the complete set of genetic info encoded in base pairs
Definition: Open Reading Frame (ORF)
portion of the genome that codes for a protein
Defintion: Regulatory Regions
control gene expression of ORF
the coding portion of a gene tat produces a functional product (eg. mRNA)
DNA regions within a gene that is not translated into protein
What is the central dogma of Omics?
A DNA open reading frame has multiple introns and exons and can code for multiple RNAs via transcritpion. These RNAs can be translated into multiple proteins which can then undergo an array of post-translational modifications
What was the previously believed central dogma of omics?
1 gene--> 1 RNA--> 1 protein
study of the genome
the complete set of RNA molecules produced in a given cell or organism-unlike the genome, it can vary with external conditions
all the proteins present in a cell at any given time--more difficult to read than the transcriptome
a mutation; a change in the sequence of DNA; a single nucleotide polymorphism (SNP) can lead to genetic variation; don't necessarily know the functional consquences of all polymorphisms; can be good/indifferent or bad
When DNA is transcribed, what does a thymine residue code for?
Uracil on the RNA
What do A, T, C, and G stand for and to which base pair do they pair?
Adenine, thymine, cytosine, and guanine, respecitvely.
C and G pair together, A and T (U) pair together
If the DNA sequence to be transcribed is ATCG, what will the mRNA sequence be?
Schematically, on which side of a polypeptide chain is the N-terminus usually drawn?
N-terminus is normally on the left while the C is on the right
Which amino acids have positively charged side chains?
Arginine, Histidine, Lysine
Which amino acids have negatively charged side chains?
Aspartic acid and glutamic acid
Which amino acids have a polar, yet uncharged side chain?
Ser, Thr, Asn, Gln
The side chain of glycine is simply a hydrogen. Does this residue help/hinder alpha helices?
Hinders. Glycine causes a kink in the alpha helix
What is unique about the side chain of proline?
The three carbons of the proline side chain covalently interact with the nitrogen that forms an integral part of the backbone, forming a ring
Which amino acids have a hydrophobic side chain?
Alanine, Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan, Tyrosine, and Valine
What determines the primary structure of a polypeptide?
The squence of a chain of AAs
What are the two most common secondary structures?
Alpha helices and beta sheets
What does it mean to say that a beta sheet is antiparallel?
The polypeptide interacts with itself so that strands forming the beta sheet are running opposite each other, such that N-termini and C-termini alternate
What is a tertiary structure?
It occurs when certain attractions are present between alpha helices and beta sheets; it is the overall 3D structure of a single polypeptide chain
What is the difference between a domain and a motif?
A domain is a discrete portion of the same polypeptide that has a discrete function. It is generally larger and found in a class of proteins.
A motif can be determined from the primary sequence and is generally smaller. It's discrete function can be found in many proteins because it is more common, broad, and less specialized than a domain
What is the difference between a quaternary structure and an aggregation?
While a quaternary structure forms through controlled folding processes, an aggregation forms through an uncontrolled process of proteins sticking together and is usually pathological
What organelle is hematoxylin stain used to identify?
The nucleus; stains it blue
What is the stain eosin used to identify?
Red blood cells (orange) and collagen fibers (pink)
It also stains the cytoplasm an intense pink when numerous mitochondria are present
What tissue is Masson's trichrome most commonly used in?
The connective tissue. It stains the cartilage blu-green and the muscle fibers red
What tissue is Mallory's Trichrome most commonly used in?
Connective tissue; it stains keratin orange, cartilage blue, bone matrix a deep blue, and muscle fibers red
What does PAS stand for, and what cellular structure is it most commonly used to identify?
Periodic acid shift: The basement membrane and carbohydrates; it stains glycogen and other carbohydrates a magenta color; Used to stain sugars
What is an antigen?
most simply, it's the protein of interest
Why are secondary antibodies used to identify antigens?
The secondary antibody is a marker-coupled antibody that is directed against a primary antibody; it is used to amplify the signal
What is the primary function of the nucleus?
To maintain the integrity of the cell by regulating gene expression
Histologically, what is the main difference between the nucelus and mitochondria?
The nucleus may be the same shape and color as the mitochondria, but it will always be much bigger than the mitochondria
What is the function of the rough endoplasmic reticulum?
