group R GROUPS ARE NOT INVOLVED
* is a condensation reaction as it produces water upon formation
* the amino group is then referred to as an amide and the carboxylic group is now a carbonyl.
* The former amino acids are now referred to as residues

Backbone atoms of the polypeptide

all the protein atoms except the side chains

chaperone proteins

* They’re proteins that assist with protein folding
* bind to them and guide them to fold in the most energetically favored path or provide an isolated chamber where they can fold undisturbed

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Alpha helix

* common secondary structure of proteins
* forms via hydrogen bonds between N-H and C = O groups in the BACKBONE (R groups are not involved and just stick out)
* Bonds form between the n and the n + 4 residue (e.g. 1 and 5, 2 and 6)
* it’s a regular right-handed helix
* typically found when there are many similar subunits arranged right next to each other

ATP

* Adenosine Triphosphate
* principle carrier of energy in cells
* composed of adenine, ribose, and three phosphate groups

phosphodiester bond

* the linkage between the 3' carbon atom of one sugar molecule and the 5' carbon atom of another
* hold together nucleotide subunits within a DNA strand

Beta Sheet

Folding pattern found in many proteins where neighbouring regions of the polypeptide chain associate side-by-side with each other through h-bonds to give a flat rigid structure

The H-bonding occurs between the carbonyl oxygen of 1 a.a and amide hydrogen of a.a in neighbouring strand

R groups are not involved in the structure but are projected up and down

Usually contain 4-5 beta strands

Can either be anti-parallel or parallel (longer but requires extra sequences)

Protein Structure

Primary (aa sequence)

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Secondary (local folding) - ex. alpha helix, beta sheet

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Tertiary (long-range folding) - ex. 3D structure

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Quaternary (multimeric organization) - ex. more than 1 polypeptide chain (Not all proteins experience this)

Coiled Coil

Two or more alpha helices twist repeatedly around each other

They wrap around each other to minimize exposure of hydrophobic amino acid side chains to aqueous environment

Amphipathic - contains both hydrophobic (side-groups) and hydrophilic (interior groups) parts

Protein Domains

Any segment of a polypeptide chain that can fold independently into its own compact stable structure

This portion of protein often functions in a semi-independent manner within the protein

Eukaryotic proteins often have 2 or more domains connected by intrinsically disordered sequences to allow for flexibility within the protein

Protein Families

A group of proteins with common evolutionary origin

Fold similarly and have similar function

Most proteins belong to families with similar structural domains

Protein Machines

“Machines” made up of individual proteins that collaborate to perform a more complex function

Tertiary structure

proteins will generally fold into the conformation that is the **most energetically favoured**

Proteins will fold into the shape dictated by their amino acid sequence, but chaperone proteins help make the process more efficient and reliable in living cells

Backbone Model

→ Protein Model

* Shows overall organization
* Straightforward string-like model

Ribbon Model

→ Protein Model

* Shows Polypeptide bonds
* emphasizes its most conspicuous folding patterns

Wire Model

→ Protein Model

* Shows positions of all amino acid side chains
* useful to predict which A.A’s responsible for activity

Space-Filling Model

→ Protein Model

* Contoured map of proteins surface
* Shows what amino acids are exposed on surface
* Shows how protein might look like to a smaller molecule

Prion

misfolded protein

Biological Function Protein Requirements

* Well-Behaved
* Well Defined 3D conformations
* Stable

Dimer

2 Identical folded polypeptide chains form a symmetrical complex of two protein subunits

* held together by two identical binding sites

Acidic Side Chains

Aspartic Acid and Glutamic Acid

Mass Spectrometry

determines amino acid sequence of proteins that have been isolated from organisms for which the full genome sequence is known. Determines mass of every peptide fragment.

Electrophoresis

Mixture of proteins loaded onto polymer gel and subjected to an electric field. Size and net charge of polypeptides causes them to move through gel at different rates.

F.I.S.H

* Fluorescence In Situ Hybridization
* Diagnostic technique
* DNA probes are used to mark the locations of their respective nucleotide sequences
* The probes are labeled with different chemical groups that can be detected with fluorescent antibodies specific for those groups

Chromosome painting

Similar to F.I.S.H, chromosome painting uses multiple probs to mark specific sequences in chromosomes and “paints” them in a fluorescent dye

Chromatin levels of organization

The process starts with assembly of a nucleosome, which is formed when eight separate histone protein subunits attach to the DNA molecule.

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Multiple nucleosomes are coiled together and these then stack on top of each other. The end result is a fiber of packed nucleosomes known as chromatin.

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This fiber, which at this point is condensed to a thickness of 30 nanometers is then looped and further packaged using other proteins. The multiple folding allows 6 feet of DNA to fit into the nucleus of each cell of our body

The end result is that DNA are tightly packed into the familiar structures we can see through a microscope: chromosomes.

