Exam 1 Study Guide | BIOL 3510 Notes by Marin Young □ What technological development and subsequent observations led to the birth of cell biology?
1665: Robert Hooke saw dead cork cells (plant cell walls) under his early microscope and coined "cells" to describe little rooms like monks lived in
• 1838/1839: Schleiden says plants are made of cells, Schwann says animals are made of cells ○ Schwann cells, which wrap around and insulate long neurons, are named after this Schwann □ What is the cell theory?
• idea that all living things come from division of existing cells, and cells are "basic unit of life" □ What are the average sizes of cells and organelles and the resolution limits of different types of microscopes? • Eukaryotic cells: 10-50 µm
• Prokaryotic cells: 1-3 µm
microscopy (three types)
Visible-light microscopy: requires staining and fixing (usually with heat) to see well, or differential interference contrast (like phase-contrast) to see living cells with optics tricks
Epifluorescent: uses a fluorescent stain to label structures and a UV lamp to cause the dye to emit visible light; best for living cells
Confocal fluorescent: uses a laser beam instead of a UV lamp to illuminate sample, which improves image quality by helping focus on a 3-D sample •
Remember, the resolution is still 200 nm, but focus depends on sample thickness; you've probably run into this problem in labs where you can't see an entire object in focus at the same time (I'm thinking of diatoms, for anyone who took micro)
Specimen coated with a thin layer of heavy metal like gold and blasted at an angle with a beam of electrons, which bounce off the metal and onto a detector. A connected computer uses the electron pattern to determine structure based on angles of "reflection" where electrons bounced.
This is a lot more like light microscopy with electrons instead of light: a condenser focuses electrons on a thin sample, which can be stained with electron-dense chemicals like uranium acetate or lead citrate. Some areas absorb more electrons than others. The electrons go through electromagnetic objective and projector lenses and hit a detector screen to make a digital image.
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□ Compare and contrast different types of light microscopy and electron microscopy.
• See chart above!
What do images produced by the different types of microscopy look like? (Check out the relevant figures in your book).
Visible-light microscopy using stains
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Scanning electron microscopy
(Public domain, accessed via
https://en.wikipedia.org/wiki/Scanning_elec tron_microscope#/media/File:Misc_pollen.jp g)
Looks like a photograph of a 3-dimensional metal replica
Don't forget about the age old question of stoge food
Transmission electron microscopy
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Looks like a light micrograph in incredibly
□ What are the differences and similarities between prokaryotic and eukaryotic cells?
Similarities: reproduction, expression of DNA via transcription and translation, phospholipid bilayer cell membrane
Differences: presence or absence of membrane-bound organelles and nucleus, presence or absence of introns, number of origins of replication
What are the subcellular organelles/components and their general functions in cells? What cellular components are unique to plants?
• I recommend http://facstaff.cbu.edu/~seisen/EukaryoticCellStructure.htm if you need a refresher on this Don't forget about the age old question of penn state university economics
□ Be familiar with the general structure of amino acids, peptide bonds and polypeptide chains (or proteins).
Amino acids: Peptide bond:
Amino acids in a peptide are called amino acid residues
While I don’t expect you to memorize the amino acid side chains, I do expect you to be able to tell if they are nonpolar, acidic etc if the structure is provide as well as their abbreviations
Uncharged and Polar
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Lys Arg His Asp
Glu Asn Gln Tyr Ser Thr Pro Gly Ala Val
Note that all abbreviations are just the first three letters except asparagine, glutamine, and tryptophan!
