BIO3220.02 Exam 1 Review

Molecular chaperones...

- Bind & hydrolyze ATP - Undergo large-scale conformational conversions - Bury hydrophobic amino acid residues of client proteins - Prevent "promiscuous" interactions of proteins via hydrophobic interactions

Clock, Period, Bmal1

- Core transcription-translational clock components

What methods are used to determine the shape of a protein?

- Cryo EM - X-ray crystallography - Alpha fold

Predict the consequences of having a triplication of the alpha-synuclein gene

Parkinsons disease

Peptide bond angles & _______ interactions favor the formation of ______ and _____ as the common secondary structures

Peptide bond angles & backbone interactions favor the formation of alpha helix and beta sheet as the common secondary structures

What is the key change in primary structure that occurring during conversion from prion- to prion+ states? Secondary structure?

Primary structure: flat proteins rather than round, capable of stacking, alpha helix structure to beta structure conformer that is infectious Secondary structure: stacked prion proteins, change that will take place is in the N-terminal alpha helices No change in primary structure, changes in secondary structure, goes from alpha helix to beta sheet, loss of helical content and loss of sheet content

_____ protein aggregates in the brains of people with CTE. ____ protein aggregates in the brains of people with prion disease (CJD, GSS, FFI)

Prion protein aggregates in the brains of people with CTE. Tau protein aggregates in the brains of people with prion disease (CJD, GSS, FFI) - Both of these proteins do NOT have an identifiable prion domain

Name an amino acid that possesses a ring structure

Proline Tryptophan Tyrosine Phenylanine Histidine

Describe a consequence of a post-translational modification to a protein

Protein post-translational modifications (PTMs) increase the functional diversity of the proteome by the covalent addition of functional groups or proteins, proteolytic cleavage of regulatory subunits, or degradation of entire proteins

Initially, prions were described as disease causing agents. You are at a cocktail party and mention to a professor that you are learning about prions in cell biology class and she states: "those only cause neurodegenerative diseases—why are you discussing that in cell bio?!" What is your reply?

Proteins are misfolded due to problems inside cell, producing more misfolded proteins which cause problems. Evidence indicates that non-prion aggregates can also move between cells and seed the misfolding of their normal conformers. This can question the therapeutical use of transplants, and also indicates that it may be possible to bring these diseases to an early arrest by preventing cell-to-cell transmission.

Which form of Sup35 is able to terminate translation?

The soluble non prion form (to big) - Function: terminate translation of proteins - Shows biochemical behavior in vitro consistent with prion formation

What is the molecular basis of circadian rhythms controlled by light in mammals?

The suprachiasmatic nuclei (SCN) in the brain constitute the major circadian clock of mammals. The "retino-hypothalamic" tract carries info about day light dirctly to the SCN in the hypothalamus (forebrain)

This amino acid is called _____ and is special because it can be ____

This amino acid is called cystenine and is special because it can be di-sulfide bonded

What type of microscopy requires incredibly thin slices of your sample

Transmission EM

True or False Histone deactylase (HDAC) inhibitors increase gene expression by blocking an inhibitor of an inhibiting modification

True

What are the additional YFG (your fav gene) proteins appearing above ~42kDa?

Ubiquitin tagged YFG that's not being degraded

When a chain of a 76 amino acid long protein is ligated to another protein, it's tagged for degradation

What is ubiquitin

How can protein structures be determined?

X-ray crystallography

Is Hsf1 involved in neurodegenerative diseases like prion disease?

Yes - Hsf1 -/- mice die faster when infected

Describe or design an experiment that demonstrates a decrease in proteasome activity. Make it clear how you will measure activity of the proteasome.

You can decrease proteasome activity by doing a SDS Page with a normal protein and then with a protein having its proteasome blocked. The differences between the two proteins would show the decrease in proteasome activity as a protein with the proteasome blocked would not be degraded and would show an accumulation of ubiquitin proteins. Describe pulse chase to measure half life, to prepare proteasome, see increase half life, cause of blocking proteasome

What unit is used to describe the size of proteins?

kiloDaltons (kDa) 1 Dalton = 1 atomic mass unit On average amino acids are 110Da

What modifications promote open chromatin states?

- Phosphorylation - Acetylation

Summary of Heat Shock Factor 1 (Hsf1)

- Required for Thermotolerance - Trimerization needed for fxn - Phosphorylation needed for nuclear entry - Specific binding sites all over the genome (heat shock elements/hse) - Upregluates molecular chaperones & other pathways too

Why are proteins treated with SDS, reducing agent, & high temp before being loaded into a gel for electrophoresis?

