DNA: What r the Basic Properties of DNA (2): Difference in Bases of RNA vs DNA Differentiate between Pyrimidines vs.

Purines

1. Replication/Transcription/Translation 2. Transforms a genome through incorporation TAGC (U instead of for RNA)

1. TA/ U are pyrimidines ( single rings that

make 2 bonds) *U is substituted for T in RNA 2. GC ( makes 3 bonds)

3. Bases are hydrophobic and are the

interior of the strand DNA Define Exo vs Endo nuclease What is DNA ligase Name the causes of Denaturation Exonuclease: cleave the last nucleotide residue Endonuclease cleave bonds in the interior of the strand DNA ligase: enzyme that forms DNA 5-3 bonds

Differentiate between Nucleotide vs Nucleoside

(high temp, extreme pH etc) May anneal and become single stranded random coils

Nucleotide: The Bases TAGC/U + phosphate (activated) Nucleoside: base + pentose ring sugar

Chromosomal Organization Describe the Hierachical Structure of a Chromosome:

Oligonucleotide (short polymers to 30 nucleotides) Polynucleotides (long nucleotide polymers) Introns: pieces of mRNA that are spliced out (not coded) *rare in prokaryotic cells Exons: splicing that remains

Oligonucleotide Polynucleotides Introns Exons

What is a Histone what are its parts? How is H1 different? Do prokaryotic cells have histones?

Histone: functions to stabilize and condense DNA. Made of 8 proteins that form a disk around which DNA wraps two ea of : (H2A, H2B, H3, H4). H1 is a stabilizer of the last DNA turn. It wraPs around the outside of disk +DNA. prokaryotic cells do not have histones Nucleosome= histone +DNA (no H1) *onlye 1 3/4 turns of DNA Chromatosome: histone disk +DNA + H1 (2 full turns of DNA) Nucleofilament: long beaded string of chromatosomes + linker DNA

Chromosomal Organization contd. Describe the following: Nucleosome Chromatosome: nucleofilament Supercoils Chromatin

Differentiate between Euchromatin/HeteroChromatin What is in a Chromosome?

Supercoils: DNA ladders wrapped around eachother - Chromatin: packed supercoils + histones Eu= dispersed, hetero= condensed; During division becomes condensed. Chromosome= looped condensed DNA of centromere + telomere

DNA structure: DNA conformations - B-DNA - A-DNA - Z-DNA What is DNA Bending: Explain Cruciforms Triple-Strands;. In what groove? What are Hoogsteen Pairs, function, example? Quadruplex DNA: 4 strands; seen when and where?

crazy Z = L-handed - B-DNA: normal (Watson/Crick) - A-DNA (DNA-RNA hybrid during transcription) - Z-DNA (Left handed coil, like during transcription) DNA bending: *bending occurs when enzymes bind and due to photochemical damage Cruciforms: intrachain H-bonds occur, these form a function in control of replication and transcription. Triple-Strands:3rd strand H-bonded in major groove forming Hoogsteen Pairs. form in regions with many purine bases. Function in control of transcription etc.. confers stability on telomeres.( Eg.termination of fetal Hb transcription) Quadruplex: seen at telomeres during recombination Inverted repeats: palindromeread the same front to back and vice versa Mirror repeats: same sequence towards each other Direct repeats: sequences that continually repeat Structural Gene: eukaryotic DNA that actually codes for protein, the rest take part in regulation and development Replication = making daughter DNA strands Transcription= de-coding the message (making mRNA) Topoisomerase: Enzymes that wind and unwind DNA by Catalyzing breaking and rejoining of dna strand. - Type I: relieves supercoil by breaking one strand *also contains ligase - Type II: breaks both strands and reseals *requires ATP - Type III (gyrases): act to relieve supercoiling at transcription bubble Note: not a Helicase. Helicase is responsible for opening the replication bubble. Nalidixic Acid: inhibits bacterial gyrase (useful for acidic environment of urethra) - Gyrase is the bacterial analog to topoisomerase

Special DNA Sequences: Be able to explain each, maybe give an example. Inverted repeats Mirror repeats Direct repeats What is a Structural Gene? How is it different from the rest of the code? What is DNA replication vs DNA transcription?

DNA replication Topoisomerases: - What is it? - What are the types?

What is an antibiotic used to treat UTIs and why? - Gyrase

Prokaryotic DNA synthesis: What are the requirements for DNA replication? What is Primer/Primase? DNA polymerase what does it do and how? - Does is have exoncuclease activity? High or Low? NOTE: adds a 5 carbon of free nucleotide to the 3-OH attached to template strand. Synthesis from 5-3 means that the new strand is being made from its free 5(C next to O-- ) end towards the 3 end (OH on 3rd C) Prokaryotic DNA REPLICATION contd. Is a template always necessary for DNA replication? Is a double-strand important?

Template, primer, nucleosides, DNA polymerase Primer = short segments of RNA synthesized by primase enzyme to provide free OH group for nucleotides. DNA Polymerase= synthesize DNA polynucleotides by polymerizing nucleotides releasing PPi *require primer +template *add nucleotides only to 3 end, therefore synthesizing in the 5 3 direction *proof-reads through exonuclease activity (3-5 removal) *high fidelity

Yes NOTE: DNA must be in double-stranded form before replication, even in organisms that carry single-stranded DNA (its just how the machine works) Polymerases also have 5-3 exonuclease activity that removes RNA primer. Each new helix has one old and one new strand

How is RNA primer removed? What is semi conservative replication?