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Phe Trp Cys Met
□ What are the four levels of structural organization of protein and what characterizes each level? Protein structure
Primary structure: order of amino acids (Asp Lys-Gly-Tyr…)
Determined by covalent bonds in
Secondary structure: alpha helices and beta pleated sheets
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Determined by hydrogen bonds in backbone
An alpha helix has R-groups facing out and 3.6 residues per turn--the carboxyl group on residue 1 accepts a hydrogen bond from the amino group on residue 5 (NCC-NCC-NCC-NCC-NCC)
Beta sheets are accordion-folded and can be parallel (NCC-NCC-NCC backbone runs the same direction) or antiparallel
• Tertiary structure: folding into actual shapes
Determined by noncovalent interactions (hydrogen bonds, hydrophobic
interactions, ionic bonds) plus covalent disulfide bonds between R groups ○
Domains are sections of a protein that can fold independently (even without the other sections of the same protein)
Gene regions for domains can be spliced together to create new proteins, in the lab or in cells (translocation mutations)
Quaternary structure: interaction of multiple polypeptide subunits
Some proteins are a single polypeptide chain and don't really have 4ostructure ○
Proteins can be called homo___mers or hetero___mers
The blank is the number (di, tri, tetra)
Homo = identical subunits, hetero = different subunits
Rubisco (a plant protein, most abundant protein on Earth) has 8 large subunits (encoded in nuclear genome) and 8 small subunits (encoded in chloroplast genome)
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□ What are covalent bonds (both polar and nonpolar)? • Strong bonds involving sharing electrons
• In nonpolar bonds, electrons are shared fairly
• In polar bonds, electrons are shared unfairly/unequally ○ Some molecules with polar bonds are polar molecules
CH3Cl is polar because the side with the Cl has a partial negative charge and the side away from it has a partial positive charge
CCl4 is nonpolar because the polar bonds all cancel each other out: the outer chlorines have partial negative charges and the inner carbon has a partial positive charge, but there's no partially positive or negative "side"
□ What are 4 types of non-covalent bonds/forces relevant to cells? Be able to describe them.
Van der Waals interactions: all atoms are slightly attracted to each other by "induced dipoles" (polarity wobble)
○ This is most important in nonpolar amino acid residues
Hydrophobic interactions: very nonpolar particles stick together to minimize exposure to water (this is why oil and water don't mix
Hydrogen bonds: occur between an oxygen or nitrogen with a lone pair, and a hydrogen on an oxygen or nitrogen--hydrogens on carbon atoms do NOT make hydrogen bonds
Ionic or electrostatic attractions: opposites attract! Positive and negative charges are attracted to each other
□ How do proteins fold?
Domains are independently folding sections that are small enough for noncovalent interactions to pull the amino acid residues into place
• Domains then fit into each other
□ What are α helices and β sheets? How are they formed?
• Types of secondary structure formed by hydrogen bonds within the NCC backbone of a polypeptide □ What is a protein domain? What are chaperones?
• Domain: independently folding section
• Chaperone: protein that helps facilitate correct folding
□ What are binding sites, ligands, substrates, active sites, catalysts, enzymes, and allosteric proteins? • Binding site: a region of a protein with a structure adapted for binding to another molecule • Ligand: a molecule such as a hormone that binds to a receptor or other protein • Substrate: a molecule that undergoes an enzyme-catalyzed reaction
• Active site: a site where a protein's activity takes place (usually also a binding site)
Catalyst: any substance that increases a reaction rate by lowering activation energy without being changed or altering the free energy change of the reaction
• Enzymes: biological catalysts, usually proteins but sometimes RNA molecules ○ Can catalyze reactions by:
□ holding substrates near each other,
bending bond angles to help break a bond ("stabilize the transition state"--help form the very middle of a reaction step where one bond is half broken and another is half formed), or
□ temporarily holding "extra" protons or electrons to stabilize an intermediate
Allosteric protein: a protein whose activity is regulated by molecules binding at a site besides the active site (like an inhibitor deactivating a protein by binding to a special regulatory binding site)
□ How does feedback regulation work?
A product of a reaction or set of reactions inhibits the same reaction(s), usually by allosterically inhibiting an enzyme that catalyzes an earlier step
□ What are four ways protein activities are modulated in a cell?
Feedback inhibition, phosphorylation/dephosphorylation, degrading ubiquitin-tagged proteins, allosteric regulation, and possibly also controlling the amount of transcription/translation that occurs
□ What do conformational changes have to do with protein activity?
