Exam 2 Cell Biology

Front 1

Chloroplast

Back 1

Inner membrane: transporters for phosphate and sucrose precursors.

Stroma: enzymes that catalyze CO2 fixation and starch synthesis

Thylakoid membrane: absorption of light by chlorophyll, synthesis of ATP4-, NADPH, and electron transport

Intermembrane space

Outer membrane: permeable to small molecules

Front 2

Photosynthesis Stage 1

Back 2

Light absorption generation of high-energy electron, O2 formation.

Light is absorbed by chlorophylls attached to proteins in the thylakoid membrane. Chlorophylls consist of a porphyrin ring attached to a long hydrocarbon side chain. Mg2+ ion (instead of Fe2+) and transfer electron to quinone (Q).

H2O acts as an electron donor in PSII.

H2O ----> O2 + 4 H+ + 4e-

Front 3

Photosynthesis Stage 2

Back 3

Electron transport formation of proton-motive force.

e- move from Q through electron carriers until they reach NADP+. NADP+ is reduced to NADP in PSI.

2 H2O + 2 NADP+ ---> 2 H+ + 2 NADPH + O2

Front 4

Photosynthesis Stage 3

Back 4

Synthesis of ATP.

Protons move down their concentration gradient from the thylakoid lumen to the stroma through the F0F1 complex (ATP synthase), which couples proton movement to the synthesis of ATP from ADP and Pi.

Front 5

Photosynthesis Stage 4

Back 5

Carbon Fixation

NADPH and ATP generated by the second and third stages provide the energy and electrons to drive the synthesis of six-carbon sugars from CO2 and H2O.

6 CO2 + 18 ATP + 12 NADPH + 12 H2O ---> C6H12O6 + 18 ADP + 18 Pi + 12 NADP + 6 H+.

Only stage that can occur in the dark.

Front 6

Prokaryotes ATP Synthase

Back 6

Hi [H+] in extracellular

ATP synthase in Plasma membrane

ADP + Pi ---> ATP in Cytoplasm

Front 7

Mitochondria ATP Synthase

Back 7

Hi [H+] in intermembrane space

ATP synthase in inner membrane

ADP + Pi ---> ATP in Matrix

Front 8

Choroplast

Back 8

Hi [H+] in Thylakoid Lumen

ATP synthase in Thylakoid membrane

ADP + Pi ---> ATP in Stroma

Front 9

In photosynthetic electron transport, H2O is the overall electron donor and NADP+ is the overall electron acceptor.

Back 9

Super excited electrons in photosystem 1 reaction center have so much potential energy they move down electron transport to make NADPH.

Front 10

Light absorption causes transient elevations in potential energy of reaction center electrons. This drives electron transport from H2O to NADP+.

Back 10

Electron deficient (+charge) reaction center in PS2 is such a strong electron acceptor that it draws electrons from the splitting of water.

Front 11

Absorption of Light Energy

Back 11

Light radiates as photons moving in waves.

Photons have specific amount of energy.

Photons with shorter wavelengths have more energy that photons moving with longer wavelengths.

Pigment molecules absorb the energy in photons at specific wavelengths.

There are several different pigment molecules within each photosystem to absorb different wavelengths of energy.

Front 12

When light is absorbed, electrons become excited

Back 12

When a photon strikes a pigment, its energy is transferred to an electron in the pigment molecule

Chlorophylls a and b have a ring structure in "head" to absorb light and the tail anchors chlorophyll in thylakoid membrane.

An excited electron will rise to a higher energy shell with greater potential energy. It will release this potential energy when it moves back to it's original state as heat or fluorescence at a longer wavelength. This obeys the 1st law of thermodynamics.

Front 13

Resonance Energy transfer focuses energy at the reaction center

Back 13

Resonating pigment molecules transfer energy to adjacent pigments closer to the reaction center. Pigments closer to the reaction center also absorb longer wavelength light energy.

Focused energy from multiple pigment molecules is absorbed by electrons in the reaction center chlorophyll (Abs max is 680 or 710 nm), which transfer these excited electrons to an electron acceptor in the electron transport chain.

Front 14

Sequence of linear electron transport

First step

Back 14

Light harvesting complex funnels energy to reaction center chlorophyll in PS1.

Excited electrons leave reaction center and travel through REDOX couples through ferridoxin and NADP reductase to make NADPH

Reaction center chlorophyll is left with a positive charge.

Front 15

Sequence of linear electron transport

Second step

Back 15

Light harvesting complex funnels energy to electrons in Photosystem II reaction center chlorophyll

Excited electrons leave chlorophyll and are picked up by plastiquinone

Plastiquinone picks up protons from stroma and moves within the bilayer to cytochrome b6/f complex.