It forms an interconnected network of tubules in vesicles within cells; its primary function is to syntehsize proteins
What is the function of the ribosomes?
This is located on the RER and is the site of protein translation
What are the function of the smooth ER?
sythensizes lipids and steroids, metabolizes carbohydrates and steroids, reguates calcium concentration, drug detoxification, and attachment of receptors on cell membrane proteins
What is the function of glycogen in the cell?
Globs of protein are stored in the form of glycogen in the cell; stains very dark
Histologically, how are lysosomes and peroxisomes different?
Lysosomes are much darker in color than peroxisomes
What is the function of lysosomes?
They contain enzymes that break up materials within the cell and cellular debris
What is the function of the golgi complex?
It processes and packages macromolecules such as proteins and lipids after their synthesis and before they make their way to their destination
What is the defining characteristic of GAs under the microscope?
They are stacked complexes
What is the function of the 2 centrioles in the cell?
They are involved in the organization of the mitotic spindle and the completion of cytokinesis
What is the function of the brush border located on cilia?
To increased the surface area and absorption of microvilli
What is the primary function of the peroxisomes?
participates in the metabolism of fatty acids and rids the cell of toxic peroxides
What is the primary function of the mitochondria?
It generates most of the cell's ATP supply, although it is also involved in cel signaling, cellular diffusion, cell death and the control of the cell cycle and cell growth
When vesicles are transported within the cell, which face of the GA do they enter?
Enter in on the cis face and are transported out of the trans face
What is the primary secretion of goblet cells?
What is the pH of bile?
At what pH is blood considered to be in acidosis?
below 6.9 (normal range is 7.35 to 7.45)
What is the pH of the cytoplasm?
What is the pH of the lysosome and why?
4.8 to 5.5
These are the optimal pHs for the enzymes within the lysosome
What is the pH of the stomach?
Anywhere from 1.0 to 4.0
Which amino acid has a pKa between 6 and 6.5?
Which amino acids have a pKa between 10 and 12?
Lysine and Arginine
What is the approximate pKa of both aspartate and glutamate?
What does the point pKa signify?
The point at which 50% of the molecules are uncharged and 50% are charged
Given that you know the pKa of a given compound, how would you draw the pH curve?
First, plot the pKa on the x axis. Then use intuition to decide when the compound will be positively charged and when it will be negatively charged
What are the 7 post-translational protein modifcations?
Formation of a disulfide bond
What is acetylation?
The addition of an acetyl group to either the N-terminus or amino residues
What is glycosylation?
The addition of a sugar residue to either a N or an O
What is lipoylation?
the addition of a lipid moiety onto a cysteine residue
What enzyme is involved in proteolysis?
Proteases cut the polypeptide backbone to produce a functional protein
What residues would you add a phosphate group to if you wanted to promote cell signaling?
Ser, Tyr, or Thr
What happens to an alpha-helix when an alanine group is substituted in?
Nothing. Alanine is small and likes to be in alpha helices
Poly-Ala forms an alpha helix
What happens to an alpha helix (or a beta sheet) when a proline is substituted in?
It is kinked. Proline is never found in an alpha helix or beta sheet. It is often found in random coils and turns.
Poly-Pro forms a PPII helix
What does Poly-gly form?
When insulin is first formed, before it is activated, what is it called?
How many modifications must preproinsulin undergo before it becomes a functional insulin molecule?
2. First the leader signaling sequence must be cleaved and the disulfide bonds must be formed, then the C chain must be removed
Which is faster acting, an insulin dimer or an insulin hexamer?
An insulin dimer. The insulin hexamer is longer-lasting
Definition: Fibrillar proteins
Repeating globular units (alpha and beta) of tubulin producing long twisted fibers
What is a protofilament?
The "vertical" line of tubulin heterodimers. 13 of these come together to forma microtubule with a hollow center (lumen) that is approximately 10 nm thick
What is the lumen?
The hollow center of the microtubule
Where are you most likely to find the hydrophobic portion of an integral membrane protein?
On the outside of the protein, facing the plasma membrane
What are the defining characteristics of an intrinsically disordered protein?
no predictable secondary structure
No hydrophobic core
A high net carge (causes the different parts of the strand to repulse each other)
Long repetitive sequences (ie >50 Gly)
Easily chopped up into small fragments by proteolysis
Many of these are transcription factors
Make up 30% of proteins in eukaryotes
What are 5 ways to classify proteins based on function?
