BIOL 4100 Exam 4 Petrov

Cells and organelles are what?

Cells are mobile (immune cells migrate)
Organelles form organelle networks (intricate)—networks are dynamic and interact with each other. The cytoskeleton is responsible for organelle placement and mobility

Describe the cytoskeleton

Cytoskeleton: complex network of interlinking filaments and tubules that extend throughout the cytoplasm of a cell. Cytoskeleton provides structural framework that determines cell shape, positions of organelles, and general organization of the cytoplasm

Cytoskeleton also responsible for movement of entire cells and internal transport of organelles

Not rigid, but dynamic structure that is continually reorganized as cells move and change shape

Describe the 3 main components of the cytoskeleton

Actin filaments: define cell shape and necessary for cell locomotion

Microtubules: position membrane enclosed organelles, mediate and form mitotic spindle during cell division

Intermediate filaments: provide mechanical strength

What are molecular motors?

proteins that convert energy of ATP hydrolysis to mechanical work

What are the main functions of the cytoskeleton?

1. Spatial organization of the contents of a cell: defines cell shape, organelle trafficking, divides chromosomes during cell division

2. Connects the components of the cell physically and biochemically to each other and the cell surface: structural support, mechanical stress

3. Generates coordinated forces that enable the cell to move and/or change and/or keep shape: cell migration, chemotaxis

Describe actin filaments

Actin polymerizes to form actin filaments (microfilaments)

Microfilaments: flexible fibers organized into various structures such as bundles and 3D networks; responds to local signaling activity; polarized; myosin protein motors

Some actin filaments are alloys built out of several isoforms

Each actin monomer (globular [G] actin) has tight binding sites that mediate head-to-tail interactions w/ two other actin monomers to form filaments (filamentous [F] actin)

How is actin oriented?

All actin monomers are oriented in the same direction. Causes actin filaments to have polarity

Filament ends: plus and minus end--arrow pointed at "minus" end, barbs pointed at "plus" end

Actin polymerization and assembly

Actin polymerization is reversible.
If assembly rate is higher than disassembly, filament grows
If assembly rate is lower, the filament will shrink
At the pointed end (-) assembly is disfavored; at the barbed end (+) assembly is favored.

How does actin self polymerization work?

Nucleation: first step of actin polymerization—dimers and trimers formed, then monomers are added to either end

Oligomer instability: small oligomers (comprised of less than 4 subunits) are unstable and fall apart

KD for dimer and trimer formation is higher (lower affinity) than for monomer addition to the long filament

Actin self polymerization is limited by oligomer instability

Free actin concentration will decrease as a result of polymerization

Filaments grow until system reaches equilibrium

Actin concentration is called critical concentration

Rate of assembly = Rate of disassembly

What is treadmilling?

Phenomenon when 1 end of a filament grows in length, while the other shrinks resulting in a section if the filament seemingly "moving" across a stratum or cytosol

Describe actin structure

Cross-linked meshwork's: cortex

Antiparallel filament bundles: stress fibers

Parallel filament bundles: filopodia

Branched networks: Lamellipodium

Arp 2/3 nucleation model

1. Arp2/3 Complex: binds to sides or - end of actin filaments

2. Nucleation promotion factors: deliver G-actin subunits to anchored complex

3. G-actin monomers: added to + end of growing filament

Tip nucleation model

1. Formins: function as dimers and interact with the terminal actin subunits

2. Each Formin monomer binds to a Profilin unit attached to a G-actin monomer

3. Monomers of G-actin/Profilin: added to growing end of filament

What stabilizes actin filaments?

Tropomyosin’s: stabilize actin filaments by binding lengthwise along the groove of the filament

Capping proteins: stabilize filaments by binding to the barbed or pointed ends

What causes the destabilization of actin?

Gelsolin and Cofilin: breaks actin filaments

What is filament nucleation?

Sees the formation of an actin nucleus, which is essentially a complex of 3 actin monomers, from which an actin filament may elongate via reversible addition; the + end elongates 5-10x faster than the - end

What is critical concentration?

Equal to the concentration of free actin, which is equal to the dissociation constant (KD).
Actin filaments are not formed when the free actin conc. is below KD

Describe thermodynamics of filament depolarization for both ends

ATP hydrolysis changes thermodynamics of dissociation.
Upon hydrolysis, most energy remains stored in the filament and released upon depolymerization.
More energy is released upon filament dissociation, becomes more negative (and thus KD goes up)

How can accessory proteins regulate actin filaments?

