Biology of Cells Quiz 1

DNA helicase

unwinds helix

single-strand DNA-binding proteins (SSB)

• bind to single-stranded DNA without covering bases

• help DNA helicases by stabilizing unwound strand

• prevents formation of short hairpin structures

DNA polymerase III

• cannot start chains de novo

• replicates DNA

DNA primase

synthesizes short RNA primers

Telomerase

• prevents DNA shortening & ensures stability of genome

• recognizes tip of G-rich strand of telomere DNA repeat sequence

• elongates telomere in 5’ to 3’ direction

DNA Polymerase I

erases RNA primer & replaces with DNA

DNA ligase

joins 3’ to 5’ ends of okazaki fragments

Proteins in mismatch repair in prokaryotes & purpose

MutH nicks unmethylated GATC sequences

Proteins in mismatch repair of eukaryotes & purposes

MutS: binds specifically to mismatched base pair

MutL: scans nearby DNA for nick & triggers degradation of nicked strand

Explain proofreading

• correct nucleotide has higher affinity to moving DNA polymerase

• if incorrect base added, DNA polymerase shifts strand to exonucleolytic site and moves in 3’ to 5’ direction to wrong base

• proofreading exonuclease clips off incorrect nucleotide

• DNA polymerase shifts strand back to polymerization site and resumes replication

Explain mismatch repair in prokaryotes

• methylation on adenine residues in sequence GATC denotes template strand

• newly synthesized strand is unmethylated

• MutH nicks unmethylated GATC sequqences

• excision of mismatched DNA segment

Explain mismatch repair in eukaryotes

• does not depend on DNA methylation

• newly synthesized DNA strands are nicked

• nicks provide signal that directs mismatch proofreading system to appropriate strand

• MutS binds to mismatched pair

• MutL scans nearby DNA for nick and triggers degradation of nicked strand

RNA polymerase

• catalyzes phosphodiester bond formations to link ribonucleotides

• starts RNA chain without primer

• no proofreading

Which RNA polymerase transcribes protein-coding genes in eukaryotes?

II

General transcription factors

• only in eukaryotes

• help RNA polymerases correctly bind to promoter

transcriptional activators

• gene regulatory proteins

• bind to enhancers to help RNA pol II recruitment

chromatin-modifying enzymes

• regulate TFs/RNA pol accessibility to DNA in chromatin

• activate/inactivate assembly of transcription initiation machinery on DNA

Transcription for RNA Pol II

1. TFIID binds to TATA box and recruits others

2. Other TFs and RNA Pol II assemble transcription initiation complex and bind to promoter

3. TFIIH (contains DNA helicase) guides transcription initiation complex to start site

4. RNA synthesizes short mRNA

5. Phosphorylation by TFIIH releases RNA Pol II from promoter to move down template strand

6. Once elongation started, most TFs released from DNA template & recycled

7. RNA polymerase reaches terminator and releases DNA template & RNA chain

mRNA processing

• 5’ capping

  • add methylated guanine nucleotide on 5’ end

  • methyl cap distinguishes mRNA from other types of RNA

• 3’-polyadenylation

• splicing- removes introns & join exons together

Small ribosomal subunit function

match tRNAs to mRNA codon

Large ribosomal subunit function

catalyzes peptide bond between amino acid & polypeptide chain

Release factors

• terminates translation

• attaches to stop codon and binds to ribosome at A site

translation initiation process

• form mRNA + ribosome complex

• initiator tRNA initiates translation (always carries methionine)

• only initiator tRNA can bind to small ribosomal uunit to form pre-initiation complex

Do all proteins have methionine as first amino acid at N-terminus?

Yes when newly made

usually removed later by specific protease

Translation elongation process

• initiator tRNA in P site

• tRNA carrying next AA binds to A-site and forms complementary base pair with mRNA codon

• form peptide bond between AA at P & A site

• release polypeptide chain from tRNA at P site

• Shift tRNAs to E & P site

• move mRNA 3 nucleotides, reset ribosomes to receive next aminoacyl-tRNA

Translation termination process

• reach stop codons (UAA, UGA, UAG)

• release factor attaches to stop codon and bind to ribosome at A site

• ribosome releases mRNA & polypeptide chain and separates into small & large subunits

Separation of nucleus from cytoplasm allows for…

• DNA to be protected from enzymes that could damage them

• Concentrate proteins acting on DNA

Chromatin is comprised of…

nuclear DNA + proteins (histones + other chromosomal proteins)

2 types of chromatin

euchromatin

• less condensed

• contains protein-coding genes

heterochromatin

• highly condensed

• no protein-coding genes

Conserved DNA regions

closely similar regions between different species (exons & regulatory sequences)

Introns

non-coding sequences

Exons

short segments of protein-coding sequences

Microtubule function

• primary organizers of cytoskeleton

• position membrane-bound organelles

• direct intracellular transport

Actin filament function

• determine cell shape

• responsible for cell locomotion

Intermediate filament function

• provide mechanical strength

basic microtubule subunit

α/β-tubulin heterodimer

Each α/β-tubulin monomer binds a ….

GTP

Which monomer has GTPase activity?

