Midterm REVIEW cards Flashcards

Question

Formation of the PIC steps:

Answer 1

1) association of TFIID to the promoter 2) followed by TFIIB and TFIIA 3) RNA polymerase is recruited and The non-phosphorylated CTD (Carboxy Terminal Domain) of RNA pol II establishes contact with several GTFs 4) TFIIE and TFIIH are recruited 5) TFIIH helicase activity facilitates the opening of the double helix of DNA: Open PIC 6) RNA Polymerase II initiates transcription 7) TFIIH kinase activity phosphorylates the CTD to release RNA polymerase from the promoter

Answer 2

After the initial phosphorylation of CTD by TFIIH kinase, 1) two negative elongation factors, NELF and DSIF, associate with RNA polymerase II 2) they pause it downstream of the initiation site. 3) * At this point another kinase, CDK9/CycT (also called PTEFb (Positive Transcription Elongation Factor b) phosphorylates the CTD and NELF. 4) * RNA polymerase II with fully phosphorylated CTD is released 5) * Positive elongation factors PAF and Spt16 join the polymerase. 6) * DSIF becomes a positive elongation factor

Answer 3

The 5’ end of the short RNA is synthesized before the polymerase pauses * The short RNA forms a secondary structure called TAR. TAR holds the polymerase and has an inhibitory effect on Cdk9/CycT. * HIV1 (human Immuno-deficiency virus) encodes the protein called Tat. Tat binds to TAR. * Upon cell stress other factors release the polymerase and some Tat is made. * When bound to TAR, Tat ACTIVATES CDK9/CycT kinase and releases the paused RNA polymerase II * Now more Tat is made and viral genes are fully expressed

Answer 4

HIV is dormant but activates upon stress when the organism is weak, slowly killing T-cells and abolishing immunity * The patients die from lost immunity and any opportunistic infections The Tat protein of HIV holds RNA polymerase II until conditions for the spreading of the virus are favorable

Answer 5

At most TATA-containing and TATA-less promoters transcription initiates and proceeds in one direction * !!!Your textbook does not have a good illustration on this type of uni- directional transcription initiation. The figure shown on the next slide is from the 8th edition of the textbook. * At other promoters (called CpG islands) transcription initiates in both directions, but the polymerase stalls and falls off DNA in one of the directions and then proceeds in the other direction only

Answer 6

CpG islands occupy the promoters of about 70% percent of the genes in vertebrates. These genes are transcribed at a low rate. * CpG rich DNA contains less nucleosomes and are easier to transcribe. * At CpG islands transcription initiates at any position (not defined) within the island. * Transcription from CpG island initiates in both directions, but proceeds in the direction of the Open Reading Frame (ORF) only.

Answer 7

Linker scanning-reporter gene technique for identifying gene regulatory elements on DNA

Answer 8

You buy a plasmid that contains a reporter gene, usually a gene that encodes an enzyme (tk in figure 8-17) The tk ORF (Open Reading Frame) on the plasmid has no promoter. Upstream of the tk ORF you clone a large piece of DNA that you know is controlling YOUR gene of interest (not tk ) You systematically replace small pieces (10 bp) of the cloned regulatory DNA by a “linker”. You produce multiple plasmids with linker substitutions (9 in figure 8-17) You transform each individual plasmid in a cell culture and measure the activity of the reporter (a simple enzyme reaction of cell extract) Loss of reporter activity means that you have hit a regulatory sequence

Answer 9

- an ORF for an enzyme (Luciferase (luc) in this case) - NO promoter-proximal regulatory sequences - A multiple cloning site (MCS) for the cloning a regulatory sequence - Translation initiation elements upstream of the reporter gene - Poly A+ signal downstream of the reporter gene

Answer 10

- Eukaryotic promoter region DNA that supports high-level expression of a reporter gene (light purple) is cloned in a plasmid vector. - Overlapping linker scanning (LS) mutations (insertions of scrambled sequence) are introduced from one end of the region to the other. - Mutant plasmids are transfected separately into cultured cells. The expression/activity of the reporter gene product (thymidine kinase gene) is assayed.

Answer 11

distal regulatory elements, could be upstream or downstream of the initiation site position independent orientation independent

Answer 12

Function within a short distance (several hundred bps from the transcription initiation site Immediately upstream or downstream from the initiation site Position dependent: usually non-functional if moved

Answer 13

via bending DNA

Answer 14

Binds to a specific regulatory sequence within the condensed chromatin Interacts with chromatin-remodeling enzymes and histone acetylases that decondense the chromatin, making it accessible to RNA polymerase II and general transcription factors.

Answer 15

Bind to specific transcription-control elements in both promoter-proximal sites and distant enhancers Interact with one another and with the multisubunit Mediator complex to assemble general transcription factors and RNA polymerase II (Pol II) on promoters

Answer 16

Pol II initiates transcription. Pol II pauses after transcribing fewer than 100 nucleotides because of action of the elongation inhibitor NELF (negative elongation factor) associated with DSIF (DRB sensitivity-inducing factor).

Answer 17

Promote association of the Pol II-NELF-DSIF complex with elongation factor P-TEFb (cyclin T-CDK9 [kinase]), which releases NELF NELF release allows resumption of RNA transcription.

Answer 18

by deletion analysis of the protein

Answer 19

Transcriptional activators or repressors often have independent DNA-binding domains and effector (activation or repression) domains

Answer 20

Activators are composed of distinct functional domains. (a) Reporter-gene construct containing a lacZ reporter gene (encoding β-galactosidase) and a TATA box ligated to UASGAL, a regulatory element that contains several Gal4-binding sites. The reporter-gene construct and DNAs encoding wild-type or mutant (deleted) Gal4 introduced simultaneously into mutant (gal4) yeast cells. Activity of β-galactosidase expressed from lacZ was assayed. High activity if introduced GAL4 DNA encodes a functional protein. (b) Wild-type and mutant Gal4 — results: N-terminal end 50 amino acid deletion: Eliminates UASGAL binding. Contains DNA-binding site. C-terminal end deletions: β-galactosidase expression activation eliminated only when between 126 and 189 or more amino acids deleted from the C-terminal end. End contains activation domain. Internal deletions — Gal4 central region is not crucial for its function in this assay.

Answer 21

Motif is a sequence in a protein that, based on similarity with other known functional domains, is likely to produce a distinct 3D structure and a distinct domain. - WITHIN DOMAINS ex. zinc-finger, leu zipper These motifs are easy to recognize by bioinformatic tools

Answer 22

DOMAIN is a portion of a polypeptide with a specified function: Transactivation Domain, dimerization domain, dna binding domain, hormone binding domain. The domains are identified by deletion analysis of actual peptides

Answer 23

regularly spaced C and H amino acids have a zn2+ in middle in DNA binding domains

Answer 24

is found in protein dimerization domains: regualrily spaced L amino acid residues:

Answer 25

helix turn helix motif found in many dimerization domains

Answer 26

This means that if two proteins (green and yellow) can combine, they will have three binding sites (gg, yy, gy), three proteins (green, yellow, blue) can have six binding sites (gy, gb, yb, gg, yy, bb) These six combinations can recognize six DNA binding sites Adding an inhibitor for one of the three proteins increase the possible outcomes for activation (sort of a ‘fine tuning’)

Answer 27

Combinatorial binding to DNA-binding proteins from different classes Eukaryotes have very complex promoters and enhancers. In these, various transcription activators and repressors can bind The transcriptional activators or repressors often interact with each other and cooperate to establish a strong activator or repressor site to tightly control the expression of the target genes

Answer 28

There are positions in the genomes where large heterochromatin blocks are established and maintained We will call these positions “SILENCERS” Genes that are close to these SILENCERS are strongly repressed The repression of the genes depends on their position (close to, or within heterochromatin) relative to the silencer and does not depend on their promoters.