Electrons transfered to cytochrome b6/f complex and protons go into the lumen

Electrons picked up by plastocyanin shuttle (peripheral in lumen) which travels to photosystem I, donating electrons to (+) charged chlorophyll

Front 16

Sequence of linear electron transport

Third step

Back 16

Within photosystem 2 reaction center (+) charged chlorophyll has redox value of +1200 mV

Water/oxygen redox couple is +816 mV, so water splits to generate electrons that fill electron deficinecy in chlorophyll

Protons and oxygen generated in thylakoid lumen.

Front 17

Sequence of linear electron transport

Fourth step

Back 17

The proton gradient generated in the thylakoid lumen is used to drive ATP synthesis in the stroma.

ATP and NADPH are then consumed to make carbohydrate

Front 18

Calvin-Benson Cycle makes Carbohydrate

Back 18

Carboxylation: 3 CO2 + 3 Ribulose 1,5 Bisphosphate ---> 6 phosphoglycerate

Reduction: Energy in ATP and NADPH converts 6 phosphoglycerates to 6 glyceraldehyde 3-phosphates. One G3P is exported to cytoplasm to generate sucrose.

Rgeneration: Energy in ATP converts five G3P's into three ribulose 1,5 bisphosphates.

Front 19

Carbohydrate Synthesis

Back 19

Two glyceraldehyde 3-phosphates from chloroplast are combined in the cytoplasm to make 6 carbon sugars (fructose and glucose)

Fructose and Glucose are linked to make Sucrose in cytoplasm.

Within the stroma, two glyceraldehyde 3-phosphates can also be converted to glucoses which are then linked together to make starch.

Front 20

Signal transduction and G protein coupled receptors

Back 20

External cues perceived at plasma membrane cause changes in cellular metabolism.

Front 21

G-Protein linked pathways

Back 21

Protein Kinase A Pathway (activation of adenylyl cyclase

Protein Kinase C patway (activation of phospholipase C)

Front 22

Signal Transduction via Cyclic

Back 22

External signals activate the trimeric G protein. This causes cyclic AMP production which activates protein kinase A. PKA phosphorylates targe proteins, changing the cell.

Front 23

Trimeric G-proteins are regulated by their GTP binding state Step 1)

Back 23

1) Binding of hormone induces a conformational change in receptor.

Hormone binds receptor at extracellular domain. This changes receptor conformation at the cytoplasmic domain containing the trimeric G-protein binding site

Front 24

Trimeric G-proteins are regulated by their GTP binding state Step 2)

Back 24

2) Activated receptor binds to G-alpha subunit

Receptor now binds inactive trimeric G protein, and acts as a nucleotide exchange factor.

Front 25

Trimeric G-proteins are regulated by their GTP binding state Step 3)

Back 25

3) Activated receptor causes conformational change in G-alpha triggering dissociation of GDP

Front 26

Trimeric G-proteins are regulated by their GTP binding state Step 4)

Back 26

4) Binding of GTP to G-alpha triggers dissociation of G-alpha both from the receptor and from G-beta and G-gamma.

Trimeric G protein exchanges GDP for GTP on the alpha subunit with GTP separates from the beta/gamma subunits.

Front 27

Trimeric G-proteins are regulated by their GTP binding state Step 5)

Back 27

5) Hormone dissociates from receptor; G-alpha binds to effector, activating it.

Active alpha subunit of G complex moves laterally in plasma membranse to bind to and activate effector.

Front 28

Trimeric G-proteins are regulated by their GTP binding state Step 6)

Back 28

6) Hydrolysis of GTP to GDP causes G-alpha to dissociate from effector and ressociate with G-beta and G-gamma

The extracellular hormone dissociates from the receptor, freeing the membrane bound beta-gamma subunit, and resetting the receptor conformation. For this pathway the effector is adenylyl cyclase, which catalyzes ATP --> cyclic AMP and PPi.

Front 29

G protein are molecular switches regulated by their G state.

Back 29

Activated by nucleotide exchange: regulated by a nucleotide exchange factor; becomes G-protein-GTP

Inactivated by GTP Hydrolysis: regulated by a GTPase accelerating factor; becomes G-protein-GDP

Front 30

Activation of Protein Kinase A

Back 30

Cyclic AMP diffuses within the cytoplasm to bind regulatory subunits of protein kinase A and release the catalytic subunits of protein kinase A.

Front 31

Target Protein Phosphorylation

Back 31

The catalytic subunits of Protein Kinase A then move in cytoplasm or nucleus to phosphorylate many different target proteins.