Actin-binding proteins (accessory proteins) regulate assembly and disassembly of actin filaments by cross-linking into bundles and networks and by associations w/ other cell structures

Describe functions and relationship between Thymosin and profilin

Thymosin: gene that encodes an actin sequestering protein which will play a role in regulation of actin polymerization; also involved in cell proliferation, migration, and differentiation.

Profilin: actin-binding protein that binds actin monomers and stimulates the exchange of bound ADP for ATP; promotes actin polymerization by increasing local conc. of ATP-actin.

Relationship: 2 major sequestering proteins that maintain pool of monomeric actin (G-actin) within cells of higher eukaryotes. Thymosin prevents G-actin from joining a filament, and Profilin only supports barbed-end elongation

What are Fimbrin and Fascin?

Fimbrin (10nm): Found in cross-linked networks. Cross-linking protein that connects actin filaments to form networks (identifying quality will measure the distance between filaments linked together; larger distance = better quality)

Fascin: Found in parallel actin bundles. Cytoskeleton-associated protein recognized to function primarily in regulation of cytoskeleton structure and formation of plasma membrane protrusions. Mediates formation of filopodia.

Can actin filaments generate force w/out motor proteins?

Yes; done by actin polymerization

What are Spectrin and Ankyrin and their functions?

Spectrin: actin-binding protein that provides the structural basis for the cortical cytoskeleton in erythrocytes

Ankyrin: proteins that bind to both Spectrin-actin network and the plasma membrane by binding to spectrum and a transmembrane protein called "band 3"

Structures are initiated by the action of what nucleating proteins?

1. Arp2/3 nucleates dendric networks

2. Formins nucleates bundle formation

3. Proteins that nucleate gel-like networks are Unknown

What are tight (parallel) bundles?

Tight bundles: Actin filaments that run parallel to each other. Individual filaments have the same polarity, known as parallel bundles

Parallel bundles: held together by small actin binding proteins

Ex. Fimbrin (in intestine; facilitate nutriment diffusion and absorption) and Fascin (energy of filament formation used to exert force on the plasma membrane and grow Filopodia)

Describe contractile antiparallel bundles

Contractile bundles: actin filaments geometrically parallel to each other. Filaments have alternating polarity (antiparallel).

Held together by alpha-actinin dimers

Actomyosin: contractile complexes of actin and myosin

Actin in contractile bundles is complexed with non-muscle myosin II (NMMII)

What are cross-linked (gel-like) networks

Filamin: protein in actin networks that form flexible cross-links

Actin fibers cannot slide over each other easily, which makes mesh tougher but flexible linkers make it more elastic

Cells require actin gels formed by filamin to extend thin, sheet-like projections called Lamellipodia (gel network assembly creates force that helps propel cell forward)

What are stress fibers?

Large actin bundles. Contractile bundles, cross-linked by alpha-actinin and stabilized by tropomyosin

Stress fibers attach at focal adhesions

Talin and vinculin proteins: bridge stress fibers to the plasma membrane by interacting w/ transmembrane integrins

Describe cell-cell contacts in epithelial cells

Cell-cell contacts (adherens junctions) form a continuous adhesion belt around each cell. Contact mediated by transmembrane proteins called cadherins, which bind to cytoplasmic catenins, which anchors actin filaments to plasma membrane

Describe cell movement

Cells move in response to signals from other cells or the environment in 3 stages:
1. Extension of leading edge (branching and polymerization of actin filaments)
2. Attachment of leading edge to the substratum (involves focal adhesions)
3. Retraction of the rear of the cell into the cell body (small GTP-binding proteins of Arf and Rho families)

What is myosin?