Nucleosome

Basic structural unit of a eukaryotic chromosome composed of short length of DNA wrapped around an octameric core of histone proteins; includes a nucleosomal core particle (DNA plus histone protein) along with a segment of linker DNA that ties the core particles together

* Octamer: H2A H2B H3 and H4
* linker histone H1

Histones

Small group of abundant, highly conserved proteins rich in lysine and arginine around which DNA wraps to form nucleosomes, structures that represent the most fundamental level of chromatin packing

Non-histone proteins

Chromatin in human chromosomes are folded into loops by special nonhistone chromosomal proteins that bind to specific DNA sequences, creating a clamp at the base of each loop

Chromatin packaging and re-modelling

Chromatin remodeling complexes and histone modifying enzymes are examples of proteins that make changes to chromatin structure and alter access to DNA for replication or transcription.

Heterochromatin

Highly condensed chromatin of an interphase chromosome; generally gene-poor and transcriptionally inactive

Euchromatin

Relatively non-condensed chromatin that exists within an interphase cell. Prevalent in gene-rich areas, its less compact structure allows access for proteins involved in transcription

Direction of DNA replication

* DNA is always synthesized in the 5'-to-3' direction
* Unidirectional growth of a single strand from two points
* Unidirectional growth of two strands from one point
* Bidirectional growth from one point

Characteristics of sequences in replication origins

* easy to open, A-T rich
* Recognized by the binding of initiating proteins

Replication bubble

* Structure formed by the separation of two DNA strands by the helicase enzymes
* Specialized proteins bind to the origin site, and open up the DNA
* Replication forks are formed and continue to move in opposite directions as replication proceeds.

Origin of replication

* a particular sequence in a genome where replication is initiated.
* Single on in Bacteria Multiple eukaryotes

DNA replication in Bacteria

* A bacterial genome has a single origin of replication (unique to circular genomes) forming one replication fork
* Supercoiled DNA is unwound by DNA gyrase
* Helicase separates the DNA strands by breaking the hydrogen bonds between the nitrogenous base pairs.

DNA replication fork

* Y shaped function (though it can curve to improve efficiency of the DNA polymerase (brings them in proximity to each other)
* two per replication origin going in different directions
* asymmetrical with a leading strand synthesized continuously and a lagging strand made discontinuously.

Nucleosome core particle

* consists of DNA wound around a core of histone proteins
* each one contains a complex of eight histone proteins
* two molecules each of H2A, H2B, H3, and H4
* double stranded DNA wounds around the protein complex
* DNA is 147 nucleotide pairs long

Chromatin

* single, long, linear DNA molecule associated with proteins
* Dynamic
* tightly packaged but DNA still remains accessible for transcription, replication and repair

Retrotransposons

* DNA sequences that are made into RNA

Initiator Proteins for replication in E.Coli

* Binds to origin
* helps helicase bind
* requires ATP

DNA-only transposon

* Sequences that only exist as DNA

Mobile Genetic elements

* Regions of DNA that make copies and inserts themselves back into the sequence

Simple elements

* simple sequences that are often repeated example: AG 30 - 100 times

segment duplications

* 1000’s - 200,000’s of base pairs and duplicated

Introns

* transcribed but spliced out of the RNA (not translated)

protein-coding exons

* sequences transcribed and translated (made into proteins)
* Wilk Wilk have untranslated regions

non-repetitive DNA that is in neither introns nor exons

* A Unique Sequence that makes up about 30% of the Human Genome
* promoters (help determine how much RNA is transcribed and which cells transcribe the RNA)

Packaging of DNA in the Cell

DNA is condensed through folding and twisting about 1000 fold and is complexed with proteins.

* Forms the prokaryotic nucleoid
* In eukaryotic cells, this DNA is packed into chromosomes to save space in nucleus

Conservative versus semiconservative DNA

Conservative

* Parental Cell makes one pure daughter cell and one newly synthesized daughter cell

SemiConservative

* Parental Cell makes two daughter cells that are both half newly synthesized and half parental strand

Interphase

Long period of the cell cycle between one mitosis and the next. Includes G1, S Phase, and G2 phase

Correlation of genome size

Genome Size does NOT equal size of animal

Re-orientation of chromatin in expressed DNA

Condensed into chromosomal territory - homologous chromosomes detected by hybridization techniques and a specially marked gene that can be on or off.

Non-Packaged State

Small prokaryotic genome would occupy a considerable portion of cell volume in this state

The Human Genome

* only 1.5 % codes for a protein
* 50% of genes are repetitive DNA
* 50% repeated sequences
* 50% Unique sequences
* 3 billion base pair per genome
* one genome from one parent and one genome from another parent
* \~19,000 protein-coding genes spread across 23 pairs of chromosomes

Binding of initiator Proteins

Binds to specific DNA sequences called replication origins, prying open the DNA strands breaking the h-bonds present (AT rich segment, not a lot of h-bonding present in that region)

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A group of proteins (helicases) are attracted to the short segments of unzipped regions to carry out DNA replication. These proteins form a replication machine, in which each protein carries out a specific function.