Front 32

Protein Kinase A catalytic subunit catalyzes

Back 32

Target protein + ATP --> Target Protein-P +ADP

Ex: Phoaphorylated CREB factors elevate transcription of genes controlling gluconeogenesis.

Front 33

Inactivating the PKA Pathway

Back 33

GTP quickly hydrolyzes to GDP on the alpha subunit of G protein. The GTPase promoting factor is part of the alpha subunit.

Alpha sub-unit-GDP dissociates from the adenylyl cyclase and reassociates with the beta-gamma subunit.

Dissociation of the alpha subunit inactivates adenylyl cyclase.

Phosphodiesterase degrades cAMP

Lower cAMP concentration causes release of cAMP from PKA regulatory subunits

Regulatory subunits reattach to catalytic subunits to inhibit PKA activity.

Front 34

Protein Kinase C pathway

Back 34

GTP-alpha subunit of trimeric G protein activates phospholipase C instead of adenylyl cyclase.

Front 35

Phospholipase C activity

Back 35

Splits a phospholipid in the plasma membrane into two secondary messangers

Phosphoinositide bisphosphate --> 1,2 diacylglyccerol + inositol 1,4,5 triphosphate

Front 36

Protein Kinase C activation

Back 36

Inositol triphosphate (IP3) diffuses into cytoplasam to ER membrane where it binds to and activates calcium channels.

Calcium flows from ER lumen to cytoplasm and from extracellular o cytoplasm, binding to protein kinase C.

PKC-calcium migrates to plasma membrane.

Diacylglycerol (DAG) in plasma membrane binds to and activates PKC-Calcium

Activate PKC-Calcium at the plasma membrane phosphorylates many target proteins.

Ex: phosphorylates and inhibits glycogen synthase in liver cells.

Front 37

Inactivating Phospholipase C

Back 37

Rapid GTP hydrolysis on alpha subunit makes it detach from phospholipase C

Phospholipase C now inactive

Resetting calcium concentrations in cytoplasm and ER

Inositol triphosphate rapidly hydrolyzed to Inositol biphosphate, which closes the calcium channel on ER.

Excess Calcium in cytoplasm pumped back into the ER by calcium ATPase and out of the cell by plasma membrane calcium ATPase.

Front 38

Nucleic acids, base pair rule, and polymers of DNA and RNA

Back 38

Nucleotides have common structure of phosphate-sugar-base (DNA, RNA, ATP, GTP)

2(A-T/U) or 3 (C-G) Hydrogen bonds connect base pairs between strands

Purine (2 rings A & G) & Pyrimidine (1 ring C & T) pairing causes uniform spacing between strands

In DNA, strands run in opposite directions 5' - 3'

Front 39

Transcription: Initiation

Back 39

RNA polymerase binds DNA, unwinds double helix by breaking base pairs (hydrogen bonds), first rNTP's base pair to DNA template, polymerase catalyzes first phosphodiester bond formation.

Front 40

Transcription: Elongation

Back 40

RNA polymerase advances along DNA, continued base pairing of rNTP's to DNA and formation of phosphodiester

Front 41

Transcription: Termination

Back 41

RNA polymerase reaches stop site and releases new RNA.

Front 42

Eurkaryotic mRNA has a 5' Cap and a poly A tail

Back 42

Both added in nucleus during RNA processing

The 5' m7G-p-p-p cap is part of the recognition signal for the ribosomes in the cytoplasm.

The poly (A) tail extends the life of mRNA by protecting it from degredation by ribonucleases in the cytoplasm

Front 43

Translation from mRNA to protein within ribosomes

Back 43

messenger RNA- sequence for translation by tRNA

ribosome- machinery for translation

tRNA- translates nucleic acid language in mRNA sequence to amino acid language of proteins by delivering amino acids for peptide bond formation

Many associated protein cofactors make the process work

Front 44

Ribosomal complex is the translation machine

Back 44

small and large subunits made of ribosomal RNA and proteins

3 tRNA binding sites:

E- empty or ejection

P- peptidyl

A- acceptor or aminoacyl

Front 45

Remember triplet genetic code

Back 45

Start codon: AUG produces Methionine

Stop codon: UAA

Front 46

Transfer RNA has 3 important binding sites

Back 46

Amino acid attachment at 3' end acceptor stem mediated by aminacyl tRNA synthetase in cytoplasm

Regulatory cofactors eIF2-GTP or eEF1alpha-GTP attach near the acceptor stem (5' and 3' ends)

Messenger RNA will bind at the anticodon loop by hydrogen bond base pairing

Front 47

Translation

Back 47

preinitiation complex formation

mRNA attachment

mRNA scans along the ribosome until the start codon is in the P site

Large ribosome subunit attaches to complete initiation complex

Elongation during Translation

Termination of Translation

Front 48

Translation Cofactors Step 1)

Back 48

eIF2-GDP must undergo nucleotide exchange to eIF2-GTP to be active. Cells block this nucleotide exchange to inhibit protein synthesis. eIF2-GTP binds to tRNA-methionine.