Steps in DNA replication: *the following words must be included in your story: Replication Fork Helicase Gyrase SSB RNA primase/primer DNA polymerase I and III Leading (5-3)/Lagging strand(3-5) *opposite of template Okazaki Fragments (are synthesized in which direction?) DNA ligase

1. Replication Fork is created through 2. Gyrases reduce the number of

breaking of H-bonds by Helicases supercoils behind the fork, SSB (singlestranded binding proteins) prevent the two strands from re-annealing 3. RNA primase lays down a primer on the leading strand and many short sequences on the lagging 4. DNA pol III starts forming the leading strand from the free 5 end, pol I fills in the free 5 ends created by the primers on the lagging strand (the pieces are called okazaki fragments (5-3 pieces) 5. Primers are removed, gaps are filled by DNA polymerase and segments are joined by DNA ligase

Prokaryotic DNA polymerases What is the function of polymerases? What are the differences between types of polymerases? I/II/III Function: add nucleotides to the end of a growing DNA chain, and repair (3-5 exonuclease) Pol I/III DNA synthesis Pol I synthesizes the lagging strand I(3-5 exonuclease), removes primer (also 5-3 exonuclease) Pol II- synthesizes ribosomes? Pol III- synthesizes the leading strand Klenow Fragment: 70kDa fragment (portion) of Pol I enzyme contains the polymerization and 3-5 exonuclease activity, as opposed to the smaller fragment for 5-3 exonuclease activity. More on Prokaryotic DNA synthesis: Define Processivity: - Which of the polymerases has the highest? What is the Sliding Clamp? The ability of an enzyme to remain on its substrate - POL III has higher efficiency than either Pol I or Pol II - Sliding clamp is an associated protein that confers stability to Pol III conferring its processivity. A replisome is the replication complex consisting of primosome: primases, lipases, helicases etc.. as well as the DNA strand, polymerases etc.. it occurs at the ORI or origin *there might be more words in this section we need to know, diff variations on replisome Prokaryotic vs Eukaryotic Replication: What are the 4 major differences?

What is a klenow fragment?

What is a replisome and where does it occur?

1. Size of the DNA of eukaryotes is much

larger

2. Eukaryotic cells pack DNA, chromatin 3. Eukaryotes replication fork movement is 4. Prokaryotes have one ORi for replication
What is a replicon? (circular DNA), eukaryotic have many and replication is bidirectional. Replicon: Segments between the various ORI on a DNA. DNA can contain up to 200 ORI. much slower.

When does replication occur in eukaryotic cells? When are histones synthesized and why? What controls the cell-cycle?

DNA replication occurs during the S-phase synthesis between the two dormant G1 and G2 phases. Note: histones are also synthesized during sphase so that new DNA contains both parental and new histones. Cyclins are a substrate for enzymes called cyclin dependant kinases (Cdksusually associated with tumourous growth). That control the cell-cycle through catalyzing microtubule formation and chromatin remodeling etc.. Note: G1 and G2 are very short in prokaryotes allowing for faster division. Ie. Bacteria grows like the dickens. Polymerase link together DNA nucleosides. Sigma- high processivity, 3-5 exonuclease, leading strand Alpha- slow processivity, synthesizes lagging strand diassembles nucleosome prior to DNA replication PCNA= proliferating cell nuclear antigen (like prokaryotic sliding clamp)

How do G1 and G2 differ in pro vs eukaryotes?

DNA polymerases- Eukaryotic: What is a polymerase? - Differentiate between Polymerase sigma vs Pol alpha? What is PCNA- proliferating cell nuclear antigen? - Antigen: helper RPA- replication protein A: What are the major differences between prokaryotic and eukaryotic polymerases? Telomerases Why cant DNA be fully replicated with a replisome?

RPA: Analogous to SSB in prokaryotes, disallows the strands to anneal ?? looping of lagging strands allow both alpha and sigma to elongate and proceed in the direction of fork movement?? Bc the lagging strand cannot be synthesized at the end where the last primer is removed (gap = 8-12 nucleotides) Telomerase: reverse transcriptase uses an RNA template for DNA synthesis. Contains RNA and is therefore template and enzyme Lacks exonuclease activity and are error prone Retroviruses do the same Telomerase activity decreases with age and therefore is related to cell senescence. *telomere length is inversely proportional to age. Tumors have upregulated telomerase activity conferring immortality prone to errors in transcription

What does telomerase do? How?

How do telomerases contribute to aging?

How does telomerase differ in relation to tumors?

Name a modern day antibiotic and how it relates to reverse transcriptases: think HIV.

AZT: dideoxythymidine, is an analogue of dT and incorporates into viral genome inhibiting reverse transcriptase and cutting short viral DNA synthesis *used in the treatment of AIDS.

DNA Recombination: name the 3 types

1. Meiosis: homologous 2. Site-specific (insertion by

bacteriophage into genome)

Give an example of site-specific recombination: What is a transposon?

3. Transposition: jumping DNA

Eg. When e.coli bacteriphageinserts into a host genome at specific sites. Genes that insert and are removed seemingly on their own. jumping gene Usually carried on plasmids RNA contains Uracil instead of THymidine Also has post-translational modification such as methylated bases. RNA is single stranded with hairpin pairings that confer stability (60% of strand) mRNA 1%, tRNA 15%, rRNA 80%, small nuclear RNA (snRNA), scRNA (small cytoplasmic), mtRNA (mitochondrial), RNAi (interface plural) RNP (ribonucleoprotein particles, Ribozymes,

RNA vs DNA How do DNA and RNA bases differ? What is the common structure of RNA? What are the Types of RNA (9 types): what are their respective percentages?

mRNA: What is the general structure for eukaryotic mRNA? What is the function of the cap? what is polycistronic mRNA? Is it more common in pro or eukaryotes? What is a cistron? What protein has the shortest of all the mRNA half-lives? Why? Name two exceptions to short mRNA halflives.

5-5 cap is followed by a nontranslated leader sequence, startcodon, AA instructions, stop, nontranslated tailing sequence (usually poly A). - The cap confers stability and protects Polystronic mRNA = mRNA that encode for more than one protein, uncommon in eukaryotes? cistron = DNA segment that codes for one polypeptide. Histone mRNAs are the shortest. Lack poly A tail.