A molecular motor or a protein that converts chemical energy (ATP) to mechanical energy, generating force and movement
Myosin head converts chemical energy to work

Name that states of myosin and what they do

1. Attached: without nucleotides, myosin is tightly locked to actin (called rigor configuration--rigor mortis)

2. Released: in active muscle rigor is short-lived as it is relieved by ATP binding in the binding cleft on the back of myosin head. Induces allosteric conformational change in myosin that leads to dissociation from actin (released state)

3. Cocked state: further conformational changes in ATP binding site trigger movement of the lever arm, resulting in 5nm movement of the head

4. Force generating: initial winding binding of the myosin to the actin promotes released of inorganic phosphate (leads to tight actin binding). Triggers conformational change that puts head into conformation close to original rigor state (responsible for converting energy of ATP hydrolysis into lateral movement of myosin filament)

5. Attached state: ADP dissociation finalizes cycle as myosin returns to attached state.

Whole cycle results in ~5nm relative filament movement

Describe skeletal muscles

Bundles of muscle fibers: large cells formed by fusion of many cells during development

Myofibrils: most of the cytoplasm; bundles of thick myosin filaments and thin actin filaments

Sarcomeres: every myofibril is a chain of contractile units of sarcomeres

What is Myosin II?

The type of myosin in muscle. Has two heavy chains and two pairs of light chains

Heavy chains: globular head regions and a long alpha-helical tail. Tails twist around each other in a coiled-coil

Thick filaments: several hundred myosin molecules in a parallel staggered array. Globular heads bind actin, forming cross-bridges between thick and thin filaments

What is the sliding filament model?

A model about muscle contraction that contains myosin (thick filaments), Actin (thin filaments). The myosin head use the breakdown of ATP to attach to the active site and pull along for muscle contraction. The sarcomere contains myosin and actin.
The molecular basis for this is the binding of myosin to actin filaments, allowing myosin to function as a motor that drives filament sliding

What are myosin light chains?

Smooth muscle cells (lack regular striations) form contractive portion of the stomach, intestine, uterus, artery walls, etc. Composed of the sheets slightly elongated cells, each having a single nucleus.

What is Myosin V?

Cargo transporting myosin
Lower force generation + longer steps = efficiency

Important points:

Actin filaments and microtubules are polar
Kinesin (anterograde transporters) and dynein (retrograde transporters) are MT associated molecular motors

How is myosin manipulated directionally?

Myosin VI: walks in opposite (-) direction.

Myosin directionality is defined by lever arms and converter domain

Describe microtubules

Rigid hollow rods; dynamic structures that undergo continual assembly and disassembly. Function: cell movements and determining cell shape.

Microtubules are formed by polymerization of tubulin dimers--there are 13 protofilaments around a hollow core

Protofilaments: head-to-trail arrays of tubulin dimers arranged in parallel

Microtubules: have polarity (+ and - ends) which determine direction of movement

Microtubules are made of globular protein tubulin

Microtubular lattice is imperfect; implies that a discontinuity exists in the microtubule wall

What are the 2 types of microtubules in the cell?

1. Axonemal microtubules: form cytoskeletal part of cilia and flagella; easy to purify and used to seed microtubules in vitro polymerization assays

2. Non-axonemal microtubules: cytoplasmic microtubules

Describe microtubule polymerization

Guided by same chemical rules as polymerization of actin
At tubulin concentration above critical, + end growth is faster than on the - end
In absence of GTP hydrolysis, disassembly is faster on the + end than on - end

What is the role of GTP in microtubule polymerization?

Microtubules can undergo rapid cycles of assembly and disassembly.

GTP bound to beta tubulin is hydrolyzed to GDP after polymerization, which weakens binding affinity of tubulin dimers for each other, which causes rapid de-polymerization and loss of tubulin bound to GDP from - end

In microtubules stabilized at the - end, rapid GTP hydrolysis results in dynamic instability (alternating between cycles of growth and shrinkage)

As new GTP-bound tubulin dimers are added more rapidly than GTP is hydrolyzed, GTP cap remains at the + end and microtubule growth continues

If GTP is hydrolyzed more rapidly than new subunits are added, GDP bound tubulin at the + end of the microtubule leads to disassembly and shrinkage

How do microtubules collapse?

Microtubule catastrophe: rapid shrinkage at the + end

Rescue: recovery of GTP cap followed by microtubule growth

Microtubules can undergo transitions between rapid growth and shrinkage when free tubulin concentration is between KD (GTP) and KD (GDP).

Growing MT has GTP subunits at its end (forms a GTP cap); if nucleotide hydrolysis proceeds more rapidly than addition, the cap is lost and MT's shrink. There is relaxation upon losing the GTP cap.