Chemistry (enzymes that catalyze reactions)
Signaling (produce cellular responses to the environment)
Structure (provide support and organization to cells)
Transport (either move molecules in and out of the cells or circulate in the blood)
Storage (allow cells to utilize oxygen, nutrients, and energy)
What are some conditions that denature proteins?
Changes in pH, ionic strength, pressure, temeprature, osmotic pressure, and urea (in the kidneys)
What is the most common definition of a quaternary subunit?
A single polypeptide that encodes on mRNA
Proteins are constantly in motion. Both their small side chains roate and domains can move on hinges. What is the best method to see these motions?
NMR, but only for small proteins.
recently computer simulations have been working on it
What are the different ways that proteins can be grouped together and classifed?
Proteins can be grouped by common structural properties, common sequence, common function, common ligand, or common post-translational modification
Fibrillar proteins, integral membrane proteins, and intrinsically disordered proteins make up apprixmately 75% of all proteins in the body. What are the other 25%?
Standard soluble, globular proteins; these are the ones we think of most often
What is the primary amino sequence repeated several times in collagen?
(Note that proline and glycine don't like to be in normal secondary structures very often)
What protein best fits the description "inside-out globular proteins"?
Integral membrane proteins
In which technique do you line up specific primary sequences of proteins and do a "best possible fit" to identify similarities?
What are traditional protein families defined by?
What is an isoform of a protein?
A protein with a similar general function, but with crucial differences. An example is liver pyruvate kinase and muscle pyruvate kinase
What is a ligand?
Something that binds to a protein
What does it mean to say that proteins are modular?
The protein is made of several different domains, but these domain usually have different functions
What does it mean to say that a protein is moonlighting?
The protein has more than one function
Describe the biochemistry of Tau
Normal cells have microtubules that are phosphorylated and dephosphorylated in steady state equilibrium. In patients with Tau, a pathogenic Tau kinase is present that phosphorylates these filaments and drastically alters the equilibrium. The filaments aggregate to from paired helical filaments. Tau tissue contains no microtubules. This is often seen in patients with traumatic head injuries (athletes) and Alzheimer's patients
Describe the biochemistry behind mad cow disease in humans
Prion proteins occur in normal conditions. Upon ingesting infected cow meat, a spontaneous mutation occurs in the the prion precursor. This mutation causes uncontrolled prion aggregation to form prion fibrils (amyloids). These elongate and then you've got problems
What are some small molecules that can assist with protein folding?
Pro, Ala, Taurine, Polyols (sorbitol and glycerol) and methylamines (TMAO and betaine)
What are the 2 types of chaperones?
Heat shock proteins and chaperonins
What is the function of a heat shock protein?
Folds new and misfolded proteins (small ones)
What is the function of a chaperonin?
It is a protein folding machine. It folds large proteins using ATP. The unfolded protein binds to the chaperonin and using magic, the chaperonin produces a perfectly folded protein
What is the function of ubiquitin?
It attaches to misfolded/old proteins, which signals a proteasome to come degrade them
If a positively charged matrix is used in column chromotography, what will be the charge on the first fraction collected?
Does protein gel electrophoresis separate proteins based on charge or size?
Size, with the smaller proteins moving faster and farther
The proteins are not separated based on charge because the polyacrylamide gel swamps the charge; makes it irrelevant
When using mass spec, will unique masses be found for each primary sequence or for post-translational modifications?
Where are epithelia found in the body?
lining cavities, body surfaces, and tubes
What is the name of the membrane that epithelial cells lie on?
Basal lamina (basement membrane works too)
What is the primary function of epithelia?
To absorb, secrete, transport, excrete, protect, and sensory reception
When naming an epithelial cells, what must you take into account?
