Lectures 5-8 Flashcards
RNA = only macromolecule that acts as both information/genetic ___________ and ____________
- transmitter
- catalyst
what is a transcriptome?
sum of all RNA molecules produced in cell under given conditions
Purpose of microRNA?
bind to specific nucleotide sequences to regulate expression of genes
RNA polymerase requires (3)
- DNA template
- all 4 ribonucleosides triphosphates
- Mg2+
movement of transcription bubble requires ? of nucleic acid molecules
strand rotation
5 RNA polymerase core subunits in bacteria + extra subnit! + functions
- 2 x alpha –> assembly and building of upstream promoter elements
- beta –> main catalytic subunit
- beta prime –> responsible for DNA binding
- omega –> appears to protect polymerase from denaturation
- sigma –> directs enzyme to promoter
Typical Pol II promoter (eukaryotes) have 2 sequences in common
- initiator sequence at +1
- TATA box consensus sequence at -30
which RNA pol (1, 2 or 3) synthesize mRNA, tRNA and rRNA (+ specialized RNA)
- Pol 1 –> rRNA
- Pol 2 –> mRNA + specialized RNA
- Pol 3 –> tRNA + 5sRNA + specialized RNA
major difference between bacteria and eukaryote RNA Polymerase –> what is it?
eykaryote Pol has carboxyl terminal domain!
- CTD = many repeats of consensus heptad aa sequence
functions of
- Pol II
- TBP
- TFIIA
- TFIIB
- TFIID6
- TFIIE
- TFIIF
- TFIIH
- Pol II: catalyze RNA synthesis
- TBP: recognizes TATA box
- TFIIA: stabilize binding of TBP and TFIIB to promoter
- TFIIB: Binds to TBP + recruits Pol II-TFIIF complex
- TFIID6: required for initiator at promoters lacking TATA box
- TFIIE: recruits TFIIH + ATPase and helicase activity
- TFIIF: binds to Pol II + binds to TFIIB to prevent binding of Pol II to nonspecific DNA sequence
- TFIIH: unwinds DNA at promoter + phosphorylates Pol II (within CTD)
5’ cap = residue of ________ linked to __ terminal residue of mRNA through unusual __,__-triphosphate linkage
- occurs at __-__ bp
- 7-methyguanine
- 5’
- 5’-5’ linkage
- 20-30 bp
3 functions of 5’ cap
- protect mRNA from degradation by ribonucleases
- binds to specific cap-binding complexes of proteins
- participates in binding of mRNA to ribosome to initiate translation
poly(A) tail = string of ____________ residues added to __-end of most eukaryotic mRNAs
- adenosine residues
- 3’end
2 functions of poly(A) tail
- serves as binding site for specific proteins
- may help protect mRNA from enzymatic destruction/degradation
How is poly(A) tail added to RNA?
- both proteins are tethered to ?
1) endonuclease cleaves RNA at a point 10-30 nt 3’ downstream of sequence (5’)AAUAAA(3’)
2) synthesis of poly(A) tail by polyadenylate polymerase (80-250 nt of A) beginning at cleavage site
- Carboxyl terminal domain! poly(A) tail is also tethered to the CTD!
Pol II synthesizes RNA before or after site of cleavage/(5’)AAUAAA(3’) ?
after!
4 classes of introns? require ATP?
- group 1 and 2 –> self-splicing = no ATP and no enzymes
- spliceosome introns –> removed by large ribonucleoproein called spliceosome
- group 4 –> protein catalyzed introns are removed by enzymes
*spliceosome and group 4 require ATP!
RNA splicing for group 1 introns (usually in _________ genes): what part of what molecules attacks what?
- 3’OH of free guanosine group attacks phosphodiester bond between U and A or exon-intro junction
- 3’OH forms bond with 5’ of intron
- 3’OH of exon acts as nucleophile on 3’ end of intron to rejoin pieces
what are snRNP?
- found where?
- act as?
small nuclear ribonucleoproteins
- make up spliceosome
- act as catalysts
what marks start/end of splicing? for spliceosome splicing
GU at 5’ end of intro + AG at 3’ end
Membranes define _________ of cells/organelles + control __________
- boundaries
- traffic
membranes are permeable to (2) + impermeable to (2)
- permeable to small polar solutes + non polar compounds
- impermeable to large polar solutes and ions
what is almost all the mass of biological membranes?
polar lipids and proteins
most membrane lipids are synthesized in ? –> what carries them to destination?