GTP tubulin adopts a linear conformation (packed into cylindrical wall of MT); GTP hydrolysis changes shape of tubulin and forces the protofilaments into a curved shape

What are MAPs?

Microtubule-associated proteins: regulate dynamic behavior of microtubules (MTs can be regulated by small molecules)

Polymerase MAPs: accelerate growth by increasing incorporation of GTP-bound tubulin

De-polymerase MAPs: dissociate GTP-tubulin from + end, leading to microtubule shrinkage (catastrophe)

What is involved in microtubule catastrophe?

Kinesin-13/catastrophe factor: molecular motor associated with MTs. Bind to microtubule and slides along it (diffuses) without ATP hydrolysis. At + end, it stabilizes protofilaments in curved conformation, which promotes MT disassembly.

CLASP proteins: rescue Mts from catastrophe by stopping disassembly and restarting growth

Other MAPs can bind to + ends and mediate attachment of MTs to other structures (plasma membrane or ER) and regulate MT dynamics

Describe Gamme (Y) tubulin and its role

Gamma-tubulin: nucleates MTs (in vivo MT nucleation requires help)

2 copies of g-tubulin associated w/ a pair of accessory proteins forms: g-tubulin small complex or gamma-TuSC

7 copies of g-TuSC associate to form a spiral structure where the last g-tubulin lies under the 1st, resulting in 13 exposed g-tubulin subunits in a circular orientation that matches the orientation of 13 protofilaments in MT

In many cells, g-TuSC spiral associates with additional accessory proteins to form g-tubulin ring complex or g-TuRC

How are microtubules organized intracellularly?

In animal cells, most microtubules extend outward from the centrosome

During mitosis, they extend outward from duplicated centrosomes to form the mitotic spindle (which controls separation and distribution of chromosomes to daughter cells)

Microtubule-organizing center: centrosome

In fungi: spindle pole bodies have similar function

In plant cells: no centrosomes, so microtubules form an array of underlying the plasma membrane and function in synthesis of plant cell walls

Describe centrosomes

Initiation sites for microtubule assembly, which grow outward from negative ends anchored in the centrosome

Role of centrosomes is to initiate MT growth.

y-tubulin ring complex: complex through to bypass rate-limiting nucleation step, which speeds up MT growth

Colcemid: a drug that treats cells and microtubules disassemble and then new MT grow outward from centrosome

Centrioles: most animal cell centrosomes have a pair. Cylindrical, contains 9 triplets of MTs. Surrounded by pericentriolar material and oriented perpendicular to each other

What are the two families of motor proteins for microtubules?

Kinesin and Dynein.
Kinesin: move mainly toward the + end
Dynein: move toward the - end; two types

Describe Kinesin

Mainly anterograde transporters: kinesin and myosin evolved from a common ancestor and are structurally similar

Kinesin I: has 2 heavy chains and 2 regulatory chains

Heavy chains: alpha-helical regions that form coiled-coils (keeps 2 monomers together)

Kinesin I: moves along MTs towards + end; Kinesins that move towards + end have N-terminal motor domains

- end direction Kinesin (Kinesin-14) have C-terminal motor domains

What are the 2 types of Dynein?

Axonemal: 18 genes. very abundant in cilia (also in flagella)

Cytoplasmic (ciliary): 13 genes. 2 headed motor protein (have 3 heads in protozoans; 1 head in some algae)

Motor domains of dynein are larger than myosin or kinesin

Retrograde transporters; structurally unique

Describe the mechanochemical cycles of Kinesin

Kinesin-1: dimer of 2 nucleotide-binding domains (heads) that are connected via coiled-coil tail. The 2 Kinesin motor domains work in a coordinated manner

Kinesin is a processive molecular motor (both heads can bind ATP)

During a Kinesin step, the rear head detaches, passes the partner motor domain, and then binds to the next tubulin binding site

At the start of each step, the rear (lagging) head is complexed with ATP and is thus tightly bound with tubulin. The front (leading) head complexes with ADP and is loosely bound to tubulin

ATP hydrolysis weakens the interactions between rear head and microtubule. Simultaneously, ADP to ATP exchange strengthens interactions between the front head and the MT (causes "neck-linker" conformational change)

Upon forward movement, the rear head is now the leading head and reattaches to the tubulin

