Week One Objectives Flashcards
What is the chemical make-up of nucleic acids
Nitrogenous Base, Ribose sugar, phosphate (deoxyribose in DNA lacks 2’ carbon hydroxyl group
Watson-Crick Base Pairing
A goes with T (or U)- 2 Hydrogen bonds G goes with C- 3 Hydrogen bonds
Compare/Contrast Eukaryotic and Prokaryotic genomes
Pro circular, Euk linear Pro is tightly coiled, Euk is compacted via histones
What is the semi-conservative model of DNA replication and why is it important for inheritance?
As DNA is replicated a daughter strand is paired with a parental strand (always one old and one new)- this is the way DNA replication ensures duplication of genetic info is passed on
Describe how properties of A.A. affect protein structure
Properities of A.A. (hydrophobicity, polarity, h-bonding ability, etc) affect folding
Primary Structure of Proteins
Amino acids covalently linking through peptide bonds
Secondary Structure of Proteins
Alpha helices, and Beta sheets: form due to hydrogen bonding of backbone (not R groups)
Tertiary Structure of Proteins
interaction of R groups of helices and beta sheets (ion, hydrogen, dipole-dipole, disulfide bridges), can have multiple ways to fold but usually results in lowest energy state
Quaternary Structure
interaction of multiple domains/ tertiary structures
What are some post-translational modifications?
addition of: sugar carbs lipids phosphates acytl group
What do catalysts do to reactions?
lower activation energy to increase the RATE of reaction DOES NOT CHANGE DELTA G (net energy change of reactants to products does not change)
How does substrate and product concentration affect reactions?
Energetically: removing products or adding reactant will make the forward reaction more favorable (makes delta G more negative) Kinetically: increasing [substrate/reactant] will increase rate of rxn
Allosteric activators
-don’t compete with substrate binding site -increasings affinity for substrate -lowers Km (makes enzyme more active) -can increase Vmax
Allosteric inhibitors
-binds at place other than the active site -decreases affinity for substrate -increases Km (makes enzyme less active) -can lower Vmax
Competitive inhibitors
-compete for substrate-binding site in catalytic cleft -prevents substrate from binding -transient interaction :increasing amount of substrate can overcome competitive inhibitor, therefore DOES NOT AFFECT Vmax -Km is INCREASED (need more substrate to get to Vmax)
Non competitive inhibitors
typically binds at catalytic site (ADPspot)/active site, makes COVALENT bond with enzyme CANT OVER COME -Km DOES NOT CHANGE -decreases Vmax
Benefits of enzymes on runs
-lowers activation energy -provides proximity and orientation of reactants -ensures specificity -stabilizing transition complex -can couple energetically unfavorable rxns with favored ones
What is Vmax?
maximum reaction rate at an infinite substrate concentration (where all enzyme is bound to substrate)
Km
Michaelis constant- concentration of substrate at half of Vmax
- Describe the components of the eukaryotic DNA replication complex and their functions
•DNA polymerase- enzyme in replication, copies parental template strand in 3’ to 5’ direction, producing new strands 5’ to 3’ •Primase- makes a RNA primer so DNA polymerase has free 3’ hydroxyl group •SSB Proteins- prevent strands from re-associating and protect them from enzymes that cleave single stranded DNA •Topoisomerases 1- enzymes that break/nick phosphodiester bonds and rejoin them to relieve supercoiling tension •Topoisomerase 2- DNA-helix-passing reaction •Ligase- joins two polynucleotide chains together
- Describe the molecular mechanisms by which eukaryotic cells prevent their genomes from becoming shorter with each cell division, and how this process contributes to normal aging and cancer
• Telomerase adds short piece of DNA (called a telomere) that is lost when cell divides and DNA is replicated • Without this cell progeny will reach Hayflick limit and commit suicide • Aging= somatic cells express telomerase at low levels and decrease expression with time
Describe the structure of a gene, identifying the relative locations and functions of promoters, enhancers, transcriptional start site, introns and exons.
•Promoter (typically upstream of start point) control binding of RNA pol to DNA and identifies the start site and frequency of transcription •Enhancer Frequency of transcription is controlled by cis regulatory sequences (same strand), all regulatory sequences interact with trans-acting proteins that assist in binding and stability of RNA pol •Transcriptional Start Site •Intron/Exons – introns are taken out, exons are spliced together to make protein product
- Compare transcription in prokaryotes and eukaryotes.