The number of cell layers and the shape of the cells
Definition: Simple Epithelia
Single layer of cells found at surfaces involved in selective diffusion, absorption, and secretion
Definition: Pseudostratified Epithelia
all cells rest on the basement membrane, but the nuclei are at different levels giving it the appearance of having different layers; found in the repiratory tract and the male reproductive system
Definition: Simple Squamous Epithleium
a single layer of cells characterized by folat, scale-like cells; nuclei are flat
Definition: Cuboidal Epithelium
epithelial cells that have a cube-like shape; their width is approximately equal to their height; may exist in single layes or multilayers depending on their location in the body
Definition: Stratified Epithelium
Epithelial cells arranged in layers upon a basement membrane where only one layer is in contact with the membrane while other layers adhere to one another; named by the most superficial layer of cells (the layer closest to the surface)
Definition: Columnar epithelium
epithelia whose height are at least 4 times their width; divided into simple and stratified
Definition: Transitional epithelium
lines the surfaces of hollow organs such as the kidney, urinary bladder and accessory organs: transitional cell carcinoma of the urinary system arises from this type of tissue
Where would you be most likely to find simple squamous epithelium?
lining blood vessels, air sacs of the lungs; it permits the exchange of nutrients, wastes, and gases
Where would you be most likely to find simple cuboidal epithelium?
lining the kidney tubules and glands; it secretes and reabsorbs water and small molecules
Where would you be most likely to find simple columnar epithelium?
lining most digestive organs; it absorbs nutrients and produces mucus; often found near goblet cells
Where would you be most likely to find stratified squamous epithelium?
on the outer layer of the skin, mouth, and vaginal tract; it protects against abrasion, drying out, and infection
Where would you be most likely to find stratified cuboidal epithelium?
Lining the ducts of sweat glands; it secretes water and ions
Where would you be most likely to find stratified columnar epithelium?
Lining the epididymus, mammary glands, and the larynx; it secretes mucus
What is the difference between microvilli, cilia, and stereocilia?
Microvilli are short projections of luminal membrane containing actin filaments. They function to increase surface area of the GI tract
Cilia are motile (ATP dependent) and much larger than microvilli
Seterocilia are really long microvilli with a filamentous actin core and are found in the reproductive tract
What are the primary functions of the basal lmaina/BM?
to provide structural support
to act as a filter
to establish polarity
to control growth and differentation
What is the function of hemidesmosomes?
to link epithelial cells to the basal lamina; specifically, it links the intermediate filament proteins inside the cell to the BM
What are the anucleated epithelial cells on the outermost layer of our skin called?
What is the primary function of basal infoldings?
To increase surface area and thereby increase transport; they are associated with cells involved in active transport
What is the primary function of the zonula occludens?
to form a barrier at the apical surface of the epithelial cells
Where can the zonula occludens be found?
Connecting two epithelial cells at their most apical surface; forms a ring all the way around the cell
What is the primary function of the zonula adherens?
Links to actin cytoskeleton of 2 separate cells
What is the zonula adherins formed by?
What is the primary function of desmosomes?
To link intermediate filaments; these are spot welds and do not form a continuous barrier around the cell
What forms the desmosomes?
Definition: Gap junction
a direct connection between 2 cells that allows for the movement of molecules and ions between the cells
the thin layer of cells that lines the interior surface of blood vessels
Which epithelial cell are you most likely to find lining the interior surface of blood vessels?
What part of the cell can rearrange very quickly, be stbale and provide structure, and can asseble and disassemble in a dynamic equilibrium?
In both actin and microtubule filaments, which end are monomers most likely to be added?
The positive end
They come off of the negative end of actin and the positive end of microtubules
What is the function of the GTP cap?
to stabilize microtubules and keep them from shrinking and falling apart
On a microtubule, which is more stablizing, GTP or GDP?
GTP. When the GTP cap is removed, the GDP-tubulin dimers immediately fall apart
In terms of energy molecules utilized, what is the difference between actin and microtubules?
Microtubules use GTP wile actin uses ATP
Definition: Intermediate filaments
the third group that makes up the cytoskeleton (actin and microtubules); provide much structural support
What are the subunits that comprise an intermediate filament?
start with a protein with open N-terminus and C-terminus and lengthy alpha-helical region; 2 proteins form a coiled coil; these find another coiled coil and form a staggered tetramer (one of the coiled coils flips so that N-terminus is facing the opposite way); two tetramers pack together; 8 of these tertramers twist into a ropelike filament which is about 10 nm in diameter
What is the defect in epidermoylsis?
one of the keratin proteins has a mutation: keratin 5/14 leads to e. bullosa (fatal); keratin 1/10 leads to e. hyperkeratosis; keratin 9 leads to e. plantopalmar
Why does the defect in keratin in epidermolysis lead to severe blistering?
the defective keratin network cannot form strong ropes, rendering the hemidesmosomes incapable of doing their job; the layers of skin simply fall apart
What is the diameter and basic structure of the microfilament that makes up actin?