Answer 29

HML and HMR they are constantly repressed thanks to Rap1 and Sir proteins

Answer 30

Acetylation of the histone tails reduces their positive charge, allows for the opening of chromatin (euchromatin) and for transcription Deacetylation of histone tails reconstitutes positive charge, strengthens DNA-histone interactions, compacts chromatin(heterochromatin) and suppresses transcription

Answer 31

Two mating type loci , HML and HMR, are constantly repressed Genetic studies identified proteins that regulate this repression Rap1 binds next to the repressed loci and recruits Sir (Silent Information Region) proteins Sir proteins spread away and cover the chromatin of nearby genes Sir2 is a HISTONE DEACETYLASE. It deacetylates the histones within HML and HMR The deacetylated histones bind tighter to DNA and form heterochromatin If the genes in HML and HMR are moved away from HML and HMR loci, they become active Conclusion: Histone hypo-acetylation is necessary for positional gene repression

Answer 32

PERMANENT GENE REPRESSION Arginine retains a positive charge only, can not be acetylated. The DNA-histone interaction is strong, chromatin is compact, gene repression can not be reversed by acetylating the histones These mutants maintain permanent gene repression at telomeres.

Answer 33

CANNOT BE DEACETYLATED - CANNOT BE REPRESSED Glutamine has a neutral charge, resembles acetylated Lysine, can not be deacetylated to gain a positive charge Chromatin is decondensed, gene can not be repressed. These mutations maintain permanent gene activation at telomeres

Answer 34

Rap1 binds directly to the telomeric repeats SIR proteins are recruited by Rap1 Sir proteins spread in the sub-telomeric regions and cover them with condensed hypoacetylated nucleosomes More detailed: Many telomeres cluster at the periphery of the nucleus and form large 3D heterochromatin block RAP1 – binds HML and HMR silencer sequences that are repeated multiple times at each yeast-chromosome telomere nucleates formation of heterochromatin SIR2 – histone deacetylase that removes acetyl groups on histone tail lysines RAP1 and SIR2, 3, and 4 proteins – bind to each other SIR3 and SIR4 – bind to histone H3 and H4 N-terminal tails that are maintained in a largely nonacetylated state by SIR2 deacetylase

Answer 35

(b,c) Yeast cells labeled with: (b) a telomere-specific hybridization probe and (c) fluorescent-labeled specific anti-SIR3 antibody SIR3 – localized in the repressed telomeric heterochromatin. (RAP1, SIR2, and SIR4 also colocalize with the repressed telomeric heterochromatin.)

Answer 36

the REPRESSOR binds to the silencer region of the dna and recruits the corepressors

Answer 37

co-repressors - they bind to H3 and H4 N-terminal tails (deacetylated)

Answer 38

a histone deacetylase - removes acetyl groups on histone tail lysines

Answer 39

Position-dependent repression of genes The repression of the genes depends on their position (close to, or within heterochromatin) relative to the silencer and does not depend on their promoters these positions are called silencers If the genes in HML and HMR are moved away from HML and HMR loci, they become active Conclusion: Histone hypo-acetylation is necessary for positional gene repression

Answer 40

promotor-dependent and position-independent regulation also works via histone acetylation/deacetylation, however different enzymes and different co-repressors and co-activators are employed EX. Ume6 and Rpd3 The repressors and activators bind to promoters and enhancers and recruit the co-repressors or co-activators The co-repressors and co-activators do not spread, they stay localized at the regulatory elements In these situations the position of the genes is not a critical factor; the promotor is.

Answer 41

1) the SAGA complex - a histone hyperacetylase 2) the activation domain 3) ACTIVATION of the genes with the UAS promoter

Answer 42

1) Rpd3L - a histone deacetylase 2) the repression domain (RD) 3) Repression of the genes with URS1 promoter

Answer 43

changes in the body part of reproduction (menstration)

Answer 44

Histone acetyl transferases and a class of co-activators called chromatin remodeling complexes

Answer 45

Chromatin-Remodeling Complexes then use ATP to push nucleosomes along DNA to “open” promoters ex. SWI and SNF

Answer 46

Chromatin remodeling factors SWI/SNF (switching/sucrose non-fermenting) ATPases Move nucleosomes along the DNA by sliding or transferring them Shift nucleosomes away from the promoter/enhancer sites and gives transcription factors access to the DNA Chromatin-Remodeling Factors use ATP to move nucleosomes

Answer 47

an example of a co-activator (a major “bridging” factor between transcriptional activators, GTFs, histone modifiers and chromatin remodelers) The Mediator is a huge complex containing variety of proteins including enzymes, proteins that recognize activators, proteins that recognize GTFs and other coactivators Different mediator complexes contain proteins required for specific genes The Mediator(s) directly interacts with transcriptional activators One mediator complex can interact with multiple transcriptional activators The Mediator(s) directly interacts with GTFs

Answer 48

Cell differentiate and select the genes they express. Then the cells divide multiple times but continue to express the same genes after each cell division. DNA replication is a major disruptor of chromatin and bound transcription factors. The “memory” of gene expression is achieved mainly through reconstitution of the same chromatin structure after the passage of the replication fork We call this epigenetic memory of transcription

Answer 49

Epigenetic control of transcription is maintained by DNA methylation and methylation and acetylation of histones. We call these epigenetic marks Epigenetic marks are faithfully rebuilt during and/or soon after the passage of the replication fork

Answer 50

(Histone post-translational modifications (PTMs): Methylation, Acetylation, Phosphorylation

Answer 51

H3 (K9, K27) Me2/3

Answer 52

Methylation of CpG islands leads to condensation of chromatin Methylated DNA recruits special class of Me-DNA-biding proteins (MeBPs). MeBPs recruit factors that deacetylate histones and condense chromatin IMPORTANT: the methylation of DNA is rebuilt immediately after the passage of the replication forks

Answer 53

At the levels of transcription, RNA processing and translation

Answer 54

Short interfering RNAs, 22 bp long, that base-pair extensively, but not completely with mRNAs, especially over bases 2 to 7 at the 5' end of the miRNA (the seed sequence) this pairing inhibits translation of the target mRNA and targets it for degradation

Answer 55

microRNAs, 22 bases long, base pair extensively, but not completely (imperfect match) with mRNA, especially over bases 2 to 7 at the 5' end of the miRNA THis pairing inhibits translation of the target mRNA

Answer 56

Pre-mRNA (the primary transcript) is capped, then spliced, then polyadenylated before being exported to the cytoplasm

Answer 57

means linking two ropes without a knot A large ribonucleoprotein spliceosome complex catalyzes the joining of two exons

Answer 58

and alternative cleavage at different poly(A) sites yield a significant variety of different mRNAs from the same gene in different cell types or at different developmental stages. Some of the proteins generated by alternative splicing have drastically different activities

Answer 59

1) Alternative splicing in neurons Perception of sound Neuron connectivity 2) Sex determination in Drosophila

Answer 60

Most extreme example of regulated alternative RNA processing yet discovered Helps to specify tens of millions of different specific synaptic connections between neurons in the Drosophila brain (bottom) Dscam neuron cell-surface protein: 10 different immunoglobulin (Ig) domains (ovals) 6 different fibronectin type III domains (rectangles) One transmembrane domain (yellow) C-terminal cytoplasmic domain Fully processed mRNA contains: One of 12 Ig2 exons One of 48 Ig3 exons One of 33 Ig7 exons One of 2 transmembrane exons Alternative splicing – can generate 12 x 48 x 33 x 2 = 38,016 possible isoforms

Answer 61

Dendrites are branches that extend out of the neural cell bodies The branches of the same neuron repel each other while overlapping with branches from other neurons This is controlled by Dscam Dscam mutants show evidence of reduced dendrite self-avoidance

Answer 62

In one-third of patients with autism spectrum disorder (ASD), more skipping of cassette exons and microexons are observed than in normals Abnormalities in the frequency of splicing of alternative 5′ and 3′ splice sites are observed only rarely in ASD.