Prokaryotic Eukaryotic Transcription occurs is CYTOPLASM Transcription occurs in NUCLEUS Polymerase (with help of sigma) is able to initiate transcription without help of other proteins Requires large set of proteins that must assemble at promoter before RNA Pol can being transcription Core and regulatory elements are adjacent Core and regulatory elements can be VERY far apart Polycistronic (more than one “gene” on mRNA) Cistronic (each mRNA contains only one “gene) Translation of mRNA begins prior to transcription termination (no post transcriptional modifications) Transcription terminates before translation begins (need 5’ cap, Poly A tail, and splicing to occur) No Histones DNA needs to be “unpacked” prior to transcription
Understand the functions, structures, and synthesis of mRNAs, tRNAs, rRNAs, and miRNAs and lincRNAs
• mRNAs- messenger RNA, precursor for protein synthesis • rRNAs- ribosomal RNA, form ribonucleoprotein complexes • miRNAs- small non-coding RNAs, used for other cell processes like regulation • lincRNAs- non-coding RNAs, known functions: gene silencing by affecting chromatin formation or by antisense
Describe relationship between DNA coding strand, DNA template strand, and mRNA
coding strand is compelmentary to template strand, mRNA is complementary to template strand but T’s turn into U’s
Transcription in prokaryotes
1.RNA polymerase holoenzyme forms (core pol plus sigma) 2.RNA pol and Sigma Factor bind to promoter, causing DNA strands to unwind and separate 3.Sigma Factor is release at approx. 10 nucleotides 4.Elongation continues until RNA pol finds termination signal a. Hairpin loop b. Binding of protein Rho c. Note- termination signals are encoded in DNA and many function by forming RNA structure that destabilizes pols grip on RNA
Transcription in Eukaryotes
- transcription factors bind promoter and help RNA pol bind 2. Other factors and RNA pol assemble at promoter 3.TFIIH opens helix, looses transcription factors 4. Elongation factors associate 5. topoisomerases relieve tension in supercoiling 6. Addition of 5’ cap 7. Addition of 3’ poly A tail 8. Introns are spliced out, exons put together 9. mRNA is bound to proteins that stabilize and transport through nuclear pores in cytosol
What are the size of rRNAs?
45S is cleaved to produce 18S, 28S, 5.8S
Given a wild type mRNA sequence, apply an understanding of the codon structure of mRNAs to predict changes in protein structure due to a specific mutation
If there is a mutation that would change the amino acid pair with that codon, it will change the folding of the resulting protein
Describe the structure and variation in the genetic code
• 64 three nucleotide codons in mRNA encode for 20 amino acids, plus translation start and stop signals; most amino acids have multiple codons; codons are read 5′ to 3′ with the 5′ terminal nucleotide at the left
Describe the structure and function of tRNAs in the process of translation
• tRNAs have clover leaf type structure with two critical spots 1. Anticodon- 3 consecutive nucleotides that pairs with the complementary codon on the mRNA 2. Short single stranded region on 3’ end which recognizes and binds to specific amino acids that correspond to the codon • tRNAs are responsible for pairing the correct amino acid with the correct codon of the mRNA sequence
Understand wobble base pairing (will be on USMLE!)
• Tolerate mismatch at third position- tRNAs are constructed so that they require accurate base pairing only at the first two positions of the codon
- Describe how mRNAs are translated to proteins, including the molecular mechanisms of initiation, elongation, and termination
• Initiation- eIFs put initiator tRNA into small ribo subunit, small subunit binds mRNA at 5’ end, goes until finds AUG, eIFs dissociate and allow large ribo subunit to bind • Elongation- New tRNA is put in A site, growing chain in P site, “empty” tRNA is E site, high energy bond broken between tRNA and AA, and energy put towards making new peptide bond (elongation factors drive rxn forward) • Termination- Signaled by stop codons (UAA, UAG, UGA), tRNA doesn’t recognize those and signals ribosome to stop translating
- Compare eukaryotic and prokaryotic translation, and describe the targets of antibiotics that target translation.