- endoplasmic reticulum
- vesicles
what is the fluid mosaic model (2)
- proteins and lipids move laterally in plane of membrane = fluid
- contains different components like proteins, lipids and carbs = mosaic
what are the 3 components of the fluid mosaic model + functions
- membrane also enriched with (2) but no ?
- phospholipids: form bilayer
- proteins –> embedded/float in bilayer –> hydrophobic domains interact with fatty acyl chains
- carbs attached to proteins and lipids on extracellular surface of PM
- enriched with cholesterol and sphingolipids but no cardiolipin
is there the same distribution of lipids in inner and outer leaflet?
no! asymmetry
which phospholipid normally in inner layer? + which one is critical but in minor amount?
- inner layer: phosphatidylserine
- phosphatidylinositol = critical but minor amount
which organelle’s membrane contains low cholesterol and sphingolipid but most of cell’s cardiolipin and phosphatidylglycerol
mitochondria membrane
3 types of membrane proteins + functions + how to remove?
- integral: traverse membrane (monotopic or polytopic) –> detergent to remove!
- peripheral: associate with membrane through electrostatic interactions and H bonds with hydrophilic domains –> change in pH, chelating agents, urea or carbonate to break bond
- amphitropic: associate reversibly with membrane + interact electrostatically or lipid anchored
phospholipids can move depending on (2) –> general shape and dimension maintained but (1) allowed to change
- temperature and lipid composition
- degree of motion
Bilayer:
- below normal physiological temp = what state? + description
- above normal temperature = what state? + description
- liquid ordered/gel like –> motion is constrained, bilayer is paracrystalline and firm
- liquid disordered/liquid like –> constant thermal motion of side chains from heat
4 factors that affect flexibility of bilayer?
- temperature
- saturation of FA chain (increase packing = increase order/gel)
- length of FA chain (increase length = increase order)
- presence of sterols: interact with phospholipid’s unsat FA chain = compact VS interact with sphingolipids and phospholipids with long sat FA = fluid
can cells regulate FA content of their membranes?
yes! ex: increase sat FA at high temp
2 characteristics of lateral diffusion
- uncatalyzed and very rapid!
transbilayer diffusion/flip flop –> catalyzed? speed?
if no catalysis, very very slow
flippase vs floppase vs scramblase
- Need ATP?
- flippase: outer to inner, requires ATP
- floppase: inner to outer, requires ATP
- scramblase: moves lipid down concentration gradient depending on type of lipid –> towards equilibrium + no ATP but activated by Ca2+
can proteins move in the bilayer?
some membrane proteins are anchored to internal structures (ie spectrin) that prevent free lateral diffusion –> form fences
- usually freely mobile but motion can be restricted
what stabilizes and modulates structure of lipid rafts?
ceramide!
Lipid rafts contain (3)
hint:
- clusters of ? enriched with ?
- specific ? in inner leaflet + ? in outer leaflet
- caves
- clusters of glycospingolipids (long chain sat FA) thicker than other regions in membrane + are enriched with lipid anchored proteins and cholesterol
- specific doubly or triply anchored acylated proteins in inner leaflet + GPI anchored proteins in outer leaflet
- caveolae (little caves) –> allow membrane to form vesicle that pinches off
are lipid rafts more ordered than usual membrane?