Answer 63

Sxl is an RNA binding protein, acts as a suppressor of splicing - present in females it suppresses the splicing of sxl gene btw exons 2 and 3 and it suppresses the splicing of tra protein btw exons 1 and 2

Answer 64

Tra is an RNA binding protein, acts as an activator of splicing activates splizing of dsx

Answer 65

is a transcription activator/repressor activates transcription of genes in females encoding female parts represses transcription the transcription of those genes in males

Answer 66

Rpb1 (along with tra) form a complex that activates splicing of the Dsx gene Tra-Rbp1-Tra2 complexes interact with six exonic splicing enhancers in exon 4. Activates exon 3–4 splicing

Answer 67

The development of C. elegans is regulated by multiple lineage (LIN) determining and lethality (LET) genes Interestingly, the genes lin-4 and let-7 genes are NOT encoding for any protein They produce small RNAs that are complementary to other mRNAs The imperfect binding of the lin-4 or let-7 micro RNAs (miRNAs) to the 3’-UTR (untranslated region) of the mRNAs dramatically reduce the translation of the mRNAs

Answer 68

miRNA would IMPERFECTLY (there are mismatches) bind to the target mRNA The binding is usually at the 3’-UTR The imperfect binding leads to repression of translation siRNA are produced from cleavage of double stranded RNA They bind PERFECTLY (no mismatches) to the target RNA and cause its rapid degradation by a mechanism called RNA interference

Answer 69

Dicer gene knockout – eliminates generation of miRNAs in mammals – embryonic lethal Experiment – Dicer knockout only in limb tissue (13-day mouse embryos): (immunostained for Gd5 protein, a marker of joint formation) (left) Dicer-expressing control – normal limb development (right) Dicer-knockout in limbs (Conditional expression of Cre induces dicer gene deletion only in limb cells.) Result – fundamental pattern maintained but abnormal limb development Conclusion – Dicer (miRNA) is required for morphogenesis but not patterning of the vertebrate limb

Answer 70

miRNA transcription and processing: RNA polymerase II transcribes primary miRNA transcripts (pri-miRNA) – folds to form double strand region Nuclear double-strand RNA–specific endoribonuclease Drosha and double-strand RNA–binding protein DGCR8 (Pasha in Drosophila) bind pri-miRNA double strand regions Drosha cleaves the pri-miRNA – generates a ~70-nucleotide pre-miRNA

Answer 71

Exportin 5 – nuclear transporter transports processed pri-miRNA to the cytoplasm

Answer 72

Step 1: dsRNA is cleaved to small (21-28 bp) RNA fragments by a dsRNA-endonuclease called DICER Step 2: Small dsRNAs bind to RISC (RNA Induced Silencing Complex) and form an inactive RISC complex. Step 3: Argonaut in RISC is a RNA helicase. It unwinds dsRNA. Active RISC* is formed. Step 4: RISC*, guided by single-stranded siRNA, cleaves target mRNA (“SLICER” activity by Argonaut)

Answer 73

Argonaut (RISC): An RNA helicase Removes one of the strands of the dsRNA Leads the trimmed siRNA to the target mRNA

Answer 74

PAX6 regulates several processes during the development of mammals PAX6 is a developmentally regulated gene it encodes a transcriptional activator It contains three promoters and three transcription initiation sites It has more than three enhancers It is expressed at different times and in different organs during embryonal development It is expressed in certain parts of the brain in adult organisms The translation of PAX6 mRNAs is regulated by several miRNAs

Answer 75

3 alternative promoters

Answer 76

the gene is inactive in all cells of the body: heterochromatin

Answer 77

pioneer factors open heterochromatin at the PAX6 locus in the pancreas (P) and the lens plate (LP) In all other organs, PAX6 remains in heterochromatin

Answer 78

the pancreatic and lens/cornea enhancers activate PAX6 by bending DNA and come in proximity of the promoter The pancreatic and the lens/cornea enhancers will be occupied by multiple, but not identical transcriptional activators (enhanceosome) Transcriptional activators communicate with the GTFs, Mediator, Chromatin Remodeling Complexes and Histone Modification Complexes. Histones are acetylated Nucleosomes are displaced from the promoter region all other organs, PAX6 remains in heterochromatin

Answer 79

Mediator and GTFs communicate GTFs recruit RNA polymerase II The CTD of RNA polymerase II is phosphorylated by the TFIIH kinase The TFIIH helicase activity opens the DNA helix and the RNA polymerase starts transcribing

Answer 80

: enhaceosome exchange The Lens/cornea enhancer stops working The pancreatic enhancer continues to operate The retina enhancers start operating. They will bend DNA and come in the proximity of the promoter.

Answer 81

telencephalon and rhomboencephalon will start working in the brain

Answer 82

PAX6 can produce three different transcripts from its three promoters Alternative splicing of these transcripts produces proteins with different function. Transcript a) is missing exon 1 and the alternative exon α (alpha) Transcript b) is missing exon 0 and the alternative exon α (alpha) Transcript c) is missing exons 0 and 1-4 but retains the alternative exon α (alpha) Exons 1-4 encode a part of the DNA-binding domain of PAX6 Hence, transcripts a) and b) produce similar DNA binding proteins that differ in their DNA-binding domains and bind to different DNA elements Transcript c) has an entirely different function

Answer 83

Range ~50 to 200 bp. Binding sites for several transcription factors. Interacting proteins distally bound can produce loops in the DNA between their binding sites

Answer 84

Six overlapping binding sites bind two heterodimeric factors: Jun/ATF-2, p50/RelA (NF-κB), and 2 copies each of the monomeric transcription factors IRF-3 and IRF-7.

Answer 85

Hydrophilic signaling molecules – (small molecules [adrenaline, acetylcholine], peptides [yeast mating factors, glucagon], and proteins [insulin, growth hormone]) Cannot diffuse across the cell membrane Step 4: Bind to specific cell-surface receptor proteins – triggers receptor conformational change that activates the receptor Step 5: Activated receptor activates one or more downstream signal transduction proteins or small-molecule second messengers. Step 6: Signal transduction proteins or small-molecule second messengers activate one or more effector proteins. Step 7a: Effector – stimulates modification of specific cytosolic proteins; short-term (sec-min) changes in cellular function, metabolism, or movement Step 7b: Effector – moves into the nucleus; triggers long-term (hours-permanent) changes in gene expression Termination or down-modulation of the cellular response – Step 8: Negative feedback/feedback repression from intracellular signaling molecules Step 9: Destruction of the extracellular signal

Answer 86

Hydrophobic signaling molecules – (steroids and related molecules) Step 1: Diffuse through the plasma membrane Step 2: Bind to cytosolic receptors Step 3: Receptor-signal complex moves into the nucleus – binds transcription-control regions in DNA to activate or repress gene expression

Answer 87

- endocrine - paracrine - autocrine - membrane protein signals

Answer 88

endocrine hormones affects target cells some distance from site of synthesis transported through circulatory system ex. epinephrine and insulin

Answer 89

paracrine hormones affect target cells in close proximity some may bind to ECM affect only nearby target cells expressing the receptor ex. neurotransmitters, growth factors

Answer 90

cells respond to signals that they produce target sites are on the same cell ex. growth hormones

Answer 91

signal neighboring cells by direct contact with surface receptors.