Shortened version of the mechanochemical cycles of Kinesin

ATP hydrolysis weakens interactions between rear domain and microtubule -> change in neck-linker orientation propels rear head forwards -> rear domains bind MT

Mechanochemical cycles of dynein

Least understood molecular motor

Dynein's heavy chain forms a motor domain and tubulin interacting head. Therefore, the number of heavy chains is equal to the number of heads in dynein

Very big protein! 4k aa's long

The large motor head forms planar ring. Contains C-terminal domain and 6 AAA domains. Out of the 6, 4 carry ATP binding sequence. Only 1 has ATPase activity

Long stalk: comprised of coiled-coil extends from the head; the stalk is terminated by microtubule binding domain. In ATP bound state, the stalk is detached from the MT. ATP hydrolysis causes stalk to reattach.

Release of inorganic phosphate and ADP causes large conformation change called "power stroke", which is a conformational change that involves rotation of the head and stalk relative to the tail

Dynein makes ~8nm step

What is Tau?

mediate tubulin and actin co-alignment

Important

Actin filaments and Microtubules are polar

Molecular motors convert chemical energy into work

ATP

adenosine 5' triphosphate
main energy source that cells use for most of their work

GTP

guanosine 5' triphosphate
energy source in protein synthesis

Energy of ATP hydrolysis

ATP -> ADP + Pi
ΔG = ΔG^0 + RT ln ([ADP] [Pi] / [ATP])
NMR is used to determine metabolite concentration in the muscle (at a certain point, the muscle stops contracting)

Describe the role of microtubules in mitosis

Microtubules completely reorganize during mitosis:

The interphase MT arrays are disassembled and free tubulin subunits are reassembled into the mitotic spindle that is responsible for separation of sister chromosomes (the centrosome is also duplicated in interphase)

During prophase, the centrosomes migrate to form the 2 poles of the mitotic spindle.

As the cell enters mitosis, the rate of MT disassembly increases, resulting in shrinkage of MTs. The # of MTs emanating from the 2 centrosomes is increased.

What are the 4 types of Microtubules in the mitotic spindle?

1. Kinetochore: complex of proteins associated with the centromere, which microtubules of the spindle attach

--MTs attach to the condensed chromosomes at the centromeres, stabilizing them

2. Chromosomal Microtubules: connect to chromosome ends via chromokinesin

3. Polar MTs: not attached to chromosomes but are stabilized by overlapping with each other in the center of the cell

4. Astral MTs: extend outward from the centrosomes with the + ends anchored in the cell cortex

Describe anaphase chromosome movement

After the centrosomes move to opposite sides of the cell, the duplicated chromosomes attach to kinetochore and chromosomal MTs. Then align on the metaphase plate. Links between the sister chromatids are severed and anaphase begins.

Chromosome movement occurs in 2 steps:

Anaphase A: chromosomes move toward spindle poles along kinetochore microtubules, driven by kinesins that depolymerize and shorten the tubules

Anaphase B: spindle poles separate. Overlapping inter-polar MTs elongate and slide against one another to push the spindle poles apart.

--Positive end-directed kinesins cross-link inter-polar MTs and move toward the + end on them. Results in movement of MT into opposite direction.

--Spindle poles are also pulled apart by the Astral MTs. Cytoplasmic dynein anchored to the cell cortex moves along Astral MTs in the - end direction

The simultaneous shrinkage of astral MTs by depolymerases leads to separation of the spindle poles

What are intermediate filaments and what do they do?

Family of related proteins that function mainly in strengthening of the cytoskeleton

-Form rope-like fibers

-Respond to mechanical stress in cell

-Non-polarized

-Do not support molecular motors

Name the intermediate filament proteins expressed in different types of cells

Type I and II: keratins in epithelial cells. Diverse expression profile dependent on the cell type.

Vimentin: forms network extending out from nucleus towards cell periphery.

Neurofilament (NF) proteins: major Intermediate Filaments of many neurons; provide support for long axons

Nestins: expressed during embryonic development in some stem cells

Type V: nuclear lamins; form meshwork underlying nuclear membrane

-Lamins: 3 genes; nuclear envelope

-Orphan: 2 genes; beaded filaments, eyes only

Describe intermediate filaments structure and assembly

Have head and tail domains that determine specific functions--domains connect by alpha-helical rod domain, which plays a central role in filament assembly

Central rod domains of 2 polypeptides form a coiled-coil; monomers in the dimer in parallel orientation

--Don't have GTPase or ATPase! No hydrolysis. No + or - end!