Changing the shape of a protein can expose a previously buried active site, move an important catalytic amino acid residue into a useful place, or even get the protein out of its own way--structure determines
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amino acid residue into a useful place, or even get the protein out of its own way--structure determines function, so changing structure changes function and can turn a protein from on to off
□ What are kinases, phosphatases, and GTPases (GTP binding proteins)? How does these regulate protein activity? • Kinases are enzymes that phosphorylate (stick a phosphate group onto) other proteins/enzymes ○ This often activates the target enzyme, but some enzymes are deactivated by phosphorylation • Phosphatases are enzymes that dephosphorylate (take a phosphate group off of) a target protein
This is the opposite of what a kinase does--likewise, it deactivates most target proteins but activates some
○ This also usually releases some energy
• GTPases bind GTP and then hydrolyze it to produce GDP and Pi (phosphate)
GTPases, which include the G proteins that work with G-protein-coupled receptors, often have other functions, and they're active when bound to GTP
□ This means they deactivate themselves by hydrolyzing GTP
• All of these are means of "switching" proteins between on and off, or between one activity and another □ What are antibodies? How are these produced?
• Antibodies are small proteins that bind to foreign molecules called antigens
They're produced in the body by intron splicing and recombination of domains that have different shapes
They're produced in the lab by regularly injecting an animal with the antigen so its immune system produces antibodies against it, and then harvesting the antibodies by drawing blood
□ Be familiar with the general structure of nucleotides, phosphodiester bonds, and nucleic acids. • Two thymine nucleotides are shown, with a phosphodiester bond circled in red
• Cytosine and guanine form three hydrogen bonds; adenine and thymine form two ○ Mnemonic for base pairing: AT&T and Cingular, the cell phone companies ○ Adenine and guanine are purines, which means their structures are bicyclic ("PUGA-2") ○ Cytosine and thymine are pyrimidines
○ One purine and one pyrimidine always base-pair to keep width consistent
A DNA double helix has two antiparallel strands in a right-handed spiral with 10 bp per turn
Antiparallel: one goes 5' to 3' (has a phosphate at one end and a sugar at the other) and one goes 3' to 5' (sugar at one end, phosphate at other)
Minor and major grooves alternate because of the offset between the two strands
▪ Proteins to bind to DNA based on this pattern/shape
Compare and contrast: Purines vs pyrimidines, ribose vs deoxyribose, RNA vs DNA, heterochromatin vs euchromatin.
• A purine has two rings and is wider; a pyrimidine has one ring and is narrower
• Ribose has an -OH group on the 2' carbon; deoxyribose has a hydrogen there instead • Heterochromatin is condensed and hard to express; euchromatin is more open and easier to express □
How are these terms related to each other in the context of double stranded DNA?-- hydrogen bonds, complementary base pairing, anti-parallel, and polarity. How are complementary sequences “written” with
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complementary base pairing, anti-parallel, and polarity. How are complementary sequences “written” with respect to polarity (DNA and RNA)?
• Hydrogen bonds between bases are responsible for complementary base pairing
The polarity of DNA means there's a 5' end (the end with the phosphate group, which is attached to the 5' carbon) and a 3' end (the end with the sugar, which has an -OH group on the 3' carbon
• Antiparallel means the strands go in opposite directions (more on this above) □
What are genomes, karyotypes, chromosomes, homologous chromosome, chromatin, and epigenetic inheritance?
• Genome: the set of all genetic material in an organism
Karyotype: an image of all the mitotic chromosomes in an organism's genome, dyed different colors with fluorescent molecules
• Chromosome: a complete DNA molecule folded into a condensed structure
Homologous chromosomes: a pair of chromosomes containing the same sequence of genes, with varying alleles because one chromosome comes from each parent (meaning, they're not exactly identical)
• Chromatin: the combination of DNA and proteins (mostly histones) that resides in the nucleus of the cell
Epigenetic inheritance: passing down epigenetic ("above the genome") modifications like methylation or acetylation of certain histones
○ This allows inheritance of gene expression patterns, not just the genes themselves
Very important in cell division, since epigenetic modifications are crucial to determining what genes are expressed in a cell type
□ What three general sequence elements are needed for chromosome replication and segregation? • Telomere, replication origin, and centromere
What are the various levels of chromatin organization and what proteins and interactions lead to their formation?