Answer 92

Dexamethasone, activates the Glucocorticoid Receptor

Answer 93

Activation domain, DNA binding domain and ligand binding domain

Answer 94

Dex = Dexamethasone, activates the Glucocorticoid Receptor In the absence of Dex the Glucocorticoid Receptor is in the cytoplasm In the presence of Dex the receptor-hormone complex moves to the nucleus The control ꞵ-galactosidase is cytoplasmic regardless of the presence of Dex In more detail: (–) Hormone – receptor retained in the cytoplasm by interaction between its ligand-binding domain (LBD) and chaperone proteins (+) hormone – 1) Hormone complex diffuses through the plasma membrane and binds to the receptor ligand-binding domain. 2) Causes a conformational change that releases the chaperone proteins 3) Receptor-hormone complex translocates into the nucleus. 4) DBD binds to response elements Ligand-binding domain and an additional activation domain (AD) at the N-terminus stimulate transcription of target genes.

Answer 95

bind only a single type of hydrophilic signaling molecules All hormone receptors are highly specific for their ligands High affinity of the hormone-receptor interaction Each receptor binds only a single type of hormone The hormone changes the conformation of the receptor sending a signal to the cascade

Answer 96

small molecules: adrenaline, acetylcholine peptides: glucagon proteins: insulin, growth hormones

Answer 97

Basically - found that only 8 DISTANTLY SPACED AMINO ACIDS CONTRIBUTE 85% BINDING ENERGY As determined from the three-dimensional structure of the 1 growth hormone: 2 growth hormone receptor complex, 28 amino acids in the hormone are at the binding interface with the first receptor protein. To determine which amino acids are important in ligand-receptor binding, researchers mutated each of these amino acids one at a time to alanine and measured the effect on receptor binding. From this study, it was found that only eight amino acids on growth hormone (green) contribute 85 percent of the energy that is responsible for tight receptor binding; these amino acids are distant from each other in the primary sequence but adjacent in the folded protein. Similar studies showed that two tryptophan residues (blue) in the receptor contribute most of the energy responsible for tight binding of growth hormone, although other amino acids at the interface with the hormone (yellow) are also important

Answer 98

Hormone receptors activate Effector Proteins Often the effector proteins are enzymes that produce small molecules called second messengers Secondary messengers carry and amplify the signals from activated receptors/hormone complexes to the effector molecules

Answer 99

- small short-lived molecules - diffuse rapidly - allow enzymatic amplification

Answer 100

Ca2+ ions, cAMP, and cGMP

Answer 101

- type of water-soluble secondary messenger - Generated from ATP by adenylyl cyclase - Activates PKA

Answer 102

- type of water-soluble second messenger - Generated by guanylyl cyclase - Activates PKG and specific cation channels

Answer 103

- type of lipid-soluble second messengers - Both made from PIP2 by phospholipase C - IP3 – opens channels to release Ca2+ from the ER DAG – with Ca2+ activates PKC

Answer 104

Released from intracellular stores or transported into the cell Activates calmodulin, specific kinases (PKC), and other regulatory proteins

Answer 105

1. tyrosine (Y) kinases 2. serine (S) / threonine (T) kinases

Answer 106

Hydrophilic hormones bind to membrane receptors. These so-called G-protein coupled receptors (GPCR) activate G-proteins (GTPase switch proteins). G-proteins exchange GDP to GTP leading to a conformational change and activation. Active G-protein sends a signal G-protein has intrinsic GTPase activity, which can be accelerated by GTPase-accelerating proteins (GAPs) Once the signal is sent, the G-protein inactivates itself by hydrolyzing GTP to GDP

Answer 107

G-protein activity is regulated by exchange of GDP to GTP Association with GTP switches the protein “ON” After sending the signal, GTP is hydrolysed and the protein is switched “OFF"

Answer 108

time G protein remains in the active conformation

Answer 109

Conformation of the G protein is altered by GTP/GDP binding Inactive (GDP-bound) G protein is capable of interacting with upstream activators Active (GTP-bound) G protein is capable of interacting with downstream effector proteins The downstream effector (often an enzyme) produces SECOND MESSENGER MOLECULES Switch domain conformation – can bind and activate specific downstream effector proteins IN THE ACTIVE/ON STATE, THE SWITCHES ARE PULLED IN AND BOUND TO GTP through interactions with the backbone amide groups of the conserved Thr and Gly residues (blue and green switches in diagram) IN THE INACTIVE/OFF STATE - SWITCH i AND ii ARE RELAXED INTO OFF CONFORMATION - inhibits interaction with downstream effectors

Answer 110

(1) A receptor that contains seven membrane-spanning α helices (2) A receptor-activated heterotrimeric G protein cycling between GTP-active and GDP-inactive forms (3) A membrane-bound effector protein (4) Proteins that desensitize the signaling pathway

Answer 111

H2 receptor (histamine is ligand) - affects acid secreting cells of stomach H1 receptor (histamine is ligand) - causes symptoms of allergy Serotonin 5Ht 2a and 1a (serotonin is ligand) - causes synaptic transmission btw neurnons in CNS Angiotensin - that is also ligand - increases blood pressure B-adrenergic receptor - (epinephrine is ligand) - facilitates respiration CysLT - (Leukotrienes are ligand) - contracts smooth muscle

Answer 112

>30 different mammalian GPCRs activate Gαs activation of adenylyl cyclase production of cAMP as a second messenger. (Most cell types express one or more such GPCRs.) Adenylyl cyclase (AC) – catalyzes formation of cyclic cAMP (second messenger) bond from ATP precursor cAMP phosphodiesterase (PDE) – catalyzes hydrolysis of cyclic bond – AMP (not second messenger) Similar reactions occur for production and destruction of cGMP second messenger.

Answer 113

adenylyl cyclase - catalyzes FORMATION of cyclic cAMP (a second messenger) from ATP precursor then, PDE (phosphodiesterase) takes that cAMP and catalyzes HYDROLYSIS of the cyclic bond AMP (not second messenger)

Answer 114

Amplification: Single epinephrine molecule binding to one G protein–coupled receptor – activates up to hundreds of G proteins (amplification) Each G protein activates 1 AC until G protein hydrolyzes GTP (not amplification). AC catalyzes the synthesis of a large number of cAMP molecules while activated (amplification). Two cAMPs activate two PKA catalytic subunits (not amplification). Activated PKA phosphorylates and activates multiple target proteins (amplification). Activated target proteins: Other kinases – kinase cascade (may be multiple steps of amplification; the more steps in such the cascade, the greater the signal amplification) Enzymes – multiple products (amplification)

Answer 115

Epinephrine activation of the fight-or-flight response: Liver and skeletal muscle – increase in glucose for energy production Adipose – breakdown of triglycerides for energy production Cardiac muscle – increased heart rate – O2 and glucose transport to peripheral tissues Smooth muscle cell relaxation (not shown) – increase blood flow and air intake Intestine – fluid secretion (use your imagination)

Answer 116

Steps 1) MAP kinase in the cytosol phosphorylates and activates the kinase p90RSK, 2) p90RSK then moves into the nucleus and 3) once in the nucleus - phosphorylates the SRF (serum-response factor) transcription factor. 4) MAP kinase translocates into the nucleus and 5) directly phosphorylates TCF transcription factor already bound to the promoter of the c-fos gene. 6) Phosphorylated TCF and SRF together stimulate transcription of c-fos and other genes that contain a promoter SRE (serum response element) sequence. MAP kinases phosphorylate over 175 target proteins in the nucleus.