yes because are more thick
membrane fusion requires 6 things/steps
- triggering signal
- recognition of each other
- close apposition
- local disruption of bilayer structure (increase in curvature of lipid rafts)
- hemi-fusion (of outer leaflet)
- fusion proteins to form 1 continuous bilayer
6 steps of membrane fusion
- neurotransmitter filled vesicle approaches plasma membrane
- vSNARE (assembles on vesicle membrane) and tSNARE (assembled on target membrane) bind to each other, zipping up from amino termini and drawing the 2 membranes together
*Q-SNARE/SNAP25 are regulatory proteins that are Ca2+ induced - zipping causes curvature and lateral tension between bilayers, favoring hemifusion between outer layer
- hemifusion: outer leaflets of both membranes come into contact
- complete fusion crease a fusion pore
- pore widens; vesicle contents are released outside cell
*then, NSF proteins disassemble SNARE complex
passive vs active transporter
passive = high concentration to low
- active = low to high/against concentration gradient
4 types of passive transporters:
- simple diffusion
- facilitated diffusion
- ion channel
- ionophore-mediated ion transport
- simple diffusion: small nonpolar compounds
- facilitated diffusion: need protein for polar molecules
- ion channel: may be gated by ion or ligand
- ionophore-mediated ion transport: small molecules mask charge of ions and allow them to diffuse through bilayer
active tranporters:
- primary vs secondary
- primary: energy provided directly by chemical reaction –> driven by ATP
- secondary: couple uphill transport of 1 substrate with downhill transport of another
types of transport system:
- uniport
- cotransport
- symport
- allport
- uniport: molecules move singly one at a time in one direction
- cotransport: S1 cotransports S2 when it moves across –> symport or allport
- symport: move than 2 molecules move together in same direction
- allport: more than 1 molecule move in opposite directions
which type of transporter are glucose transporters in erythrocyte?
uniport
3 steps for glucose transport in erythrocyte
- T1: glucose binds to transporter
- conformational change to T2 –> Glucose is released inside the cell
- another conformational change to go back to T1
GLUT 1, 2 and 4 –> where and function?
GLUT 1: erythrocytes + most tissues at low level –> basal glucose uptake
GLUT 2: liver, pancreatic islets, intestine, kidney –> role in liver and kidney
GLUT 4: muscle, fat, heart –> basal glucose uptake + high turnover number
Insulin dependant glucose transport (5 steps ish)
- glucose transporters are stored in vesicles in cells
- when insulin interacts with receptors, vesicles move to surface and fuse with PM (PKB) = increase number of transporter in PM
- when decrease insulin, GLUT are removed from PM by endocytosis –> forming small vesicles
- smaller vesicles fuse with larger endosome
- patches of endosome enriched with GLUT bud off to form small vesicles
what type of transport is electroneural transport of anions?
- facilitated passive transport
- cotransport –> antiport!
CO2 = ___________ in blood –> converted to ___________ to affect ____ and allow ________________ of hemoglobin to bind and release ____
- not soluble in blood
- bicarbonate (HCO3-)
- pH
- conformational change
- O2
in erythrocytes of respiring tissues –> what enters and what leaves?
VS in lungs?
- CO2 enters
- HCO3- leaves as Cl- comes in = chloride bicarbonate exchange in protein
Lungs: - bicarbonate enters as Cl- leaves
- CO2 leaves
Describe 2 steps of active transport (examples with symport/antiport)
- S1 moves out using ATP (1st active transport)
- then, S1 flows back in = provide energy to drive cotransport of S2 to go against gradient
ex.: symport: (Na+ & glucose) and (Na+ & aa)
allport: (Na+ & K+ & ATPase) and (Na+ & H+ in kidneys)
active transport through P-type ATPase (4 steps)
- transporter binds 3 Na+ from inside the cell
- phosphorylation/use of ATP makes conformational change to P-Enz-II
- transporter releases 3 Na+ outside and binds 2 K+ from outside
- dephosphorylation leads to Enz I shape –> transporter releases 2 K+ inside
Proton transport through F-type ATPase
- F-type role?
- high proton gradient supplies what?
- F-type ATPase uses energy of ATP to pump protons across membrane
- supplies energy to drive reverse reaction –> ATP synthesis by ATP synthase
what type of ATP pump does mitochondria use?
F-Type –> energy coupling factors to synthesize ATP
what does ABC transporter stand for?
ATP binding cassette
are ATP transporter active or passive transporters?
active! use ATP to go against concentration gradient
describe process of ABC transporters
- substrate binds to transporter + ATP is bound to NGB/NBD sites
- conformational change occurs when substrate bind + when ATP hydrolyzed to ADP
- substrate diffuses away from transporter
most enzyme catalyzed reactions are regulated in order to ?
regulate metabolic pathways (that are all interconnected)
cells need a _______ __________ of energy –> __________
constant supply of energy
- homeostasis
what depends on body’s needs/metabolic pathways (2)?