• DNA wraps around specialized proteins called histones
A histone complex is an octamer with two each of H2A, H2B, H3, and H4
A 147-bp length of DNA wraps twice around each histone
complex, with a 50-ish-bp segment of linker DNA between histone complexes
○ Nucleosome = one histone complex plus its wrapped and linker DNA
If this "beads on a string" chromatin (DNA + histones) was exposed to a nuclease, the linker segments would be destroyed and yield nucleosome core particles (histone complexes with wrapped DNA)
Histone proteins contain many Arg and Lys residues (+ charges attract DNA) and are highly conserved among species
▪ H3 subunits' tails are most used for regulation
• Nucleosomes are packed into 30 nm fibers
Histone H1 (NOT part of the octamer) binds between wrapped and linker DNA to hold nucleosomes close together
○ Histone tails can also interact
• 30 nm fibers form 300 nm loops
• 300 nm loops are squished and folded together to form a mitotic (condensed) chromosome □ How do DNA binding proteins access nucleosome-wrapped DNA?
• The chromatin remodeling complex slides DNA over the histone complex to expose a different segment □ What is the relationship between the amount of DNA condensation and the level of transcription? • More condensation (tighter packing) correlates with less transcription
□ What are the main structural components of the nucleus?
• Nuclear envelope: two concentric lipid bilayers (like two cell membranes, one lining the other)
Nuclear lamina: mesh of intermediate filaments (a type of cytoskeletal filament) that supports shape of nucleus
Nuclear pores: channels through the nuclear envelope, made of proteins that selectively allow certain molecules to enter or exit
○ Controls when mRNA can leave (only when capped, tailed, and spliced)
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• Nucleolus: an important biochemical neighborhood in the nucleus where ribosomes are manufactured ○ Ribosomal genes are transcribed to RNA
Some is considered rRNA and will become part of the ribosome; some is mRNA and will be transcribed
The mRNA exits the nucleus and gets transcribed in the cytoplasm, and then the proteins synthesized re-enter the nucleus
○ The ribosomal proteins and rRNAs are assembled in the nucleolus □ What is a biochemical “neighborhood”?
An area within the nucleus with characteristics that facilitate different biochemical activities, like an area with lots of RNA polymerase that's good for transcription or an area with lots of spliceosomes for mRNA processing
□ What are characteristics unique to RNA?
• Uracil instead of thymine
• Ribose instead of deoxyribose
• Usually single-stranded and can take on many, many different structures • Sometimes has catalytic activity (ribozymes)
Sometimes capable of unconventional base-pairing, like two hydrogen bonds forming between an adenine and a cytosine--it's not perfect, but it's better than nothing
○ Stabilizes a structure just enough to still be dynamic
Can also allow different widths of base pairs: an adenine-guanine pair would be wider than a usual bp, and a cytosine-thymine pair would be narrower
□ What do polymerases do? Remember 5’ to 3’.
• Synthesize a polymer of DNA or RNA in the 5' to 3' direction • This means they read the template strand from 3' to 5'
What are the main steps in prokaryotic transcription? What is the role of the promoter (-10 and -35 sequences), sigma factor, RNA polymerase, and termination sequence?
• Initiation, elongation, and termination
There are two short promoter regions, one about 10 bp before the origin and one about 35 bp before the origin
• The sigma factor binds to both these promoter regions and helps RNA polymerase bind to the DNA • The termination sequence causes RNA polymerase to stop transcribing and leave the DNA ○ This can happen with or without proteins, but this wasn't covered in class
What are the main steps in eukaryotic transcription? What are the roles of the promoter (TATA box), general transcription factors (especially TFIID and TFIIH), RNA pol II, and the C-terminal domain of RNA pol II? • Still initiation, elongation, and termination, but a bit more complex
• The promoter/TATA box binds transcription factors
○ TFIID binds to the TATA box
• TFIIH phosphorylates the C-terminal domain/cytoplasmic tail domain of RNA pol II, which activates it
□ How are eukaryotic transcripts processed prior to exiting the nucleus? • Introns are spliced out
• A methylated GTP cap is attached to the 5' end, via a triphosphate bridge • The 3' end is polyadenylated (a long tail of 150-250 adenosine nucleotides is added) □ What is the role and composition of the spliceosome?