1. Unfertilized eggs (inactive and ready

for fast synthesis

2. Hb mRNA, reticulocyte mRNA does not

turnover (no nucleus remember).

Name two mRNA modifications that extend their half-life. What is the primary determinate of mRNA stability? tRNA: what are the two functions of tRNA? rRNA: where is it synthesized? Made up how how many parts what are they called?

Inverted methylated base (5-5 phosphate link cap) Poly A tail. Primary determinate of mRNA stability. Histones lack this tail, thus the short life. The longer the tail, the longer the life I guess.

tRNA: T shaped AA carriers 1. Activate amino acids (acceptor stem) 2. Recognize codons in mRNA (anticodon loop) rRNA: ribosome subunits Synthesized: nucleolus 2 parts: Large and SMall

Explain what each of the following is in one sentence: snRNA scRNA mtRNA RNP RNAi Ribozymes Does RNA turnover? Why or why not? How long is mRNA half-life? tRNA half life? What catalyzes this degredation?

snRNA: (small nuclear)recognizes introns on mRNA scRNA (small cytoplasmic): selects proteins for transport mtRNA: its own exclusive with one tRNA for ea. AA RNP: function in RNA processing (splicing etc..) RNAi: control cell phenotype during development Ribozymes: RNA that act as enzymes eg. RNase P that cleaves pre-tRNA to mature tRNA Yes. Bc RNA is not repaired like DNA, RNA is degraded and newly synthesized. mRNA Half-life = minutes to hours, tRNA=5 days RNA is destroyed by ribonucleases, exonucleases, endonucleases.

RNA synthesis: List the steps to RNA synthesis: Know the definitions of the following: pribnow box Primary transcript ribonuclease What are the types of post-transcriptional modification? How does transcription ensure unidirectionality? How is a promoter determined weak or strong?

1. RNA polymerase searches for promoter

site on DNA .(TATAAT box, or Pribnow box) 2. attaches promoter/ Releases subunit sigma 3. RNA polymerase creates RNA strand the primary transcript. 4. Post transcriptional modification creates mature mRNA that is then shuttled outside nucleus. a. Removal of external/internal nucleotides by ribonucleases. (degradation) b. Base modification c. Addition of nucleotides The TATAAT box does not have symmetrical bases in the complimentary strand.

Whether it is complimented by enhancers or repressors.

RNA polymerase Can RNA polymerase synthesizein 3-5 direction? Does RNA polymerase require a primer? Which is more accurate DNA or RNA polymerase? PROKARYOTIC RNA POL. What is a holoenzyme? Core enzyme? Which RNA subunit recognizes the promoter region? EUKARYOTIC RNA POL What antibiotic can distinguish between pro and eukaryotic RNA pol? What is used to detect eukaryotic RNA pol? - 4 types: pol I, pol II, pol II, and mitochondrial. What is the primary purpose of RNA pol I? What is a nuclear organizer? Rifampicin. Used to treat TB binds to B subunit in prokaryotic RNA pol. A mushroom toxin: amanitin RNA pol I: 50 % of all RNA. synthesizes only the transcript for rRNA subunits within the nucleolus at the nuclear organizer Nuclear organizer: The region within the nucleolus containing DNA that codes for rRNA. No RNA polymerase, like DNA polymerase can only make 3-5 linkages. No. DNA polymerase. RNA polymerase does not have exonuclease activity and therefore cannot proofread. Holoenzyme is the prokaryotic RNA subunit complex (2A, B, B, omega, sigma). Core enzyme = all subunits minus sigma (recall, sigma is released upon binding to promoter region) Sigma acts to recognize the DNA promoter

EUKARYOTIC RNA POL contd. Which RNA pol does the main RNA transcription? Which RNA pols are sensitive to amantin (the eukaryotic RNA pol detector)? What is the role of RNA pol III? Describe ribonuclease P. Splicing: Name the process for intron removal. What is the role of snurps in splicing? Describe alternative splicing:

RNA pol II transcribes structural genes (premRNA), 20-40% of all RNA. I no, II/III yes, mitochondrial*. No (*but sensitive to bacterial antibiotic rifampicin). RNA pol III transcribes tRNA, and small 5s rRNA. Makes up 10% of RNA activity Ribonuclease P is a ribozyme that trims the 5 end of the primary tRNA transcript.

Splicing. Note: exons are what are spliced together and kept as the primary transcript. snRNPs: Snurps are small nuclear ribonucleoproteins. They (U1RNA, U2RNA)discriminate splice sites and remove introns. A splicing is alternative when the same primary mRNA transcript is spliced differently coding for slightly different proteins. Eg. Tropomyosin.

Translation: What is a codon? Are codons necessarily specific? What is the sequence for start codon? What are the three stop codons? Is an AA necessarily in the synthesized protein if its codon exists in the transcript?

mRNA = code, tRNA = activate AAs, rRNA translates code. Nucleotide base triplet that codes for an AA. No. however some are more specific. Eg. Met, the tRNA initiator, has only 1 codon. The start codon is AUG. however the start complex begins first with the TATAAT box. UAA, UAG, UGA. Note: they all begin with U. No. modification of the transcript and eventually the mature protein can remove AAs during processing.

What are the 3 characteristics of the genetic code. mRNA conventions: - Usually write code from 5-3, the same direction it is translated. Proteins are written by convention from amino terminus to carboxyl Where is an anticodon triplet found? What is the wobble effect?

1. Degenerate (AAs have more than one 2. Universal (both pro and eukaryotes use
the same nucleotide triplets) codon)

3. Continous (codons are not separated by

spacers)

On tRNA anticodon loop. to recognize specific AAs. Many codons for each AA. The ability of an anticodon to pair with more than one codon. Eg. A single tRNA can recognize all 4 Ala codons.