6 nm; double helix
What are the 6 major types of intermediate filaments and where are they found?
Glial fibrillary acidic protein (glia)
What is the diameter and basic structure of a microtubule?
23 nm; 13 protofilaments forming a hollow tube
What is the function of cofilin?
To tighten and hold together the actin filaments (from 74 nm to 57 nm)
Specifically, what structure is profilin bound to?
The formin whiskers
What does profilin do?
It binds actin monomer and helps it bind ATP; the formin whisker then pulls the complex toward the site of the next actin addition, where profilin releases it and moves back to the original position
What does the formin dimer do?
It puts the actin monomer in the right conformational position to bind to the actin filament, thereby increasing actin buildup
How does the centrosome contribute to microtubule stability?
The negative ends bidn to the centrosome complex at the core and are stabilized while the positive ends radiate out and continue growing; the negative end is capped with gamma tubulin and accessry proteins in a gamma tubulin ring complex
What does ARP stand for?
Actin related proteins (2 and 3)
What does the ARP do?
ARP 2 and 3 combine with an activating factor (unknown) and actin monomers associate with it to form the nucleated actin filament; the ARP complex then binds to another actin filament at a 70 degree angle to form an actin meshwork
Does formin move the actin when adding actin monomers?
Negative. It simply puts the actin in the good place to bind; does this using a wobbling motion
How is capping used to cap filament length?
Uncapped filaments groaw at plus and minus ends; the capped population of filaments grows at the minus end only
What are the different types of bundling that filaments can do in a cell?
Contractile bundling (anti-parallel) is seen in stress fibers of the cells
Gel-like networking (no real order) is seen in the cell cortex
Tight parallel bundling is seen in the filopodium
What is a filamin dimer?
two associated L-shaped proteins that sit on top of the actin meshwork to further stabilize it
Which proteins are used tos tabilize actin bundles?
fimbrin (monomer); alpha-actinin (dimer); spectrin (tetramer); and filamin (dimer)
each of these connects actin, and spaces them differently according to where they are in the body
Is mysoin II more likely to enter a contractile bundle or a parrallel bundle?
More likely to enter the contractile bundle. The tight packing of parallel bundles prevents myosin II from entering
How does fimbrin help stabilize the actin filaments?
Crosslinks the actin filaments (seen in the microvilli)
What connects the actin filaments to the plasma membrane of the microvilli?
calmodulin and myosin-I act as the lateral side arms
What are the cytoskeletal elements of a neuron?
neurofilaments and microtubules
What are the top three leading causes of death in the US and Kansas?
Heart disease, cancer, and stroke
What's the leading cause of death in children under the age of 1?
What's the leading cause of death in children ages 1-4?
Unintentional injuries and congenital anomalies
What's the leading cause of death in children age 5-14?
unintentional injuries, cancer and homicides
What's the leading cause of death in young adults ages 15-24?
unintentional injuries, suicide, and homicide
What's the leading cause of death in adults, ages 25-44?
Unintentional injuries, cancer, and suicide
What's the leading cause of death in adults, ages 45-64?
cancer, heart disease and unintentional injuries
What's the leading cause of death in adults, ages 65 and older?
Heart disease and cancer
What is healthy people 2010?
A set of health objectives for the nation to achieve over the first decade of the new century
What is Healthy people 2010 based on?
Initiatives pursued over the past two decades and the surgeon generals 1979 report
What is a protein binding site?
a well-defined region that binds ligands, can stabilize folded proteins and can change protein conformation; this region has a specific AA sequence that allows the binding to occur
What are the results of a mutation in the primary AA sequence of a binding site?
a change in affinity and specificity
How tightly a ligand is bound
What is Kd?
The dissociation constant; the point at which 50% of the protein/enzyme is bound to substrate
What is the equation for Kd?
given in M
What are the units for the association constant?
When [A]=[AB], what can we say about Kd?
What is the equilibrium constant equation?
What can we say about the affinity of a protein for a ligand if the Kd shifts to the right?