β-tubulin

Protofilament

• α/β-tubulin heterodimers joined end to end

• structural polarity

assembly of microtubule

• α and β-tubulin bind noncovalently > α/β-tubulin heterodimer

• α/β-tubulin heterodimers assemble into protofilaments

• 13 protofilaments laterally associate (between same subunits) into hollow cylindrical microtubule

Longitudinal and lateral interactions between α/β-tubulin heterodimers generate the _______ of the microtubule

helical lattice

Subunit loss in microtubules occurs… because …

• microtubule ends

• each α/β-tubulin subunit stabilized by multiple contacts within lattice

Location of nucleation of microtubules

• microtubule-organizing center (MTOC)

• in most animals, MTOC is centrosome

Centrosome

• composed of protein-rich (fibrous) matrix

• centrioles organize centrosome matrix

Centriole

short cylindrical structure made of modified microtubules + many accessory proteins

Microtubules are nucleated by…

• protein complex containing γ-tubulin

• centrosome contains >50 copies of γ-tubulin ring complexes (γ-TuRCs)

• γ-TuRCs efficiently nucleate microtubule growth

How are microtubules nucleated at centrosome?

• with - ends anchored and + ends extending outwards toward periphery

• star-like (astral) pattern

Microtubule elongation

• unphosphorylated stathmin binds α/β-tubulin dimers & prevents their addition to + end of microtubules

• reduces effective concentration of free α/β-tubulin available for polymerization

• normally, rate of extension > rate of GTP hydrolysis

• Decrease in free α/β-tubulin concentration = decreased rate of extension

• Rate of GTP hydrolysis catches up and hydrolyzes GTP cap into GDP so there is no more cap

• Triggers catastrophe

Catastrophe

growing microtubule loses GTP cap and rapidly starts to shrink

Rescue

shrinking microtubule regains a GTP-rich end and starts to grow again

Dynamic instability

repeated switching between growth and shrinkage

Tubulin and actin exist in 2 nucleotide states

T-form: GTP/ATP

D-form: GDP/ADP

Free tubulin and actin subunits are mostly in ___ form because…

T

nucleotide hydrolysis slow in free subunits but accelerated after incorporation into filament (only become D form after added to microtubule)

Microtubule stabilization

• Once cytoskeletal filament formed, proteins bind along sides & change stability & mechanical properties

• Micortubule-associated proteins (MAPs)

  • bind microtubule to stabilize them

  • promote microtubule formation by stabilizing small tubulin oligomers during early polymerization

• tau allows microtubules to form closely packed bundles because of its shorter projecting domains than other MAPs

Actin filament soluble subunit

G actin monomer

each bound to ATP

Actin filament assembly

• assemble head to tail

• structural polarity

• faster-growing + end and slower growing - end

• flexible compared to microtubules

Phases of actin assembly

• nucleation (lag phase)

  • small, stable nucleus must form before filament growth (slow step)

• polymerization (growth phase)

  • actin filaments elongate rapidly by adding subunits

• steady state (equilibrium phase)

  • rate of subunit addition = rate of subunit dissociation

Critical concentration (Cc)

• concentration of free subunits left in solution at steady state

• [free actin]>Cc= polymerization

• [free actin]<Cc= disassembly until equilibrium

Location of nucleation of actin filaments

plasma membrane

Highest density of actin filaments found where?

cell periphery

What is cell cortex and its function?

• actin-rich layer under plasma membrane

• helps control cell shape & surface movement

Arp2/3 complex

• promotes actin nucleation

• arp2/d nucleated filaments grow rapidly at + ends

In most non-muscle cells, _____ of actin is F-actin and ____ of actin is soluble (G) actin

50%

50%

Actin binding proteins

• bind actin monomers and regulate filament formation

• thymosin

  • binds actin monomers and keeps them inactive

  • thymosin-bound actin cannot be added to + or - ends of filament

• profilin

  • binds actin monomers

  • prevents addition to - end

  • promotes addition to + end

• compete for binding to actin monomers

arp2/3 complex role in dynamics

• nucleates new actin filaments

• remains associated with + ends

capZ

binds and caps + end (prevents further growth or shrinkage & stabilizes filament)

tropomyosin

binds along actin filament and blocks other actin-binding proteins from binding

cofilin

• binds to ADP-actin instead of ATP-actin

• changes twist of actin and weakens contact between actin subunits, promotes filament severing & depolymerization

• newly-formed actin filaments have more ATP actin and are less sensitive to cofilin

blue= heterochromatin

white= euchromatin

Wobble base

• some tRNAs only require accurate base-pairing at first two positions of codon

• tolerate mismatch (wobble base) at third position

Treadmilling

• behavior of actin filaments

• actin subunits added at + end and removed from - end at same rate

• filament appears to move through cytoplasm with little change in length

Treadmilling & dynamic instability allow cells to do what?

rapidly remodel cytoskeleton

Intermediate filaments

• not present in all cell types

• in many cells, form extensive network in cytoplasm and provide mechanical strength

Nuclear lamins

• type of intermediate filament

• form meshwork under inner nuclear membrane & support nucleus + anchor chromatin

Intermediate filament soluble subunit

tetramer

Structure of intermediate filament

• elongated molecules with central alpha-helical domain

• no structural polarity

• 2 monomers —> parallel coiled-coil dimer

• 2 dimers associate antiparallel —> tetramer

• 8 tetramers associate laterally —> intermediate filament

Accessory proteins function

• crosslink intermediate filaments and connect them to other cytoskeletal systems

Filaggrin

• accessory protein

• bundles keratin filaments

Plectin

• accessory protein

• links & bundles vimentin & other intermediate filaments

SUN-KASH complexes

connect cytoskeleton to the nucleus across nuclear envelope