Answer 117

Step 1: Ligand binding induces receptor activation conformational change. Step 2: Activated receptor binds to trimeric G protein. Step 3: Activated receptor GEF activity stimulates Gα subunit release of GDP. Step 4: GTP binding changes Gα conformation Dissociates Gβγ (Gβγ subunit activates other effector enzymes in some pathways (b)) Activates Gα Step 5: Gα·GTP activates effector enzyme. Step 6: Gα intrinsic GTPase activity hydrolyzes GTP to GDP – dissociates Gα and turns off effector enzyme. (G protein active for minutes or less)

Answer 118

1) RTK activation leads to the auto-phosphorylation of the RTK intracellular domain 2) The phosphorylated intracellular domain interacts with adaptor proteins (GRB2, Sos) through the so called SH2 and SH3 domains (standing for Src-Homology Domains) 3) Sos activates Ras (binding of GTP) 4) Ras activates Raf (MAP Kinase Kinase Kinase) 5) Raf activates MEK (MAP Kinase Kinase) 6) MEK activated ERC (MAP Kinase)

Answer 119

Receptor tyrosine kinases activate Ras via adapter proteins. 1) Binding of FGF hormone causes receptor dimerization, kinase activation, and phosphorylation of cytosilic receptor tyrosine kinases A specific phosphotyrosine on an activated, ligand-bound receptor provides a specific site for binding the cytosolic adapter protein GRB2 SH2 domain. 2) GRB2 SH3 domains specifically bind proline domains on SOS, recruiting SOS to the membrane to interact with Ras∙GDP (tethered to the cytosolic surface of the plasma membrane by a hydrophobic farnesyl anchor). (Binding of GRB2 and Sos couples receptor to inactive Ras) (3) The SOS guanine nucleotide exchange factor (GEF) activity promotes release of GDP and formation of active Ras∙GTP. Sos promotes dissociation of GDP from Ras; GTP binds and active Ras dissociates from Sos

Answer 120

Part of Grb2 protein involved in Ras activation SH2 domain associates with phospho-tyrosine present on ligand-activated receptors of the RTK

Answer 121

part of Grb2 in the Ras activation domain binds specifically to proline-rich sequence in Sos

Answer 122

is a protein involved in ras activation - Has SH2 and SH3 domains - Joins active receptor (RTK) to cytoplasmic Sos (brining it closer to the plasma membrane)

Answer 123

Sos is a GEF involved in Ras activation Sos gets recruited by SH3 domain of Grb2 to be close to the plasma membrane, where it can promote dissociation of GDP from Ras

Answer 124

Stimulated cell: Step 1: Active RTK receptor activates formation of active Ras·GTP. (Ras activated by the exchange of GDP to GTP) Step 2: Ras·GTP triggers the downstream kinase cascade by interacting with the Raf N-terminal regulatory domain, which results in dephosphorylation of one of the serines, release of 14-3-3, and activation of the Raf kinase activity. (Active Ras recruits, binds, and activates Raf) Step 3: Ras GTP hydrolysis to Ras·GDP releases active Raf. Step 4: Raf phosphorylates and activates MEK. Step 5: MEK, a dual-specificity protein kinase, phosphorylates MAP kinase (and ERK kinase) on both tyrosine and serine/threonine residues. Step 6: MAP kinase phosphorylates many different proteins in different cells, including nuclear transcription factors, that mediate cellular responses.

Answer 125

PI-3 kinase! aka Phosphoinositide Signaling Pathway

Answer 126

RTKs and cytokine receptors can initiate the IP3/DAG signaling pathway by activating phospholipase Cγ. RTKs and cytokine receptors can initiate another phosphoinositide pathway by activating PI-3 kinase formation of PI 3-phosphate, which bind PH domains in various proteins to form signaling complexes that activate PKB. Activated PKB promotes survival of many cells by directly inactivating pro-apoptotic proteins and synthesis of pro-apoptotic proteins.

Answer 127

PhosphoInositol 3,4,- biphosphate (IP2) PhosphoInositol 4,5,- biphosphate (IP2) PhosphoInositol 3,4,5- triphosphate (IP3

Answer 128

Full activation is achieved by phosphorylation of the membrane-bound PKB by two other kinases, 3-phosphoinositide dependent protein kinase 1 and 2 (PDK1 and PDK2)

Answer 129

Unstimulated cells: PKB is in the cytosol with its PH domain bound to and inhibiting its catalytic kinase domain. Hormone stimulated cells: Inactive PKB is in the the cytosol of unstimulated cells Step 1: Hormone stimulation leads to activation of PI-3 kinase, which forms PI 3-phosphates (phosphorylates 3 position of PI to form PI 3,4 bisphosphates) Step 2: The 3-phosphate group binds the PH domains of PKB (partially active) and PDK2, docking both to the membrane and partially activating PKB Step 3: PDK1 phosphorylation of the PKB activation loop serine and PDK2 phosphorylation of a PKB C-terminus serine fully activates PKB activity, which induces many cellular responses.

Answer 130

All G protein–coupled receptors have: The same orientation in the membrane – N-terminus outside, C-terminus in cytosol Contain seven transmembrane α-helical regions (H1–H7) Have four extracellular segments (E1–E4) Four cytosolic segments (C1–C4)

Answer 131

Ligand binding causes movement of the receptor TM5 and TM6 helices and changes in the C3 loop to create a surface that forms extensive interactions parts of Gαs Gαs small conformational change Lengthening the α5 helix creates a large surface consisting of the N-terminal α-helical segments αN and α5 that bind mainly to receptor TM5 and TM6. Triggers release of GDP and Gβγ (c) Binding of epinephrine to the side chains of multiple amino acids in the interior of the β2-adrenergic receptor

Answer 132

Forster resonance energy transfer (FRET) technique: Wavelength of emitted fluorescence changes when two fluorescent proteins interact. Reveals kinetics of G protein activation Mechanism of detection of protein interactions IN VIVO One protein is tagged with Gα-cyan fluorescent protein (CFP) Another protein is tagged with Gβ-yellow fluorescent protein (YFP, excited by CFP (cyan fluorescent protein) when alpha subunit is bound to beta subunit On the left: The two proteins are close to each other (INACTIVE RECEPTOR) Then the light emitted by CFP (blue) excites YFP and WE OBSERVE YELLOW LIGHT On the right: The two proteins are away from each other (ACTIVE RECEPTOR) We observe only the light emitted by CFP (blue) WE ONLY OBSERVE CYAN LIGHT

Answer 133

PGE and adenosine are inhibitory hormones and they bind to the inhibitory receptor which activates G protein complex and binds and causes the inactivation of adenylyl cyclase which causes the down regulation/lack of production of cAMP (seesaw looking diagram)

Answer 134

Epinephrine and Glucagon these are stimulatory hormones that bind to receptors causes activation of a stimulatory G protein complex that binds and activates adenylyl cyclase and causes production of cAMP (seesaw looking diagram)