What is the siginificance of inosine (I)?

Inosine is a modified base often found in the 5 (last)position of an anticodon that allows for less stringent base pairing. Eg. UorI, Gor I, A or I etc 3 end is first base read in the codon. High conservation of this codon is necessary because of stop codons. (recall all stop codons start with C or U) Activation of AA requires aminoacylation by aminoacyle tRNA synthetase. Each AA has its own specific enzyme for activation. 2 reactions: 1. Activation of AA with ATP 2. Transfer of aminoacyl group to tRNA Prokarytoes: fMet-tRNAfmet (formyl Met) Eukaryotes: met-tRNA Termination = stop codon. No associated AA. Pro: Shine-Delgarno sequence, Eu: Kozak sequence UAC. Recall that AUG is the start codon, therefore the initator AA will have the anticodon to the start sequence. 1. A (aminoacyl/entrance site) 2. P (peptidyl transferase binds 2 AAs) 3. E (exit site) Read: 5-3, formed: amino to carboxyl. N to C terminus.

Why doesnt the wobble effect occur at the 3 end?

How are amino acids activated? Note: aminoacyle tRNA synthetase are highly selective. proofread Initiator tRNA, pro vs eukaryotes: Protein synthesis is initiated by the formation of? Terminated by? *non-formylated Met exists inside the protein and is not an initiator in prokaryotes Name the leader sequence small subunit rRNA binds to on the mRNA transcript? What is the anticodon on met-tRNA? How many sites are in rRNA? Name them. In which direction is mRNA read? Protein formed?

Common antibiotics that inhibit protein synthesis: Briefly describe mechanism and common use. 1. STREPTOMYOCIN 2. TETRACYCLINES (group) 3. PUROMYCIN* 4. ERYTHROMYCIN 5. CYCLOHEXIMIDE* 6. RICIN

Amino Acids: Describe Structural vs. dynamic proteins. What are 2 specific characteristics of AAs in living organisms? What is the general structure of AAs?

Note: I have included clinical use for my personal reference 1. First line antibiotic for TB. Prevent binding of fmet-tRNA to rRNA small subunit in prokaryotes. Mutations can confer resistance (ie. MDR-TB) 2. Cholera, rosacea, acne. Inhibits aminoacyl-tRNA binding to mRNAribosome complex. 3. Used only in research, toxic to both pro and eukaryotes. Is an aminoacyl-tRNA analog that irreversibly binds to A-site. Stops elongation. 4. Used in case of penicillin resistance (2nd line). Inhibits peptidyl transferase in prokaryotes 5. Used only in research. Inhibits peptidyl transferase in eukaryotes only. 6. Lethal to humans. Castor bean toxin. Removes adenine from rRNA Structural: matrix for bone and CT = form. Dynamic = movement. So everything else. (enzymes, transport, regulation etc) 1. made of -amino acids (contain central alpha carbon). 2. AAs are L-chiral. That is, have a left handed chiral center. -C has 4 attachements: - Carboxyl group (COOH/ COO-) - Amino group (NH2/ NH3+) - H atom - R side chain AAs can act as buffers due to ability to hold and release protons (H+s) Glycine: has 2 Hs (R-group is an H) Peptide bond: primary amino group catalyzed to primary carboxyl group of another AA. Reaction releases H2O through release of Hgroup on -C. Very strong (50% double bond character) In one plane field (planar) Rotation does not occur around carbonylNitrogen however bonds around -C can rotate during folding. Protein: >50 polymers Peptide: <50 AAs Polypeptide: small protein of < 50 peptides. -

What is a pH function of AAs? - Which AA is the exception?

Peptide Bond: Define a peptide bond. What does peptide formation release?

Characteristics of peptide bond: Differentiate between protein vs. peptide vs. polypeptide

Chemical and charge properties of amino acids and proteins: Formation of some AAs are derivations of other AAs. Give 2 examples.

Cystine: derived from two cysteines. (derived through thiol oxidation forming a S-S bond) Glutathion: derived from 3 amino acids. Glu-CysGly. Functions as electron donor(reduces) to repair oxidative damage. *S-S bridge is used to stabilize protein structure and in Redox reactions. (as in cystine and glutathione) 1. Ionizable groups influence protein properties 2. Hydrophobic/non-polar sidechains, found on the surface of proteins, critical for protein folding. Primary: its amino acid sequence Secondary: local folding of the amino acid sequence; -helix and -pleated sheets, turns, and random coils. Tertiary: 3-D structure of the protein.bonds: electrostatic (salt bridges btw and + AA chains) , hydrophobic, H-bond (between AAs), disulfide. Quarternary: association of discrete polypeptide units functioning as a multi-unit protein. Formed by homomultimers (of the same polypeptide) and heteromultimers Hydrophobic chains prevent interactions with water. The more hydrophobic chains, the more stable the protein is. -helix: H-bond Right handed helix, side chain project outwards -pleated sheets: two chains aligned in parallel. Side chains project about and below. -turn, reverses the direction of the polypeptide chain to form compact proteins

Why are disulfide bridges important? What are two group/side chain functions of AAs and why?

Protein structure: structure determines function Differentiate primary vs secondary vs tertiary and quarternary structure.

How does hydrophobicity confer stability to a protein?

Describe an -helix/ -pleated sheets

Globular vs Fibrillar proteins?

Random coildrandom functioning binding sites Globular: tertiary structure resembling globes Fibrillar: structure resembles rods

What are the advantages of quarternary structure?

Stability, genetically economical (same gne many proteins), efficiency (no transport necessary) , cooperativity (facilitation) Primary structure contains properties to spontaneously fold but is limited to short range. Facilitation by other proteins called chaperones to create domains. Final conformation is called native structure

What types of interactions initiate protein folding?

What is a proteins native structure?