Dimers associate in staggered antiparallel fashion to form tetramers, which assemble end-to-end to form protofilaments (8 of which wind together to form a filament). All contingent on if they have no distinct ends! Non-polar.

More stable and don't have the dynamic behavior of actin filaments or microtubules.

Phosphorylation can regulate assembly and disassembly

Define Desmosomes and Hemidesmosomes

Intermediate filaments form a cytoplasmic network in most cells (ring around nucleus to plasma membrane). Epithelial cells have specialized cell contracts:

Desmosomes: Junctions between adjacent cells (bring two cells together)

--Keratin filaments: attach to dense protein plaques on the cytoplasmic side. This attachment is mediated by desmoplakin

----Plakin: large family w/ proteins that connect different cytoskeleton components together

Hemidesmosomes: junctions between epithelial cells and underlying connective tissue

--Ketatin filaments: linked to interns by different plakins (plectin)

Plectin: binds actin filaments and MTs, forming bridges between them and IFs. Increased mechanical stability of cell

Plectin, IFs, and MTs: do not exist isolated from each other! All interlink

More on intermediate filaments

-Some cells in culture don't make cytoplasmic IFs.

-Injection of cultured cells with antibody against vimentin disrupts IF networks without affecting cell growth or movement.

-Primary role of IFs: probably to strengthen cytoskeleton of cells in the tissues of multicellular organisms.

What might explain lack of intermediate filaments in culture dish cells?

Lack of mechanical stress

What are 4E-BPs and how do they regulate translation?

Global regulation

Numerous 4E-Bps that act in mRNA specific manner.

4E-BP is an important regulator of overall translation levels in cells. By binding eIF4E, 4E-BP impairs recruitment of the 40S ribosomal subunit to the cap structure present at the 5′-end of all eukaryotic cellular mRNAs. 4E-BP activity is controlled by TOR (Target of Rapamycin).

4E-BP: eLF4E binding protein! Binds eIF4E and prevents binding of eIF4G

Phosphorylated 4E-BP loses affinity to eIF4E. Conseqeuntly, eIF4G can then bind eIF4E and eIF4F is being formed. Leads to activation of translation

eIF4E binding proteins (4E-BP) can regulate global translation or translation of specific mRNAs

Describe mechanism of translational suppression in unfertilized oocyte

Translational regulation is very important during early development. Majority of mRNAs have been remade during oogenesis and stored in inactive form.
Many such mRNAs have short poly-A tails; long enough to stabilize them but not enough for translation.
Initially, these mRNAs carried long poly-A tails that were shortened by a repressor protein. The lengthening of the poly-A tail occurs upon fertilization.
Extending poly-A tails allows the binding of poly-A binding protein (PABP), which will then stimulate translation.

How does spatial organization of translation impact embryogenesis?

Localization to specific regions of eggs or embryos is important in developing, allowing proteins to be synthesized at appropriate sites.

Ex. Spatial localization of the key mRNA is responsible for development of posterior anterior axis

How does eIF2 phosphorylation inhibit translation?

(eIF=eukaryotic initiation factors)

Phosphorylation of eIF2 leads to translational shut off and translation of mRNAs with upstream ORFs.

Both eIF2 and eIF2B can be phosphorylated by regulatory protein kinases. These phosphorylations inhibit the exchange of bound GDP for GTP, thereby inhibiting initiation of translation.

Example: If mammalian cells are subjected to stress or starved of growth factors, protein kinases that phosphorylate eIF2 and eIF2B become activated, inhibiting further protein synthesis

What are uORFs? How do they regulate translation?

uORF: upstream open-reading frames

Open-reading frames: stretch of nucleotide sequence that does not contain stop codons and can encode a polypeptide

uORFs can regulate eukaryotic gene expression. Translation of the uORF typically inhibits downstream expression of the primary ORF.

non-function sequences in the 5' UTR that can trap a ribosome and thereby block translation. Regulation of how efficiently these are recognized allows regulation of translation initiation

Why does translation of mRNAs w/ uORFs increase when eIF2 is phosphorylated?