• Similar process • prokaryotic translation can occur during transcription (eukaryotic can’t) • Ribosome sizes are different • Prokaryotic ribosomes don’t need a 5’ cap to begin translation
What does 2,3-BPG do? and what happens to the oxygen saturation curve?
2,3 BPG decreases hemoglobins affinity for oxygen by altering its structure, shifting curve to right
Glycosylation of hemoglobin
noenzymatic, irreversible glycosylation related to amount of hemoglobin related to amount of glucose in blood (can cause aggregation)
Sickle cell anemia vs disease vs. carrier
Sickle cell allele has valine at position 6 in beta chain Anemia- HbSS Carrier- HbSA Disease- HbS[not S or A] *more aggregation occurs in deoxygenated state
Why would a patient with sickle cell anemia present with jaundice?
-sickled hemoglobin results in a lot of hemolysis -liver needs to work harder produces excess bilirubin -liver is not able to excrete bilirubin
How does pH affect the protonated state of an “R” group and an A.A.?
if pH < pKa is acidic form (protonated)
if pH > pka is basic form (deprotonated)
What are the role of chaperones and how do they affect cancer?
Chaperones help to fold proteins correctly.
New cancer drugs target chaperones
Cancer cells have many misfolded proteins and have increased levels of chaperones
By inhibiting chaperones, the cell well have to many misfolded proteins and need to commit suicide
What are amyloid fibers?
increase in beta sheets leads to self-association and the formation of amyloid fibers
Histone Regulation
Tails of histones are modified by acetylation (by adding acetyl group the overall positive charge on lysing is removing making it more difficult to neutralize the negative charge on DNA.
HATS- histone acetyltransferases- makes euchromatin -open to transcription
HDAC-histone deacetyltransferases-makes heterochromatin- closed to transcription
*HDACs are upregualted in cancer cells to silence tumor suppressor gene, HDAC inhibitor are used as anticancer drugs
Describe relationship between vitamins, cofactors, apoenzymes, and holoenzymes
Vitamins- act as cofactors for enzymes
Cofactors- binds to active site of enzyme, can be bound for lifetime of the enzymes or can bin transiently like other substrate or product
Apoenzymes- enyme without its cofactor
Holoenzymes- enzyme + cofactor (able to convert substrate to product)
Describe chemical basis of
Thymine dimers
Deamination
Depurinaton
and what the cell does to repair these damages
Thymine dimers- heat excited pryimidines and adjacent bases form covalent dimers (nucleotide excision repair)
Deamination- removal of NH3 (coverts cytosine to uracil) (base excision repair)
Depurination- looses guanine or adenine (base excision repair)
What causes double stranded DNA breaks?
ionizing radiation, oxidizing agents, replication errors, certain metabolic cellular products
What are homologous and non-homologus end joining?
Non-homologus end joining- (most common), creates overhangs, fills in, ligates together (loss of sequence)- occurs in G1 phase, utilizing Ku protein for end recognition of double stranded breaks
Homolgous end joining- untilizes undamaged homolgous chromosome and general recombination mechanisms - no loss of sequence, more work for cell, RAD 51 protein mediates
What is strand directed mistmatch repair and how does it work?
- Recognizes mis matched base
- MutS binds specifically to a mismatched base
- Mut L looks for nick (newly made strand) upstream and triggers degradation of the nicked strand all the way back through the mis match
- DNA pol goes back to repair
Base Excision Repair
single base repair, recognizes wrong base
- glycosylase takes out base
- Ap endonuclease and phosphodiesterase removes sugar and phosphate
- DNA pol adds new nucleotides, ligase seals nick
Nucleotide Excision Repair
- recognizes T dimers
- excision nuclease takes out problem area
- DNA pol adds new nucleotides, ligase seals nick
What is Benzo[a]prene ? and how does it work?
Pro-carcinogen produced by cigarette smoke
Needs oxidation to be carcinogenic
Binds covalently with guanine residues in DNA (bulky adducts, interrupts G-C base pairing and distorts double helix)
Results in G to A transisition mutation (purine to purine)
*p53 tumor suppressor gene is mutant in most cancers (espcially lung cancers)
How does glucose enter the cell? What is there relative affinity?