Chaperones: When do chaperones first bind to the protein? What happens each time a protein crosses a membrane? What are functions that require protein chaperones?

They bind as the protein is synthesized to prevent aggregation/precipitation. They become unfolded. Refolding of proteins after crossing membranes or with temperature increases. Facilitate transport into mit. And through ER. Protein assembly. Cells cultured at higher temperatures have greater percentages of chaperones bc more refolding is needed to retain native protein structure.

Why are chaperones called cellular heat shock proteins? Denaturation: Always causes loss of function Caused by?

pH, ionic strength, temp, (solvent, denaturants such as urea, detergents, guanidine) *weaken AA interactions. Denaturation of improper folding triggers enxymatic breakdown and phagocytosis of protein.

Explain the molecular reasoning for development of Alzheimers and Creutfeldt-Jakob disease.

Alzheimers: proteins form abnormally due to incorrect cleavage of extracellular transmembrane portions of . These form spontaneous aggregates called amyloid which are not phagocytized. Creutzfeld-Jakob (CJD): fibrous amyloid plaques develop in the brain. Thought to be of prion origin causing insolubility of protein. Characterized by causing nervous system dysfunction such as ataxia, paralysis as wella s dementia. *prion protein normally has 3 a-helix and 2 bstrands, altered protein exhibits transformation of 2 a-helixes into b-strands which then form fibers. 1. Functional role within blood: high concentration (eg. RBC) 2. Carried by blood function outside it *disease may be caused by increase or decrease in the amt of ea. Of these in the blood Serum = blood plasma without clotting factors (no fibrinogen, rbcs?, or platelets?) Electrophoresis: separating charged particles within a gel, usually done at ph 8.6, proteins move towards (+) or (_) poles. Gel is then scanned to show protein peaks. Albumin, alpha/beta globins, gamma globins

Blood proteins: 2 types of blood proteins:

Define serum: Proteins from blood serum can be used to diagnose disease through separation based on net charges, what is this process called? Name some of the major gel peaks:

What is the most abundant blood plasma protein? What is its function? What is the clinical sign for hypoalbuminemia? Name some examples of disorders that cause hypoalbuminemia:

Albumin makes up almost 50% Function: to maintain osmotic pressure and to transport molecules such as bilirubin, FAs, and hormones. Edema bc oncotic pressure out of the tissues is not great enough. Liver dysfunction (Hepatitis C, alcoholism, etc..), Kwashiorker disease (protein malnutrition in children), hypoproteinemia from renal and bowl diseases.

Common gel protein electrophoresis patterns: Normal Immediate response Delayed response Hypogammaglobinemia Hepatic cirrhosis Monoclonal gammopathy Nephritic Protein-losing

Define the following: - Antibody, antigen, epitope, hapten Describe the structure of IgG molecules in terms on constant and variable regions. How does the composition of the hypervariable region account for antibody specificity? What is the role of the constant region? What do disulfide bonds do? What is an immunoglobulin fold? Define the Hinge region of Ig.

Normal Immediate response: stress or inflammation - Delayed response: infection, increase in immunoglobulins - Hypogammaglobinemia: immunosuppressive diseases - Hepatic cirrhosis: reduction in albumin increase in gamma (to retain osmolarity) - Monoclonal gammopathy: gamma myelomas - Nephritic: inflammation + low synthesis of small proteins that are lost at kidney - Protein-losing: loss of albumin and gamma (stress and nephritic issues) Epitope: specific portion of antigen to which the antibody binds Hapten: a small molecule which by itself does not produce an immune response

Ig: 2 heavy chains: constant region (Ch and Cl) are highly conserved sequences 2 Light chains: variable region epitope binding side (Vh and Vl) responsible for specificity - Hypervariable region: multiple N-terminal loop part of light chain that aids in diversity of epitope binding. *2 regions connected through S-S bonds that stabilize the domains A structural motif seen in Ig and elsewhere Area btw Ch1 and Ch2 connected through an SS bond can swivel to accommodate conformations IG function: - To activate the complement-binding site system. An 11 protein cascade system that eventually cause cell lysis. - Initiates a conformational change cascade within the Ig itself to trigger cascade. - Amplification of the signal also occurs (Bcell system?) Activated B-cell experiences oncogene translation or limitless promotion (loss of stop codon) and over produces antibody, therefore attacking various organ systems.

Describe the general biological function of Ig: After antigen binds to Ig what happens?

What is the molecular mechanism of multiple myeloma?

What determines the different classes of immunoglobulins? Describe the functions and different classes: IgG: o o What does it look like? what are the cleavages it undergoes?

5 main classes: - The H chain classes are differentiated into IgG, iGA, IgM, IgE, IgD. - Two isotypes of L chain- kappa and lambda. IgG = most abundant, 75% Looks like : Y Gamma variations in light chain Undergoes enzymatic cleavage into functional subunits Papain:3 fragments = 2 antigen binding fragments, Fc- complement fragment control Ig catabolism. Pepsin: 1 function unit, and 1 proteolyzed Fc region. Can bind 2 antigens. Looks like: Y or 2Ys joined by a J chain Serves as early defense mechanism prevent pathogen adherence IgA binds and then phagocytosis occurs Normally found in locations with environmental contact (tears, mucous, saliva,) First antibody made by B-cell and is presented on its surface (first line defense) Primary antibody in a fetus Can be found as a Y monomer or 5Y pentamer o Note: although there are 10 active sites only 5 actually work. Primarily in immune surveillance of mucosal membranes: intestine/lung Induces release of histamines allergic reaction Anaphalaxis associated Exists only as a Y monomer Also present on cell surface of B-cell Exists only as a monomer Less than 1% of total Igs

IgA: o o o

What does it look like? What are the two major subtypes? Where is it normally found?

IgA: IgM: -

IgM: o o

What other cell type is it associated with? What does it look like?