Answer 135

Hydrophilic signaling molecules (hormones or peptides) activate Receptor Tyrosine Kinases (RTKs), which phosphorylate downstream targets and initiate a signal transduction cascade RKTs have a different mechanism of action as compared to GPCRs Usually the hormone or peptide leads to dimerization of two kinase polypeptides The dimerization of RTKs leads to cytoplasmic domain autophosphorylation, which recruits proteins that initiate intracellular signaling pathways

Answer 136

1) receptor associated kinases 2) cytosolic kinases 3) protein subunit dissociation pathways 4) protein cleavage pathways

Answer 137

Many receptor cytosolic domains are protein kinases or tightly associate with a cytosolic kinase. (1) Ligand binding causes receptor dimerization which activates kinase activity: Kinases directly phosphorylate and activate transcription factors or other signaling proteins. (2) Many receptors also activate small GTP-binding “switch” proteins such as Ras. The Ras signal transduction pathway and others activate a kinase cascade, in which one kinase phosphorylates and thus activates (or occasionally inhibits) the activity of multiple copies of another kinase for signal amplification. G-protein pathway kinases phosphorylate multiple protein targets, including cell type-specific transcription factors.

Answer 138

Seven membrane-spanning segments receptors activate GTP-binding Gα proteins. Gα-GTP activates specific kinases or other signaling proteins.

Answer 139

Several signaling pathways cause disassembly of a multiprotein complex in the cytosol, which releases a transcription factor that translocates into the nucleus.

Answer 140

irreversible signaling Proteolytic cleavage of a receptor releases an active transcription factor.

Answer 141

The binding sites for the factor and the gene's epigenetic state and on the presence of master transcription factors and other nuclear proteins

Answer 142

are soluble or membrane-bound peptide or protein hormones: Growth factors: NGF - nerve GF PDGF - platelet-derived GF FGF - fibroblast GF EGF - epidermal GF

Answer 143

RTK activation mechanism: 1) (–) ligand: RTKs generally exist as monomers with poorly active kinases. (2) (+) ligand: Two ligands binding to two RTK extracellular domains homodimerizes the receptors, bringing together two poorly active cytosolic domain kinases, which phosphorylate each other on an activation loop tyrosine residue. The phosphorylated loop moves out of the kinase catalytic site, increasing ATP and/or the protein substrate binding. (3) The activated kinase phosphorylates several tyrosine residues in the receptor’s cytosolic domain, which provides docking sites for SH2 and other binding domains on downstream signal-transducing proteins.

Answer 144

(a) (–) EGF: β-hairpin from domain II that forms the “dimerization arm” binds to domain IV of the same receptor molecule, inhibiting dimerization. (b) (+) EGF: EGF binds domains I and III, inducing an extracellular domain conformational change that exposes the domain II dimerization arm. (c) Dimerization arms interactions dimerize two identical ligand-bound receptor monomers.

Answer 145

The formation of an asymmetric kinase domain dimer The EGF receptor kinase domain is activated by phosphorylation of an activation loop tyrosine. Inactive, monomeric state: activation loop binding in the kinase active site inhibits kinase activation. Receptor activation mechanism: The dimer is stabilized by interactions between the juxtamembrane segments of the two receptors. Dimerization generates an asymmetric kinase dimer in which the donor kinase C-terminal C-lobe binds the acceptor kinase N-terminal N-lobe. Interactions cause a conformational change that removes the activation loop from the acceptor kinase site, activating its kinase activity. The active acceptor kinase phosphorylates cytosolic domain tyrosine residues on both receptors

Answer 146

a sequence of ordered events these events are controlled by CDKs consists of G1, S, G2 and mitosis phases

Answer 147

Cyclin Dependent Kinases (CDK) The key regulatory subunits of CDKs are called Cyclins Other regulatory mechanisms involve synthesis and degradation of CDK, inhibitors or activating or inhibitory phosphorylation of the CDK

Answer 148

Each Cyclin acts at a specific stage of the cell cycle and sets up the transition to the next stage, then gets degraded

Answer 149

there are surveillance mechanisms that check for the proper progression through the cell cycle. If anything goes wrong, these mechanisms, called checkpoints, hold the cycle until repair is done

Answer 150

The decision to divide is tightly controlled at the end of G1. Once this decision is made, the cell must divide or die.

Answer 151

Make sure that the stage of the cell cycle is completed act as brakes on cell cycle progression when events are not completed or when DNA is damaged

Answer 152

models for studying the cell cycle unicellular organisms, easy to grow and synchronize rapid cell cycle (2-4 hrs) cell cycle stage visualized by the morphology of the cell can be grown as haploid cells (amazingly simple genetics) conditional loss of function (ts) mutants display distinct morphology

Answer 153

G1 – growth to size large enough to form viable bud; irreversible commitment to progression through cell cycle is made at the G1 START point; bud begins to emerge S – DNA replication (1n to 2n in haploid organism) G2 – less distinct stage in yeast than in higher eukaryotes M – intranuclear spindle; nuclear envelope does not break down in yeast

Answer 154

cell cycle mutations are easily generated and recognized by differences in cell size G1 grow longer before cell division. G1 START point – irreversible commitment to cell division S − nuclear envelope does not breakdown

Answer 155

Model organisms have revealed that: A few master cell cycle regulatory proteins control and coordinate processes such as DNA replication initiation and entry into mitosis. Master regulators and the proteins that control them are highly conserved across eukaryotic cells

Answer 156

progesterone stimulates maturation of Xenopus oocytes Model cell − large enough for biochemical characterization of cell extracts Progesterone treatment causes G2-arrested Xenopus oocytes to enter meiosis I, a modified form of mitosis. Homologous chromosome segregation and asymmetric cell division expels one set of chromosomes into the first polar body. Oocyte arrests in metaphase II to yield an egg. When fertilized by sperm, egg proceeds through meiosis II anaphase and a highly asymmetric cell division expels one chromatid of each chromosome into a second polar body. The haploid female and sperm pronuclei fuse to produce a diploid nucleus. Diploid zygote proceeds through cell cycle to divide by mitosis into two cells. 11 more synchronous divisions produce blastula.

Answer 157

HeLa Kyoto cells seen under phase contrast microscopy were filmed as they underwent nitosis. HeLa Kyoto cells − are flat during interphase but round up during mitosis and cell division. Daughter cells flatten again. Normal primary human cells and other mammalian cells have a finite life span when cultured in vitro. Normal human cells divide 25–50 times before undergoing replicative senescence. Immortalized cell lines, some of tumor origin such as the HeLa cells, harbor genetic alterations that enable them to escape replicative senescence and become immortal.

Answer 158

Mating pheromone arrests the cells in late G1 Nocodazole disrupts mitotic spindle – cells arrest in pro-metaphase

Answer 159

Hyproxyurea and nocodazole

Answer 160

Anti mitotic agent used in yeast and humans that reversibly interferes with polymerization of microtubules it disrupts the mitotic spindle

Answer 161

a cell synchrony agent used in yeast cells that arrests the cells in late G1

Answer 162

aka thymidine is an antimetabolite medication that inhibits the synthesis of dNTPs - CAUSES CELL AREST IN EARLY S PHASE USED IN MAMMALIAN CELL CULTURE

Answer 163

the amount of cellular DNA

Answer 164

Fix cells by Ethanol (Permeabilizes and preserves cells) Degrade all RNA (RNAse) Soak the cells with a DNA staining dye (propidium Iodine) Wash the cells Run cell suspension through a capillary tube, one cell at a time Use lasers to detect the amount of stain (indirect measure of the amount of DNA) in each cell Plot the results

Answer 165

Researchers use multiple tools to study the cell cycle. DNA in yeast cells stained with propidium iodide fluorescent dye can be quantified by flow cytometry. DNA content analysis shows three populations of cells: cells with unreplicated DNA (1C), cells with replicated DNA (2C), and cells in the process of DNA replication (S phase).