- precursor levels
- energy requirements
cells/organisms maintain a _________ _________ ______ –> what does it mean? ex.:
- dynamic steady state
- intermediates are formed and consumed at equal rates
- ex.: rate of glucose entry in cells is regulated
when a transient perturbation alters rate of formation/consumption of a metabolite –> what happens?
compensating changes in enzyme activities return system to steady state
cells regulate their metabolism either by (2)
- changing number of a specific enzyme molecule
- changing activity of existing molecules
6 principles to regulate changing number of a specific molecules (in regulation of enzymes)
- extracellular signal
- transcriptional regulation
- mRNA stability
- mRNA translation
- Protein stability
- enzyme localization
describe:
1. extracellular signal
2. transcriptional regulation (3)
3. mRNA stability
- extracellular signal: conformational change –> leads to signaling pathway
- transcriptional regulation:
- activation of transcription factors in signaling pathway
- TF phosphorylation/dephosphorylation
- TF interactions with ligand - mRNA stability: resistance to degradation –> rate of synthesis vs degradation
describe:
4. mRNA translation
5. Protein stability
6. enzyme localization
- mRNA translation: rate of translation into proteins
- Protein stability: balance between rate of synthesis and degradation
- enzyme localization: segregation or sequestration of enzyme = limiting factor
4 principles of regulation of enzymes to change the activity of existing molecules
- changes of levels of substrate
- enzyme binding allosteric effectors/ligands
- covalent modification (phosphorylation/dephosphorylation)
- interaction with regulatory protein that can regulate enzyme’s activity
pathways in opposite directions are favored/not favored simultaneously?
not favored
common regulatory mechanisms at organism level (5)
- pathways in opposite directions are not favored simultaneously
- maximizes product utilization
- ability to partition metabolites between alternative pathways
- draws on fuel/product best suited for the need
- slows down synthetic pathways when products accumulate = homeostasis
various _______ activate or inactivate _________ ________ that act in ________ to regulate ______ _____________
- signals
- transcription factors
- nucleus
- gene expression
changes in transcriptome lead to changes in (2)
proteome and metabolome of cell
in some tRNA, anticodons include a ____________ at __ position –> forms _______ bond with (3 nt) –> creates?
- inosinate (I)
- 1st position
- weak H-bonds
- A, U, C
which 2 nt at first position so that the binding is specific/only 1 codon recognized? vs which other. 2nt so binding is less specific/2 different codons may be read
- specific = C and A
- non specific = U and G
what sequence (3 nt) at 3’ end of amino acid arm?
CCA
what does T(Y)C arm do?
interacts with large subunit rRNA
5 examples of post-translational modification for proteins
- acetylation of N-terminal residues
- enzymatic removal of formyl group from 1st residue OR removal of Met/other residues
- removal of signal sequences/other regions
- forming disulfite links
- attaching carbs
E1 vs E2 vs E3 for attachment of ubiquitin to target proteins
- E1 = activating enzyme
E2 = conjugating enzyme
E3 = ligating enzyme
what esterifies aa to corresponding tRNA?
aminoacyl tRNA synthetase
what are the 2 types of met?
Met and fMet
A vs P vs E site
Aminoacyl site: where aa added to tRNA
Peptide site: aa added to polypeptide chain
Exit site: tRNA released from ribosome
release factors replace (2) by (1) resulting in tRNA dissociation
- Ribosome recycle factor (RRF) and EF-G
- with IF3
what serves to define template vs non-template strand?
promoter
where is TATA box? bacteria vs eukaryotes?
bacteria: -10
eukaryotes: -30
how many subunits does RNA Pol II has in humans?
12!
synthesis of 5’ cap carried out by (#) enzymes tethered to the _____ of Pol II –> cap remain tethered through association with ___ ___________ _____________
- 4 enzymes
- CTD
- Cap binding complex (CBC)
exception: _________ genes don’t have introns
histone!
U1 and U2 bind to which site? for spliceosome splicing?
U1 –> donor site (5’ splice site)
U2 –> branching site
ATP required for spliceosome splicing?
only for spliceosome assembly