IgE o o o Primarily found where? What disease process is it associated with? What does it look like? associated with what other cell?

IgE: IgD: -

igD o o

Hemoglobin and myoglobin: - briefly define the following: Globular vs fibrillar proteins: Globin fold motif: Hemoglobin vs Myoglobin Heme

Globular vs fibrillar proteins: globular = sphere like and soluble, fibrillar= looks like fiber, insoluble Globin fold motif: 8 -helices, highly conserved throughout evolution although AA series conservation is very low. Hemoglobin vs Myoglobin: Hb = tetrameric o2 binding transport protein, Mg= monomeric muscle protein that binds a single o2 Heme: iron- porphyrin common to both Hb and Mg, found in other various metalloproteins such as peroxides etc.. Primary sequence point mutation change in Hb changing glu to Val in one helice. Creates a hydrophobic pathch promoting aggregation and thus changes in RBC membrane. 4 pyrrole rings and a fe2+ Resides in the hydrophobic core of Hb and mg. 2 axial positions coordinated by His residue - when bound protected from oxidation - *genetic mutations can compromise this state - Oxidized Fe cannot transport O2 Hb: -

what is the sickle cell anemia mutation?

General Structure of heme: What is the significance of the His residue in heme?

how are Hb and mg related? What are the chain types that hb consists of?

Basically 4 myoglobins that posses hydrophobic patches that assist quarternary structure Typically composed of o 2 chains o 2 chains

How does the expressions of these change over time?

Expression of ea of these chains changes over development into 5 different subtypes

Fetal is predominantly Adult has both and

O2 binding and release by Hb and Mg: Describe the difference btw: - Tense state: Relaxed state:

Tense state: si deoxy state, ferrous ion stands out of the plane. Relaxed state:oxygen binds and ferrous ion moves to center of porphyrin ring. Energetically more favorable state. As ea. O2 binds, conformation changes so that o2 binding becomes more and more favorable. Conversely, as o2 is dropped off in tissues, o2 affinity decreases as ea. O2 is removed. (curve is sigmoidal) *binding of 4th O2 requires no E!! *Myoglobin does not experience cooperativity and thus has a linear dissociation curve

Describe binding Cooperativity of Hb:

What is the shape of the myoglobin dissociation curve and why?

What is BPG/DPG?

How is it relevant clinically? How does it contribute to fetal development?

2,3, diphoshoglycerate is produced in high quantities in RBCs, has a high affinity for deoxygenated state of Hb. Bind subunits together. Thus, lowers Hb o2 affinity. (considered a metabolic byproduct and o2 dropoff molecule) Clinical: - Hypoxia and high altitude adaptation. - oxygen trap of anticoagulant acidcitrate-dextrose decreases DPG synthesis - Fetal Hb does not bind DPG very well shifting dissociation curve to the left so that o2 can be taken from the mother. When Hb is in R state, certain factors contribute to decreasing o2 affinity of Hb. - Increase in pH, DPG, metabolites, low o2 concentration, high CO2, high temp 1. Can be chemically bound to Hb 2. Can be coupled to H+ transport through carbonic anhydrase present in RBCs. (releases HCO3- and H+ from CO2 and H2O) Blow off too much CO2 cause a Left shift to Hb droping off less O2 to brain tissues and ultimately fainting.

Describe the Bohr effect:

What are the 2 ways Co2 is transported?

Why do people pass out when they hyperventilate for too long?

Describe how Diabetes is related to Hb.

Non- enzymatic reaction of Hb with glucose forms gycosylated Hb A1c. is a very slow process but glycosylatedd HbA1c is used as a marker of the progression of diabetes.

Many genetic mutations of Hb are known. Are usually named by place of discovery. What is thalessemia? Thalassemia: General term attributed to mutations that one or more of the globin genes is not expressed properly. - Eg. / etc

Hemoglobinopathies: *mutations occur spontaneously but must be kept over multiple DNA cycles in order to permanently incorporate into the genome. Mutagen is an inducer of mutations. Give some examples: Silent vs expressed mutation:

Mutagens: - Oxidative deamination oxidative damage - Apurination/Apyrimidation-loss of a base type - O6-methyl guanosine (highly mutagenic and can cause other modifications to occur) - Cyclobutane forms T-T dimmers that block DNA polymerase. Silent: mutations that are not expressed in the phenotype Expressed: - Conservative: AA is changed to a similar AA - Non-conservativeB: AA is changed to diff AA - InvariantAA: change occurs at active site

Give examples of different types of mutations: - Conservative - Non-conservative - Invariant AA Categorized as: - Missense - Nonsense - Read-through - Frameshift - Transition - Transversion - Triplet expansion

Missense: change in expressed AA Nonsense: early termination (stop codon) Read-through: loss of stop codon Frameshift: insertion/deletion of nucleotide Transition: a purine-pyramidine pair substituted for another Transversion: pair flips chains (becpmes pyramidine-purine) Triplet expansion: addition of GC series in promoter regions.

Give an example of a triplet expansion disease: What is a hemoglobinopathy? Describe molecular basis of hemoglobinopathy categories:

Fragile X: primary defect in initation or elongation of transcription. - Conditions of abnormal Hb that result in anemia

Unstable Hb structure: mutations of AA residues at helix-formation sites. Precipitate as Heinz Bodies Increased or decreases O2 affinity: Increases heme oxidation: Mathemoglobin; oxidation of ferrous ion, higher than 10% show cyanosis. Imbalance of chain synthesis or Changes in chain properties: thalaseemias

- thalaseemia: - Homo vs hetero zygous

- thalaseemia: fetal -globin continues synthesis after birth. - Heterozygous: some adult Hb do not require treatment - Homozygous: severely anemic during 1st year of life requiring blood transfusions. (Cooleys disease) o This can result in Fe overload and death btw ages of 15-25 yrs.