Answer 166

Isolated temperature sensitive (ts) mutants not growing at higher temperature (arrest with certain morphology) Treat yeast w mutagen Grow colonies (23*C) Check which colonies do not grow at higher temperature (these are called TS - temperature sensitive mutants) Check morphology of TS mutants (the morphology tells you at which state of the cell cycle the mutated gene works) Isolate and analyze the gene

Answer 167

temperature sensitive cell division cycle mutants In ts mutants grown at restrictive temperature, cell morphology (presence and size of bud) indicates at which stage of the cell cycle the mutants gene works. Position in the cell cycle can be determined by morphology

Answer 168

mitosis at size larger than normal delayed progress through cell cycle (fission yeast)

Answer 169

mitosis at size smaller than normal advanced progress through cell cycle

Answer 170

Cyclin levels rise during interphase peak at mitosis and drop before anaphase

Answer 171

samples collected at different times post-fertilization (every 10 min) identified a protein that was continuously synthesized and periodically destroyed cyclin destruction occurred just prior to onset of mitotic cleavage (anaphase)

Answer 172

cdc28, cdc2, Cdk1 and MPF (cdk1)

Answer 173

Early G1 − no cyclin − CDKs are active Mid-G1 − G1/S phase CDKs activate transcription of genes required for DNA replication S phase − initiated by SCF ubiquitin-protein ligase ubiquitinylation of S phase CDKs inhibitors, marking them for degradation by proteasomes. S phase CDKs activate DNA replication. Late G2, mitotic CDKs trigger entry into mitosis. M Anaphase − anaphase-promoting complex (APC/C) ubiquitinylates: The anaphase inhibitor protein securin, marking it for degradation by proteasomes, activating securin to degrade cohesin complexes linking the sister chromatids. Separation of sister chromatids initiates anaphase. Mitotic cyclins, targeting them for degradation by proteasomes. The resulting drop in mitotic CDK activity and protein phosphatase activity results in chromosome decondensation, reassembly of nuclear membranes around the daughter-cell nuclei, and cytokinesis.

Answer 174

CDKs are active only in the stages of the cell cycle they trigger: G1/S phase CDKs are most active at the G1–S phase transition to trigger entry into the cell cycle. S phase CDKs are most active during S phase. Mitotic CDKs are most active during mitosis.

Answer 175

The anaphase promoting complex ubiquitin protein ligase - ubiquitinylates proteins, including cyclins, targeting them for degredation by proteosomes - catalyzes two key cell cycle transitions: anaphase and exit from mitosis

Answer 176

The activity of the different cyclin-CDK complexes varies in a manner that is very similar, but not exactly identical to the accumulation of the corresponding cyclins: Conclusion: There are other regulators of CDK activity

Answer 177

1) Kinases and Phosphatases 2) inhibitory proteins 3) ubiquitin-protein ligases

Answer 178

CDK inhibitors (INKs, CIPs, KIPs) associate with CDK/Cyclin complexes and inhibit them until the previous stage of the cycle is completed. After the completion, the active CDK/Cyclins phosphorylate the CDK inhibitors. These inhibitors are then degraded. The inhibitor degradation provides a clean molecular trigger of the next stage of the cycle. Next, the cyclins from the previous stage are degraded via protein ubiquitination. The protein-ubiquitin ligases are: SCF - works at the transition from G1 to S phases APF - works at the onset of mitosis (anaphase)

Answer 179

A protein ubiquitin ligase works at the transition from G1 to S phases Degrades phosphorylated S phase inhibitor

Answer 180

a protein ubiquitin ligase used during metaphase/anaphase transition AND at the mitotic exit/end of anaphase degrades securin degrades mitotic cyclins

Answer 181

Kinases and Phosphatases Inhibitory proteins ubiquitin-protein ligases and Phosphoserine/Threonine Binding proteins and modular domains

Answer 182

Wee1 kinase phosphorylates Y (tyrosine) and T(threonine) residues on CDK and inhibits the mitotic kinases in S-phase. The dual-specificity Wee1 kinase inhibits mitotic cyclin-CDK activity by phosphorylation of CDK T14 and Y15 until DNA replication is completed

Answer 183

removes the inhibitory phosphorylation of Y and T residues previously phosphorylated by Wee1 and activates Mitotic CDKs When DNA replication is completed, the dual-specificity Cdc25 phosphatase is activated (by an unknown mechanism) and dephosphorylates CDK T14 and Y15 , reversing the inhibitory effect of Wee1 and activating cyclin-CDK activity.

Answer 184

They activate mitotic CDKs

Answer 185

Phosphorylates Cdc25, activating its CDK-activating phosphatase activity Phosphorylates Wee1, inhibiting its CDK-inhibiting kinase activity

Answer 186

inhibits Wee1 activity when a cell has reached an appropriate size to divide

Answer 187

The decision whether to divide or not is made in late G1 phase: RESTRICTION (or START) point This decision is tightly regulated Cells that never divide (highly differentiated cells) arrest before this START point in G0

Answer 188

Whi5 SBF Rb E2F

Answer 189

Whi5 and SBF! Whi5 is on top of SBF and it is a transcriptional repressor - inhibits SBF activation of the CLN1/2 cyclin gene transcription Nutrient availability allows translation of Cln3 mRNA, activating Cln3-CDK activity. Cln3-CDK phosphorylates Whi5, promoting its export from the nucleus. When 50 percent of Whi5 has been exported, cells transition through START. Active SBF induces transcription of CLN1 and CLN2 and other genes whose products are needed for DNA replication. G1/S phase CDKs further phosphorylate Whi5, promoting further CLN1 and CLN2 transcription. High levels of G1/S phase CDKs initiate DNA replication, bud formation, and spindle pole body duplication

Answer 190

G1 − The E2F transcription factor and its regulator Rb control the G1–S Phase transition Rb binds and inhibits E2F activity. Growth factors (mitogens) stimulate G1 CDK activity. Cyclin D–CDK4/6 complexes phosphorylate Rb, releasing E2F. E2F stimulates transcription of early response genes encoding cyclin E, CDK2, and E2F. Cyclin E–CDK2 further phosphorylates Rb, initiating a positive feedback loop that leads to a rapid rise in the expression and activity of both E2F and cyclin E–CDK2. G1/S phase cyclin E/A-CDK2 CDKs support cell passage through the restriction point and initiation of DNA replication and centrosome duplication.

Answer 191

No! All cyclins have been degraded at the end of mitosis by the APC

Answer 192

to increased synthesis of cyclin Cln3 in early G1

Answer 193

regulator of E2F transcription factor binds to E2F and suppreses the transcription of cyclin E or cyclin A in mammals in early G1

Answer 194

Cyclin D (CDK4/6)

Answer 195

Sic1 is an S phase inhibitor (it's attatched to S phase CDK/cyclin) it prevents initiation of DNA replication ununtil the cells have completed all G1 events 1) G1/S phase CDKs assembled in late G1 phosphorylate Sic1 at multiple sites, marking it for degredation 2) SCF ubiquitin-protein ligase ubiquitinylation of SCF and subsequent proteasomal degradation 3) The active S phase CDKs trigger initiation of DNA synthesis by phosphorylating and recruiting MCM helicase activators to DNA replication origins.