- To promote unfolding - To disrupt non-covalent bonds - To make the protein neg charged - To break disulfide bonds

Two proteins are near each other in solution. Which side chains are likely to interact with each other?

- Tryptophan & Phenylalanine (hydrophobic effect/van deer walls since both are nonpolar) - Lysine & Aspartic Acid (electrostatic interaction since +/- charged)

What are the three tenets of cellular theory?

1) All living organisms consist of one or more cells 2) A cell is the basic unit of structure for all organisms 3) All cells arise from pre-existing cells

What is a gene? Describe the structure of a gene in the genomic context. What parts of the gene are made into RNA and what parts are not?

A gene is an inherited unit of DNA and each one usually codes for a particular protein or function. A gene consists of a enhancer/silencer, a promoter, a 5' untraslated region, a start codon, open reading frame, a stop codon, and a 3' untranslated region, a terminator, and an enhancer/silencer

Both mice and humans have approximately this many genes

About 3,500

What are the 2 most common folds?

Alpha helix & Beta sheet

_____ are ordered aggregates (clumps) of protein

Amyloids are ordered aggregates (clumps) of protein - Beta sheet rich - Stain with Congo Red & other dyes

What is circadian rhythm? Give an example

An internal process that regulates ones sleep-wake cycle, 24hr rhythm **must persist in the absence of zeitgeber (time giver) ex) sleep cycle, bowel movements, secreting melatonin

Name an amino acid that bears a negative charge at physiological pH

Aspartic acid Glutamic acid

Your PhD advisor tells you to study a novel protein called np33. Describe how you would GFP-tag this protein and how that could help you study it's cell biological properties (like where it goes inside or outside of a cell)

Attach GFP to gene promoter in order to cause expression of glowing colors showing this particular protein under UV light.

Infectious prions typically convert alpha helices into this type of secondary protein structure

Beta sheets

_______ _______ is a simple eukaryotic cell

Brewers yeast

What is NOT a downside of electron microscopy?

Can only be used on living samples

Predict the phenotype of an XX female that's unable to do X-inactivation

Dead 💀 - Lethal to have a duplicate copy of a chromosome

Mass spectrometry is useful for...

Determining the size of a protein Determining post translational modifications on proteins

Predict the consequence of this increased gene dosage of extra chr.21

Down Syndrome

Which model organism is pictured?

Drosophila melanogaster (fruit fly)

Name the following model systems: Drosophila melanogaster Mus musculus Arabadopsis thalania Caenorhabditis elegans Saccharomyces cerevisiae Escherichia coli

Drosophila melanogaster - Fruit fly Mus musculus - Mouse Arabadopsis thalania - Plant Caenorhabditis elegans - Roundworm Saccharomyces cerevisiae - Yeast Escherichia coli - Bacteria

Which enzyme is responsible for maintaning an active pool of ubiquitin?

E1

What specific chaperone helps regulate prion formation in yeast

Hsp104

Whats an example of a protein chaperone?

Hsp60/GroEL-ES

This amino acid is _____________

Hydrophilic (Arginine)

Is tryptophan hydrophobic of hydrophilic?

Hydrophobic

During folding, what type of amino acid residue prefers to aggregate together and become "buried" within a protein

Hydrophobic amino acids

Name the amino acid

Its NOT an amino acid, its a nucleotide (no =O-)

Why does PrP from a prion-sick animal migrate with so many bands, with or without PK? (Western Blot)

Its a glycoprotein

________ __________ is the cheapest and most accessible type of microscope

Light microscopy

The one-letter abbreviation for this amino acid is "K"

Lysine

What is the amino acid residue that ubiquitin can be ligated to

Lysine

Name an amino acid that bears a positive charge at physiological pH

Lysine Arginine Histidine

What hydrophobic amino acids is found on the N terminus of a protein

Methionine

Which amino acid is the first amino acid in almost every known protein?

Methionine (Met, M)

What modifications promote closed chromatin states?

Methylation

Molecular chaperone _____ uses energy from _____ hydrolysis to fold proteins

Molecular chaperone Hsp70 uses energy from ATP hydrolysis to fold proteins

What is a molecular chaperone? Give an example.

Molecular chaperones: auxiliary protein that protect and stabilize folding proteins ex) Hsp70

Could you breed a Chinese & Indian muntjac?

No - Need good alignment of chromosomes for meiosis - Chromsome # and size varies

What would the luciferase activity look like in an SCN derived from a Bmal1 knockout mice?

Non 24hr rhythm - Little to no rhythm

How can these small post translational modifications be measured?

1) SDS Page 2) Western Blotting - Acetyl lysine antibody - Phosphoserine antibody 3) Mass spectrometry

About 90% of what we know about cell biology came from studying what number of organisms?