-thalassemia:

Barts Hb?

-thalassemia: - 2 copies of the gene ( and ) on ea. Chromosome, several levels of deficiency. - 2/4 defective = mild anemia - = Hb H disease with sever hemolytic anemia - 4/4 = hydrops fatalis, no Hb at all. Barts Hb: - Synthesis of 4 gamma chains does form tetramers however exhibits too high of an affinity for O2 and is useless. Carbonmonoxide poisoning: -CO increases affinity for O2 to HB slowly suffocating the individual. High O2 pressure (hyperbaric chambers) are used to treat this. (carboxy-Hb)

Review HbS: sickle cell (val glut) Give an example of an acquired Hb opathy:

Fibrillar Proteins: Collagen type I has 4 AAs that make it unique. What are they? Obviously these AAs include posttranslational modifications. What 3 enzymes are responsible for these? 4 components are required for these enzymes to work. What are they? In all collagens what AAs occur every 3rd residue? AAs: Gly, Pro, HydroxyPro, HydroxyLys

1. Lysyl hydroxylase (lys to OH-Lys) 2. Prolyl-4-hydroxylase (Pro to OH-pro) 3. Prolyl-3-hydroxylase (4-OH-Pro to 3OH-Pro) Components: 1. Fe2+, O2, -ketoglutarate, ascorbic acid Gly, and Pro 1. Gly- the smallest AA fits into small holes in the core of the structure 2. Pro- is a helix breaker, does not allow regular alpha-helices (polyproline type II helix) 3. OH groups react further to form crosslinks and eventually the 3 braided strand of Collagen. polyproline type II helix: - L-handed - Ea. Peptide bond is perpendicular to the axis - 3 member super helices formed by Gly (this upser helix is R-handed) Hyp residues allow for interchain H-bonds, higher degree of Hyp = more stability and resistance to degredation with temperature. Varies by species. Random for structural tissue strength Meshwork for filtering Covalent cross-links for meshes. Some Lys are converted to allysine by enzyme lysyl amino oxidase (copper dependent) which then spontaneously reacts with non-modified Lys of another collagen chain. Collagen fiber termini form globular ends called telopeptides, usually extracellularly cleaved in order to form poly-fibers.

What are the structural consequences of this unique sequence? What special helix is made? What are its characteristics?

Which AA varies in the collagen structure in different species and why? *PS> this is why UV causes wrinkles.

Collagen has 2 major structural arrangements, what are they? How are mesh-works formed? Describe the mechanism.

What region of collagen is considered of globular character, what are they called, what happens to them?

Describe the steps in the biosynthesis of collagen. *collagen is formed from fibroblasts Explain how ea one comes about and what defines it. 1. 2. 3. 4. Preprocollagen Procollagen Tropocollagen Collagen

5. Translation of 3--chains occur on

ribosomes in RER. To form preprocollagen (pre-refers to signal peptide) 6. Signal peptidases cleave signal peptide forming procollagen 7. Hydroxylation modifications occur in ER lumen by 3 enzymes, the glycosylation 8. Intra and inter chain bond formations begin to occur and triple helix is formed inside RER, procollagen then sent to golgi for end glycosylation 9. Procollagen is excreted via exocytosis 10. Telopeptides are cleaved by procollagen peptidadese = tropocollagen 11. Fibers self-assembleinto fibrils and fibers in quarter staggered configurations and stabilized by cross-links. Fibrillar (I-II, V), network-forming (Type IV), Fibrillar associated (IX, XII) etc..

3 major categories of Collagen types: name them. What are the three major diseases associated with collagen? Explain their general mechanism.

1. Scurvy: ascorbic acidic deficiency

(collagen enzymes dont work right). Usually osteoporosis, fragile capillaries (bruising), loss of teeth and bleeding gums. 2. Osteogenesis imperfect: Debilitating genetic disorder. Unstable procollagen helices (lack of Gy residues), brittle bones 3. Ehler-Danlos Syndrome: group of disorders referring to weak CT. hyperextensibility, impaired joint mechanics, etc.. Elastin provides elasticity to tissues through coils that are relatively distensible, insoluble in water. They are critical to arteries and vessels- think AORTA. No secondary structure, unordered coils with mobile AA residues. Modified lys residues (allysine) are found. Catabolised by lysyle amino oxidase. (also seen in collagen crosslinks) These react to form heterocyclic structure of desmosine an isodesmosine to form crosslinks. With age, more cross-links form diminishing elastins distensability properties. Scarring =

What is Elastin? why is it special? Talk about elastins general structure:

Aging does what to elastin? What about scarring?

same thing.

Intracellular fibrillar proteins contribute to cytoskeleton: name them. (3) Microfilaments are found mostly where? What are they?

Microtubules: what is there general structure, what are they made of? Cells use microtubules for which general processes?

1. Microtubules 2. Microfilaments 3. Associated filament binding proteins (Eg. Microtubule associated proteins such as kinesin, dynein) Microfilaments are found in muscle: - Actin: polymerization of globular (Gactin) units - Myosin: motor protein, produces movement through ATP dependent binding to Actin. Microtubules: - Hollow tubes made from polymerized alpha and beta-tubulin monomers. Reinforce cytoskeleton, construct cilia/flagella, separate chromosomes during mitosis. *trafficking

Define (+) and (-) microtubule ends and their general properties. What are Kinesins and Dyneins, why are they cool?

(+) = fast-growing end. Most likely seen oriented towards cell periphery which modulate. Anterograde trafficking. (-) = retrograde vesicular trafficking (think neurons) Microtubules provide cellular train tracks for ATP dependent transport of organelles/vesicles. Kinesins: move vesicles to (+) end: anterograde *has two heads that bind to microtubule, and tail for transport Dyneins: move to (-) end: retrograde *eg. Endocytosed molecules.