The affinity is weaker
If it shifts to the left, it's stronger
Definition: Negative Cooperativity
binding one ligand makes subsequent binding more difficult;
A protein exhibiting negative cooperativity will NEVER reach 100% saturation (bound)
On the log curve, also a less steep slope because binding is slower
Will the curve of an enzyme exhibiting positive cooperativity by steeper or more shallow than one not exhibiting this?
Figured this out by taking the weighted average of the low affinity and high affinity sites
When does cooperativity occur?
When a single polypeptide chain has identical binding sites, and the binding of one ligand changes the affinity of the next ligand binding
Definition: Positive cooperativity
When the binding of one ligand causes the second to bind more tightly
Does allostery affect the Kd?
Yes, it shifts it, depending on whether the allostery is positive or inhibitory
When does allostery occur?
When a protein has binding sites for 2 different types of ligands, and the binding of one ligand changes the affinity for the other
What happens to the Kd with inhibitory allostery?
It shifts to the right because the affinity for the second ligand becomes weaker
Definiton: Inhibitory Allostery
Neither ligand wants to bind as much when the other ligand is already bound
What happens to the Kd with enhancing allostery?
It shifts to the left because the affinity is strengthened by the binding of the first ligand
Why does nature need allostery?
REGULATION! it allows the cell to adapt to changing conditions
How is allostery used in metabolism?
Changing nutrients allows the reaction rates to be turned up or down
How is allostery used in DNA transcription?
Can change transcriptome/proteome in response to the environment
What is the key difference between allostery and cooperativity?
Allostery involves 2 different ligands (one is usually an effector) at 2 different binding sites; cooperativity involves identical binding sites and the same ligands
Does cooperativity affect the Kd?
No, it merely changes the slope
the rate of NEW cases of disease in a susceptible population in a given period of time
What is the equation to determine incidence?
incidence=(# New cases of disease)/(# at risk in population in a given time period)
ALL cases of disease (new and old) in a susceptible population; depends on incidence and duration of disease
What is the equation to determine prevalence?
prevalence= (# new and old cases of disease)/(#people in pop. at that point in time)
What is the equation to determine mortality rate?
(total # deaths in a period)/(# of people in pop. mid-period)
Definition: Standard Deviation
How spread out things (data points) are in a population
Definition: Standard Error of the Mean
How spread out is the sample mean
What kind of hypothesis do you use in statistics?
Null hypothesis--assume that the opposite of what you wish to prove is true; the hypothesis of no difference
What is the p-value?
A measure of how likely it is that what we have observed could occur randomnly; use a p-value of 0.05
If you have a normal distribution and continuous data, what type of test should you run to disprove your hypothesis?
If the data is categorical and the distribution is not normal, what type of test should you run to disprove your hypothesis?
statistical test used to compare the mean values between 2 different groups
Definition: chi-square test
statistical test used to compare proportions between 2 different groups
Definition: Confidence intervals
A range of plausible values for the difference; if a 95% confidence interval for a difference does not contain 0, then the p-value is less than 0.05
If a 95% CI for a ratio does not contain 1, what do we know about the p-value?
it is less the 0.05
How similar are the primary sequences of hemoglobin and myoglobin?
~18% identical and ~38% similarity
Secondary and tertiary structures are very similar; they bind the same prosthetic group (heme) and the same ligand
How are hemoglobin and myoglobin's quaternary structure different?
Mb is a monomer
Hb is a tetramer with 2 alpha subunits and 2 other subunits (usually beta, but doesn't have to be)
Which has the greater affinity for oxygen and why?
Mb does, needs to steal it from Hb to take to muscles
What happens to the oxygen binding curve at high altitudes?
There is a reduction in the amount of oxygenated blood leaving the lungs; more of it is reduced
What happens to the oxygen binding curve during exercise?
There is an increase in the gaseous pressure of oxygen in the reduced blood returning from the tissues; becoming acidotic
What happens to the oxygen binding curve in methemoglobinemia?
There is a decrease in the percent saturation of the oxygenated blood leaving the lungs
What is methemoglobinemia?
A disorder characterized by the presence of higher than normal level of methemoglobin (metHb) in the blood. This metHb has an extremely high affinity to oxygen, resulting in almost no oxygen delivery to the tissues