6 model organisms ex) fruit fly, mouse, bacteria, plants etc.

You're preparing a slide you viewed on a fluorescent microscope. You use a fluorophore that's excited 568nm. What might be the wavelength of its emission?

640nm

What group of proteins helps assist with the proper folding of other proteins and helps them to avoid misfolding

Chaperones

What specific kind of microscope is able to take "layers" of images and stack them to provide a more 3D picture

Confocal microscopy

Techniques for examining proteins in gels

Coomassie stain vs Western blot: - Coomassie is good for seeing everything there (general separation of proteins) - Western blotting is good for use on antibody to recognize a specific protein - Semi-denaturing agarose is good for looking at prions

Given its large size, what technique would you want to use to get a structural insights into the proteasome?

Cryo EM

Name the amino acid

Cysteine

Which amino acid is capable of forming a di-sulfide bond?

Cysteine

What is the difference between the cytosol and cytoplasm?

Cytosol: only the fluid in the cell (no organelles) Cytoplasm: includes everything but the nucleus and is the term for the cytosol PLUS the organelles suspended in the cytosol

How are cytosolic proteins turned over (degraded) in cells?

Cytosolic proteins are marked with ubiquitin which signal for the proteasomes to destroy them.

True or Flase: The function of Hsf1 in mediating the heat shock response is to repress chaperones gene expression

False

What type of microscopy gives high resolution images of surfaces and 3D structures

Fluorescence microscopy

Which amino acids appear to promote prion states?

Glutamine & Asparagine

This di-peptide is made of

Glycine

What's the name of the consensus sequence of DNA that Hsf1 binds to

Heat shock element (HSE)

How are molecular chaperone levels increased during times of need?

The levels of molecular chaperons increase during the times of need through the action of heat shock proteins. These HSPs are increased in the cell during stress conditions and inturn they increase the molecular chaperon levels.

What method is used to measure protein turn over?

Pulse chase

What type of experiment is used to measure protein half life and turn over

Pulse chase

What microscopic method was used to make this picture?

Scanning EM

What type of microscope captured this image?

Scanning EM

Name two uncharged, polar amino acids

Serine Threonine Tyrosine Asparagine Glutamine

Name at least two amino acids that can be phosphorylated

Serine Threonine Tyrosine

The molecular clock is in the _____ synchronizes clock in all cells through behavioral & hormonal regulation

The molecular clock is in the SCN synchronizes clock in all cells through behavioral & hormonal regulation

Describe the structure and function of the proteasome in as much detail as you can.

Structure: made up of two subcomplexes: a catalytic core particle (CP; also known as the 20S proteasome) and one or two terminal 19S regulatory particle(s) (RP) that serves as a proteasome activator Function: plays a central role in the regulation of proteins that control cell-cycle progression and apoptosis

________ is a yeast prion that is a translation termination factor

Sup35

What is the normal function of Sup35? How is this function altered when it enters the prion + state?

Sup35 normal function in yeast is as a translation termination factor by breaking apart ribosomes to stop translation. This protein is capable of adopting the prion formation; while this does not cause disease, it does weaken the translation termination causing it to not work as faithfully. Prion + aggregated (clumps)

How can we test whether CTE brains contain infectious Tau prions?

Test on lab mice

The N-end rule

The amino acid composition of the N-terminus of a protein is predictive of its half life - Stabilizing residues: methionine, glycine, alanine - Destabilizing residues: tyrosine, glutamine There are many enzymes targeting the N-terminus of proteins - Arginylation, acetylation etc.

What is the "central dogma of molecular biology"?

The central dogma of molecular biology is a theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein.

How can one transcription factor regulate the expression of many different genes?

The genes all share a conserved (the same) promoter

Draw or describe chromosomes and how DNA is packaged with histone proteins and how they are regulated through post-translational modifications. What techniques can be used to determine post-translational modifications?

• Chromosomes are DNA molecules tightly packed and highly condensed using histone proteins to wrap around.• Each strand of DNA is wrapped around a histone two times, making a nucleosome, which then coil on each other to wrap even tighter.• Eukaryotes have a set of chromosomes with one linear piece of DNA per chromosome; each with two free ends. They have a telomere on each end, and a centromere in the center.• Post-translation modifications are used to regulate these histone proteins. Acetylation promotes gene expression, methylation inhibits gene expression, and phosphorylation activated gene expression.• Post translational modifications to histones can be determined by a "biological code" which is then read by a protein called a "code-reader". This tells the cells what has been modified. Methylation, Acetylation, Phosphorylation etc.