Intermediate Filaments: what are they? What are their general categories? What are the two types of keratin? What is the general structure of keratin?

- are named such bc of their size relative to microtubules and microfilaments. - they are exclusively structural - 2 general categories: Keratins, Lamins (note: lamins were not discussed in our book) Soft Keratins = internal structures Hard Keratins = hair, skin, claws -helix dimer that forms coiled-coils (recall, collagen is a triple helix of helices). dimers are assembled into multimers stabilized by disulfide bridges. - modified by reducing agents that break disulfide bonds (such as temp/ straighteners/curling irons) - re-shaped by oxidizing agents (like water/rain!) Nuff said. Remember enzymes are proteins that speed up / Slow down reactions. (otherwise takes years) Attributes: Specific (regulate), reversible/irreversible, competitive/non-competitive. Domains: - Prodomain - Catalytic - Substrate Binding - Transmembrane Zymogen: inactive proform of an enzyme

Why can girls straighten and curl their hair? (boys can too.. you just probably shouldnt.)

If youre in medical school you better know the difference between an enzyme and a substrate. Function of enzymes? Enzyme attributes: Enzyme Domains:

What is a zymogen? General Regulators of enzyme activity: (4)

1. Substrate availability (amount and

location) Eg. Alzeihmers: overproduction of specific cleavage enzyme which disallows substrate for other enzyme. excess cleavage and amyloid precipitate 2. Enzme availability (synthesis) - Cyclooxygenase turn arachidonic acid into prostaglandins (inflammation). Downregulation of this enzyme (aspirin) decreases inflammation 3. Enzyme activation (non-zymogen) - Activation of trypsinogen by enterokinase. See acute pancreatitis. 4. enzyme inhibitors, endogenous vs exogenous. - Endogenous tend to be non-specific. -

Specific eg. In lecture were given for each of these and are included briefly above.

Define the following: 1. oxidoreductases: 2. transferases: 3. hydrolases: 4. isomerases: 5. lyases: 6. ligases:

1. oxidoreductases: transfer hydrogen or oxygen from one substrate to another (e.g. oxidases) 2. transferases: transfer a specific group (for example a phosphate) from one substrate to another (e.g. kinases). 3. hydrolases: catalyze hydrolysis of a substrate (e.g. digestive enzymes) 4. isomerases: change the molecular form of a substrate 5. lyases: remove or add a group to a substrate in a non-hydrolyticalway (e.g. carboxylases) 6. ligases: join two molecules through the formation of bonds between carbonand O, S, N (e.g. citric acid synthetase)

What are Proteases? What do they do? Name the 4 major classes of proteases. There is an example of ea.

Proteases: hydrolases that cleave peptide bonds

Give some examples of proteases functions:

1. 2. 3. 4.

Serine proteases (trypsin) Aspartyle proteases (HIV) Metalloproteases (matrix) Cystein proteases (caspases)

-maturation of viral proteins (HIV proteases) - food digestion (pepsin, trypsin etc..) - intracellular protein degradation (macrophages) -Orchestrating biochemical cascades (eg. ACE) -Apotosis - activation of complement ECM remodeling

Course objective: illustrate protease significance in CT components. 3 examples: 1. Physiological ECM remodeling 2. Pathological remodeling 3. Cancer Metalloproteases: what do they do? Eg. Of Ehlers-Danlos syndrome is a mutation in the ADAMTS family of metalloproteinases.

1. Physiological ECM remodeling:

breaking and remodeling of tissues (eg. Endochondral ossification) 2. Pathological remodeling: tissues broken down during disease (eg. Arthritis) 3. Cancer: cells use proteases to invade tissues Metalloproteases: use Zn to coordinate and activate H2O molecule, mediate turnover of matrix molecules. (Development, Maintenance, Reproduction)

Endongenous inhibitor of metalloproteinases: eg. -2 macroglobulins (produced by the liver) and selective inhibitors TIMPS? Drug Development: methods, pitfalls, safety - Selectivity - Tissue distribution of target enzyme - Reversible vs. irreversible.

Post translational modifications: Signal Peptides: describe where they are and their sequence. Describe the steps for which signal peptides are involved. 1. Signal peptide 2. Signal recognition particle 3. Docking protein 4. Translocon 5. Signal peptidadeses Signal peptide: 15-30 AA at amino terminus. Contains hydrophobic AAs, is + charged and contains cleavage site for signal peptidase.

1. Signal peptide emerges from a

translating sequence

2. Signal recognition particle binds 3. Ribosome moves to ER membrane

and binds to docking protein, and halts synthesis

4. translocon opens a hole and inserts 5. after termination signal

peptide+ribosome into ER lumen peptidadses cleave signal peptide.

Formation of insulin is similar to collagen Describe the formation of insulin. Preproinsulin still contains signal peptide After cleavage become proinsulin Proinsulin folds forming disulfide bonds Transported to golgi and packaged into secretory vesicles Then proteolytically modified by proteases cleaving specific residues and forming mature active insulin Posttranslationally. What is Phosphorylation? Distinguish between a Kinase and phosphatase. What is glycosylation? What is a Proteoglycan? Aggrecan? When and why does cartilage fluoresce? Phosphorylation is a covalent alteration which may activate or inactivate an enzyme Kinases: add phosphate group Phosphatases: remove phosphate groups. Glycosylation: addition of carbohydrates to proteins to change stability, solubility, and physical bulk, may also act as recognition peptides/targeting, function modification. Proteoglycans: contain protein and carbohydrates (similar to glycoprotein) but primarily carbohydrate Aggrecan: is a large negatively charged particle to which H2O binds, single protein core with extensive sugar chains. Primary component of cartilage. Fluoresces with age due to Advanced Glycation End (AGE) Products, or cross-link formation.

Do protein modification occur posttranslationally or posttranscriptionally?