• Made of catalytic RNAs and proteins
• Pulls out introns in a lariat (lasso) shape and splices/joins exons together □ What is the advantage of alternative splicing?
Multiple different polypeptides can be synthesized to express a single gene, depending on the combination and order of exons spliced into the final mRNA
□ How is eukaryotic transcription regulated?
Enhancer regions (located upstream/before the TATA box and gene to transcribe) bind activator proteins, which help the mediator protein set up the RNA polymerase complex
○ Activator proteins bind specifically to the DNA sequence of their enhancer regions
• Repressor proteins can get in the way of RNA polymerase assembly and transcription Repressor proteins bind to the silencer region
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○ Repressor proteins bind to the silencer region
Note that neither enhancers nor repressors bind to the promoter region; transcription factors and RNA polymerase do
Transcription is regulated by a complex "team," with various specialized roles that help in different ways (and some that instead slow progress, just like teams in real life)
Transcription factors, histone modifiers, and the chromatin remodeling complex can all bind to DNA sequences or to the mediator
Regulation with many variables or inputs is called combinatorial control and allows fine-tuning the rate of transcription
Some regulatory proteins allow the expression of genes that make other regulatory proteins that allow the expression of genes that do all kinds of other stuff and make more regulatory proteins that…and so forth ○ These are sometimes called linchpin/keystone transcription factors
In Drosophila (fruit flies), the protein ey is normally expressed in the eye region but can make a replica eye on the fly's leg if expressed in the leg
MyoD causes a chicken fibroblast to become a muscle cell line, which is useful for growing flavorless chicken meat in the lab
□ What are codons, the genetic code, redundancy, and reading frames?
• Codon: group of three consecutive nucleotides in mRNA
• Genetic code: each codon corresponds to a specific amino acid
• Redundancy: some amino acids are represented by multiple codons
○ But not the other way around--amino acids are a function of codons, if you like math
Reading frames: where each group of three nucleotides starts (123 456 789 vs 234 567 890; like the placement of bar lines to divide measures of music, if you like)
□ What are the important structural features of a tRNA? How is it charged? How is the charging checked? • The anticodon loop is complementary to the mRNA codon
• The 3' end carries an amino acid corresponding to the codon complementary to the tRNA's anticodon • There are two other loops to make an overall clover-shaped or t-shaped structure • A tRNA is "charged" with an amino acid by an aminoacyl-tRNA synthetase
Charging is checked two ways: the amino acid should fit in the synthesis site and not fit in the editing site of the aminoacyl-tRNA synthetase
What are ribosomes made of? What are the roles of the large and small subunit? What are the E, P, and A sites?
• Many proteins and several catalytic rRNA molecules
• Large subunit catalyzes peptide bond formation
• Small subunit binds mRNA
A (aminoacyl-tRNA-binding) site is where a tRNA enters and brings an amino acid into position to bond with the one before it (on the tRNA in the P site)
P (peptidyl-tRNA-binding) site is where the amino acid leaves the tRNA so that the next amino acid can form a peptide bond to its carboxyl group
• E (exit) site is where the tRNA leaves without its amino acid
• I highly recommend watching an animation of this process
What are the steps in translation including initiation, elongation, and termination? What are the roles of the initiator tRNA, MET-tRNA, release factors, and GTP hydrolysis in translation?