Glucose enters the cell by facilitated diffusion through GLUT transporters
GLUT 1 & 3: most cells/ hight affinity (Kd= 1 mM)
GLUT 2: liver and pancrease/ low affinity (Kd= 15-20)
GLUT 4: insulin induced/ medium affinity (Kd= 5)
SGLT: intestinal/ kidney (secondary active transport with Na+ gradient)
Note: if blood [glucose] is > Kd constant, cells will be utilizing those transporters
Michaelis- Menten Plots
Lineweaver-Burk Plots
For M.M.:
If line shifts to the left- it is an activator
If line shifts to the right- it is an inhibitor
If line goes from sigmoidal to hyperbolic it is allosteric activator/inhibitor
For L.B.:
(y-intercept is inverse Vmax, x-intercept is inverse Km)
Inhibitor (slope increases)
Competitive inhibition: Y intercept stays the same
Non competitive inhibition: X intercept stays thesame
Closer line is to 0 the bigger it gets
Hexokinase vs. glucokinase
First regulatory step in glycolysis
Hexokinase: found in most cells, low Km
Glucokinase: found in liver, high Km
Regulated by products/reactants- makes glucose 6-phophate which also inhibits it
What are the inhibitor and activators of phosphofructokinase-1 (PFK1)
Activators: AMP, and Fructose 2-6, bisphophate
Inhibitors: ATP, and citrate
Note** fructose 2,6 bisphophate is made from fructose 6-phosphate when there is access (PFK1’s reactant)
What are the three regulatory steps in glycolysis?
- hexokinase/glucokinase
- phosphofructorkinase
- Pyruvate kinase
Tryptophan operon
No trp= operon on -> makes trp
lots of trp -> binds to repressor -> makes repressor active and bind to DNA -> no trp
Lac Operon
Lactose present:
Lactose present inceases allactose, allactose binds to repressor and removes from operator
Glucose present:
Glucose decreases [cAMP], cAMP no longer binds CAP(gene activator), CAP dissociates from DNA turning off operon
*In order for operon to be ON, needs to be lactose and no glucose
What is epigenetics?
heritable, reversible changes in genome (NO CHANGE IN SEQUENCE)
X inactivation in females
two XX chromosomes
one is randomly silenced, my single RNA (XIST)
all progeny will have the same X chromosome silenced
(direct inheritance of the pattern of chromosome condensation)
Imprinting
differential expression of a gene allele depending on parental origin
imprinting is created in parental germ cells-
if paternally imprinted- both alleles in male germ line are methylated
TPP
Reactions: decarboxylase
Precursor: Thaimine
From foods: meat, legumes
Deficiency: Beri Beri (headache malaise, peripheral neurpathy, heart failure)
Lipoate
Reaction: acetyl transferase
No vitamin precusor
Wide spread in diet, no know deficiencies
Coenzyme A
Reactions: acetyl group acceptor (acyltransferase)
Wide spread in diet
no known deficiencies
FAD
Rxn :Electron acceptor (oxidizes lipoate)
Precursor: Riboflavin
Found in: mushrooms, organ meats, legumes
Deficiency: cheilosis, glossitits, keratitis
NAD
Rxn: electron acceptor (oxidized FADH2)
Precursor: Niacin
Found in: fortified grains, meats
Deficiency: Pellagra 3D’s
dermatits, diarrhea, dementia
Biotin
Rxns: carboxylase Rxns
wide spread in diet
can be free or protein bound
regeneratable
Deficiency- raw eggs, Aviden binds to biotin
Ascorbic Acid
Reactions: Hydroxylase
Vitamin C, no precursor/ not modified by body
Deficiency- Scury
slow wound healing, irritability, apathy, anemia
PLP
Rxns: transaminases
Precursor: B6 vitamins
Found in: fortified cerals, meat, bananas, rice
Deficiency: peripheral neuropathy, siezures/ anemia in infants
Test: If siezures resolve with pyridoxal but no pryidoxine then it is a pyridoxine oxidase deficiency
If either one works then it was an antiquitin deficiency
Hydrophobic side chains (intertior protein structure
Alanine
Proline
Valine
Leucine
Isolucine
Tyrptophaon
Phenyalanine
Tyrosine (hydrophobic stacking with rings)
Uncharged and can form H bonds
Asparagine
glutamine
serine
threonine
tryosine
cysteine
Oxidation results in disculfide bond
cysteine
Charge-charge (depends on pH)
Aspartate
Glutamate
Arginine
Lysine
Histidine