Low eIF2 or initiator tRNA availability leads to uORF bypass and increased protein production

GCN2 (one type of eIF2 kinases in humans): activated by binding of uncharged tRNA. Activated in response to amino acid starvation. Leads to activation of translation of ATF4 mRNA which contains 3 uORF

What is codon usage and how does it affect translation?

The effectiveness of translation is influenced by the nature of the codons used.

The genetic code is degenerate (most amino acids encoded by several codons). Such codons are called synonymous codons. tRNA abundance directly impacts the rate at which the corresponding codon is read.

The codons are not equivalent as tRNAs present at different concentrations. Highly expressed genes preferably carry synonymous codons that are decoded by abundant tRNAs.

Codon usage dictates elongation speed.

^ rate of elongation defines a rate at which start site is cleared, thus controlling initiation rate.

On highly translated mRNAs, elongation speed controls overall translation rates.

What is translational recoding?

Programmed ribosomal frame-shifting.

Phenomena when open reading frame is changed during elongation.

During frameshift, ribosome slides by 1 or 2 nts into either 5' or 3' direction and then resumes translation in the new ORF. Shift by 1 nt shift is the most common frameshift.

Slippage towards 5' end of mRNA is called -1 or -2 frame-shifting. Slippage towards 3' end is called +1 or +2.

Frameshift occurs in a defined place on mRNA when ribosome encounters a specific mRNA element called frameshift signal.

Frame-shifting sites could be predicted computationally (allows production of 2 proteins from 1 mRNA)

How do viruses utilize translation recoding to control gene expression?

Recoding uses less space, therefore allowing small genome.
Example: HIV encodes Gag protein (a capsid protein) in the 0 frame and polymerase in the -1 frame. Polymerase could only be expressed as Gag-Pol fusion. Frame-shifting efficiency ensures correct Gag to Gag-Pol ration necessary for proper virion assembly.

Describe how translation of ferritin mRNA is regulated

Ferritin: stores excess iron.

-Ferritin mRNA carries IRE in the 5' UTR. In low iron conditions, IRP binds to IRE. Bound IRP blocks scanning and thus inhibits translation. This will then inhibit ferritin production and therefore more iron is available for iron-requiring enzymes.

-When iron is abundant IRP, IRP binds iron. Complex with iron adopts inactive conformation and could not bind IRE. Thus, translation of ferritin is activated and excess of iron is sequestered.

Describe the mechanism that controls stability of transferrin receptor mRNA

Iron is transported through the body in the complex with protein called transferrin. Import of transferrin into the cell occurs by transferrin receptor (TfR) via receptor induced endocytosis.

TfR mRNA contains IRE elements. However, these elements are located in the 3' UTR. They contain destabilizing AU-rich stem. In high iron condition, IRP does not bind mRNA. AU element is exposed and mRNA is rapidly degraded. Thus, TfR is not synthesized.

In low iron, IRP binds IRE. AU-element is protected, and mRNA is stabilized. Thus TfR mRNA is translated.

What differences and similarities are there between miRNA and siRNA?

siRNA: Small interfering RNAs

--Produced from double-stranded RNAs by nuclease Dicer. siRNA often require perfect complementarity between guide RNA and target RNA. Therefore, siRNAs tend to regulate a specific or small number of target mRNAs

miRNA: MicroRNAs

--Transcribed by RNA pol II, then cleaved by nucleases Drosha and Dicer. miRNA often do not require perfect complementarity and thus potentially can regulate larger mRNA pool.

What is the NMD pathway and its mechanism?

NMD pathway: Nonsense-mediated decay
Recognizes and degrades incorrectly spliced mRNAs.

Stop codons are also called nonsense codons because they do not encode an amino acid.

Damaged and incorrectly processed mRNAs leads to production of the aberrant proteins that are deleterious to the cell. Thus, mRNAs are continuously surveyed in the cell. mRNAs surveyed during translation.

What are No-go and No-stop decay pathways?

No-stop decay:

Incorrect splicing might remove stop codon and 3' UTR. In this case, ribosome will continue translation past last exon and will encounter poly-A tail. AAA codon encodes lysine, thus poly-A will be translated. This will displace poly-A binding protein and will target mRNA for degradation.

No-go decay:

During translation, ribosomes must unfold mRNA structure. mRNA mis-folding might stall the ribosome. Such mRNAs are recognized by Dom34-Hsb1 and targeted to degradation.