• Initiation: the initiator tRNA is a methionyl-tRNA; it and some initiation factors bind to the small subunit ○ Then mRNA binds
Then the small subunit carries MET-tRNA down the mRNA until they find a start codon (complementary to the MET-tRNA's anticodon)
○ And THEN the large subunit can bind, with the MET-tRNA ending up in the P site • Elongation: an aminoacyl-tRNA enters the A site
○ Its anticodon is complementary to the codon facing the A site
At about the same time, the newest amino acid forms a peptide bond to the previous amino acid, and the large subunit translocates (moves a bit towards the 3' end of the mRNA)
Forming a peptide bond to the previous amino acid requires that previous amino acid to break its bond to its tRNA, which ends up in the E site when the large subunit moves
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its bond to its tRNA, which ends up in the E site when the large subunit moves
▪ The amino acid just added is still attached to its tRNA, which is now in the P site ▪ The A site is briefly empty until another aminoacyl-tRNA comes in
○ The small subunit translocates as the tRNA in the E site leaves
○ GTP hydrolysis provides the energy for these bonds to form and translocations to occur ▪ Elongation factors like EF-Tu help with this
Termination: a release factor, which acts a lot like an aminoacyl-tRNA, enters the A site and binds to the stop codon
When the large subunit translocates and the last amino acid leaves its tRNA, the release factor ends up in the P site, which causes the ribosome to disassemble and release the new polypeptide
□ What do proteasomes, ubiquitin, and proteases have to do with protein degradation? • Proteasome: protein complex with two cylindrical caps providing channels to a protease core
Ubiquitin: attaches to target proteins, recognized by proteasome caps, causes target proteins to enter proteasome
Protease: responsible for protein degradation by hydrolyzing peptide bonds in the proteins that enter the proteasome's core
□ X-ray crystallography?
• Make a pure, solid crystal of a protein and shoot X-rays through it
• The diffraction pattern can be mathematically analyzed to find atomic structures • This is how Rosalind Franklin (NOT Watson and Crick) figured out that DNA was a double helix □ Nuclear magnetic resonance spectroscopy?
Applying a strong magnetic field to a protein sample causes measurable atomic vibrations in patterns depending on how close hydrogen atoms are to each other
This only works for fairly small proteins--it's the same NMR used in organic chem, and it gets really complicated really fast
□ Use of antibodies in immunoprecipitation and as molecular tags?
• Antibodies for a specific protein will selectively bind to that protein
• This can cause it to precipitate (like agglutination in blood typing), which isolates the protein
If the antibodies are covalently bound to radioactive labels or fluorescent dye molecules, they work as molecular tags to visualize protein locations
□ Restriction nuclease digestion?
I'm not sure we talked about this in class, but it's a method of slicing up DNA in a way that leaves "sticky ends" that can anneal to each other
• Combining various DNA molecules with the same sticky ends can create a big recombinant DNA molecule □ Electrophoresis?
DNA goes in wells at the negative end of the gel and is electrostatically attracted to the positive end of the gel
Smaller molecules move faster, so in a period of time, the DNA molecules will be spread out over the gel according to their size
Combining this with restriction nuclease digestion allows restriction fragment length polymorphism, or DNA fingerprinting
□ Hybridization and northern and southern blotting?
I'll update this study guide if these topics are covered in class on Tuesday the 9th, but otherwise, I don't believe they'll be tested
□ Genome editing by nucleases?
• CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats
Basically, this is a thing bacteria have to "train" their nucleases to target virus sequences for chopping up, and molecular biologists figured out how to use this to target specific sequences for opening in a way that lets them insert more DNA
The explanation in the lecture notes is…fun. Rather than try to recap that, I highly recommend this article: http://gizmodo.com/everything-you-need-to-know-about-crispr-the-new-tool-1702114381 •
The video embedded is also nice--here's a direct link to start at the beginning:
Fair warning: this starts with an overview of basic DNA structure, so you may want to skip forward a
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Fair warning: this starts with an overview of basic DNA structure, so you may want to skip forward a bit (or enjoy the confidence boost from knowing what they're talking about)
□ That's all for now!
Congratulations on making it through Dr. Chapman's giant test review! I hope reading this for you is as helpful as making it was for me. Happy studying, and good luck!
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