Front 49

Translation Cofactors Step 2)

Back 49

eIF1, eIF3, eIF1A are bound to small ribosome subunit. eIF1 and eIF3 bind at E site, eIF1A binds and blocks A site to prevent premature tRNA binding. eIF5 now binds at E site and transfer RNA with methionine and eIF2-GTP binds at P site to complete preinitiation complex.

Front 50

Translation Cofactors Step 3)

Back 50

eIF4 subunits bind to 5' cap (eIF4E) and poly A tail (eIF4G) of mRNA forming a circular mRNA complex. eIF4B binds and activates eIF4A helicase activity (ATP hydrolysis), which unwinds mRNA secondary structure at 5' end.

Front 51

Translation Cofactors Step 4)

Back 51

mRNA then attached to the preintiation complex. eIF3 mediates this attachment.

Front 52

Translation Cofactors Step 5)

Back 52

eIF4A dependent ATP hydrolysis slides the mRNA along the small ribosome until the start codon on mRNA is in the P site.

Front 53

Translation Cofactors Step 6)

Back 53

Recognition of the start codon by the tRNA causes GTP hydrolysis on eIF2 (activated by the GTPase activating protein eIF5). This irreversible step prevents further scanning and keeps the start codon at the P site.

eIF4E and eIF4G/4A/4B stay conneced while ATP hydrolysis on eIF4A moves the small subunit along the mRNA. This forms an expanding loop of mRNA between eIF4E and eIF4G

Front 54

Translation Cofactors Step 7)

Back 54

eIF5B-GTP mediates the attachment of the large ribosome subunit and the displacement of eIF1, eIF2-GDP, eIF3, 3IF5, complex of eIF4A, B, G, E from ribosome.

Large ribosome subunit attaches to complete the initiation complex.

Front 55

Translation Cofactors Step 8)

Back 55

GTP hydrolysis on eIF5B completes ribosome assembly(irreversible step). eIF5B-GDP and eIF1A then dissociates.

Front 56

tRNA enters A site

Back 56

a pool of tRNA's with attached amino acid, elongation factor (eEF1a-GTP) have access to the A site

Correct base pair match (hydrogen bond formation) between mRNA codon and tRNA anticodon promotes GTP hydrolysis. (important proofreading step)

Front 57

Ribosome tightly binds tRNA

Back 57

GTP hydrolysis to GDP and Pi changes ribosome conformation. (the twist step of the twist/return sequence)

Releases eEF1a-GDP and positons 3' ends of tRNAs in P and A sites to catalyze peptide bond formation.

Front 58

Peptide bond formation

Back 58

amino acid in P site breaks from 3' end of tRNA

peptide bond forms between carboxyl on P site amino acid and amine on A site amino acid

polypeptide now on tRNA in A site

Front 59

Ribosome shifts over mRNA and tRNA

Back 59

eEF2-GTP binds next to the A site

Ribosome translocates 3 bases down mRNA

tRNA that was in P site Now in E site

tRNA that was in A site NOW in P site

No tRNA in the A site

eEF2-GTP hydrolysis to eEF2-GDP prevents the ribosome from going backwards along the mRNA. This is irreversible and is the Return step of the twist/return sequence

eEF2-GDP then released

Front 60

Cycle repeats

Back 60

New tRNA binds in A site and base pairing promotes GTP hydrolysis on eEF1a

The twist step of the twist/return cycle promotes peptide formation but also ejects the tRNA in the E site

Front 61

Termination of Translation step 1)

Back 61

Stop codon on mRNA enters the A site

eRF1 binds directly to stop codon

eRF3-GTP binds to eRF1

Front 62

Termination of Translation step 2)

Back 62

GTP hydrolysis release the new protein

GTP on eRF3 hydrolyzes to GDP and Pi within the A site. This cleaves peptide from P site-tRNA and releases the new PROTEIN.

Front 63

Termination of Translation step 3)

Back 63

Small ribosome subunit recycled for more translation

ABCE1 uses ATP hydroysis to dissociate the large and small ribosome subunits, eRF1 and eRF3-GDP

initiation factors (eIF1, eIF1a, eIF3) bind to small ribosome subunit in preparation for new translation

Front 64

Polysomes and mRNA circularization

Back 64

Multiple ribosomes scan the mRNA to increase efficiency of translation

Eventually the 3' polyA tail is degraded. This breaks the circle and the initiation cofactors no longer attach a new small ribosome subunit.