Answer 196

G1/S phase CDK phosphorylation of a single optimal Sic1 site would cause a sluggish G1/S phase transition. G1/S phase CDKs accumulation during G1 would cause slow progressive degradation of Sic1 and initiation of S phase.  slow Slc1 degradation  slow S phase CDK activation  sluggish G1 – S phase transition (c) Six suboptimal phosphorylation sites ensure that Sic1 is fully phosphorylated and targeted for destruction by SCF only when G1/S phase CDKs have reached high levels. This ensures that Sic1 degradation and DNA synthesis initiation occurs rapidly, when G1/S phase CDKs have accomplished all their other G1 tasks. Six sub-optimal sites of phosphorylation of Sic1  fast Sic1 degradation  fast S phase CDK activation  optimized G1 – S phase transition

Answer 197

Step 1: Exit from mitosis and early G1 the origin recognition complex (ORC) is recruited and associated with all DNA replication origins Step 2: Late G1 CDK activity is still low and the Cdc6 and Cdt1 MCM loading factors associate with ORC and load the INACTIVE MCM replicative helicase complex onto each DNA replication origin. The Pre-replication complex is formed Step 3: DDK and S phase CDKs are activated. They phosphorylate: MCM → activation of helicase fires up the origin of DNA replication Cdc6 and Cdt1 → degradation (after firing there will be no loading of MCM on the newly replicated origins) CDC45, Sld2 → Recruit GINS to MCM to facilitate elongation replication. Step 2: Late G1 - DDK and S phase CDKs are activated. DDK phosphorylates two subunits of the MCM helicase. S phase CDKs phosphorylate Sld2 and Sld3. These phosphorylation events promote recruitment of Cdc45-Sld3 complex and the GINS complex MCM helicase activators to sites of replication initiation, which leads MCM helicases to unwind DNA. (S phase CDKs prevent reloading of MCM helicases by phosphorylating Cdc6 and Cdt1, promoting their release from the replication origins and degradation by SCF.) S phase CDKs also phosphorylate MCM helicases, causing the helicases to disengage from the DNA when replication is complete and export from the nucleus. Step 3: DNA polymerases recruited to origins initiate DNA replication

Answer 198

They load MCM onto the origin of replication

Answer 199

Checkpoints ensure that the next stage of the cell cycle does not initiate prior to the completion of the preceding one. Checkpoint pathways arrest the cell cycle progression in response to DNA damage or improper spindle assembly. so we don't make bad cells

Answer 200

- event sensors - a signaling pathway - and effector that halts cell cycle progression

Answer 201

1) Damaged DNA activates ATM, ATR (protein kinases) and DNA-PK 2) DNA-PK, ATM and ATR phosphorylate and activate the chicks! (Chk1, Ck2) and MK2 (Chk1 and Chk2 are checkpoint kinases) 3) Chk1 and Chk2 inhibit/block cdc25, which induce DNA repair machinery! (cdc25 normally would activate mitotic CDKs but now that it's inhibited, it can't) 4) This inhibits mitotic CDKs and causes cell cycle arrest

Answer 202

Cells require TROPHIC FACTORS! that bind to surface receptors to repress apoptosis for survival

Answer 203

they degrade cellular proteins and apoptosis involves the activation of caspase protease

Answer 204

Bone morphogenic proteins - expressed by interdigital cells - induce apoptosis (Dominant negative type I BMP receptor blocks BMP signal and apoptosis (like in the diagram with the hands and one's webbed) (BMPs – not expressed in duck interdigital cells) Conclusion: BMP signaling mediates interdigital cell death in the embryonic limb.

Answer 205

MESSY CELL DEATH Different than apoptosis cell rupture, cellular contents pouring out leading to inflammatory response, auto-immunity (messy cell death)

Answer 206

Necrosis: cell rupture, cellular contents pouring out leading to inflammatory response, auto-immunity (messy cell death) Apoptosis: cell membrane remains intact, cell is dismantled and packaged into apoptotic bodies, cellular contents within vesicles, absorbed by macrophages and recycled (clean cell death)

Answer 207

Mild convolution Chromatin campaction and margination Condensation of cytoplasm Breakup of nuclear envelope Nuclear fragmentation Blebbing Cell fragmentation INTER_NUCLEOSOMAL DNA FRAGMENTATION IS A FEATURE OF APOPTOTIC CELLS Apoptotic bodies are produced Phagocytised Apoptotic stimuli triggers DNA degrading nucleases that cleave DNA between nucleosomes Degraded chromatin is packaged into apoptotic bodies The apoptotic bodies contain less that 2n DNA as compared to normal living cells

Answer 208

- Flow cytometry and COMET assay

Answer 209

You detect these apoptotic bodies as a broad peak to the left of the C1 peak since they have much LESS DNA THAN NORMAL CELLS

Answer 210

Cells are imbedded in agarose Electric current is passed through the agarose Similarly to gel electrophoresis, DNA fragments will move because of their charge Large fragments of DNA (intact chromosomes) do not move Fragmented chromosomes leave the cell and produce a “comet-like” tail. The detection of such tails is indicative of apoptosis.

Answer 211

S. Brenner: introduced Caenorhabditis elegans as a model organism to study development Small nematode roundworm, ~1.2 mm long, transparent Consists of 1090 somatic cells produced during development Of these, 959 cells survive, 131 “doomed” cells die by apoptosis

Answer 212

determined complete cell lineage map

Answer 213

cell death genes

Answer 214

R. Horvitz isolated > 4000 cell lineage (lin) mutants that do not kill some of the 131 cells He postulated that the mutant genes must be responsible for the killing Conclusion: Cell death is genetically controlled - Led to search for cell death (CED) genes

Answer 215

ced-1 mutant: apoptotic cells are not engulfed and digested (arrows) ced-1/ced-3 double mutant: apoptotic cells do not appear They do not die!!! ced-3 is necessary for apoptosis (pro-apoptotic gene)

Answer 216

Initiator caspases: process and activate effector caspases - casp9 (intrinsic) - casp8 and 10 (extrinsic) Effector (executioner) caspases: cleave specific cellular proteins leading to apoptosis - caspase 3, 6, 7 these caspases digest the proteins of the cell and activate nucleases that degrade DNA and chromatin

Answer 217

The release of Cyt C is regulated by pro-apoptotic and anti-apoptotic proteins Pro-apoptotic proteins: Bak, Bax, Boc Pro-survival proteins: Bcl-2, Bcl-xl All these proteins are homologous to Bcl-2 and contain Bcl-2 Homology domains (BH1, BH2, BH3, BH4) BH3-only proteins: (Bad, Bim, Puma) Regulate pro-survival proteins

Answer 218

Bad, Bim and Puma - regulate pro-survival proteins (Bcl-2)

Answer 219

Play central anti-apoptotic (pro-survival) or pro-apoptotic roles in apoptosis Contain one or more functional Bcl-2 homology domains (BH1–4) All participate in oligomeric interactions. Three classes – one anti-apoptotic and two pro-apoptotic: Only some BH3-only proteins contain a transmembrane (TM) domain that anchors other Bcl-2 family proteins in the outer mitochondrial membrane. Pro-apoptotic proteins Bax and Bak form pores in the outer mitochondrial membrane large enough for cytochrome c escape to the cytoplasm.

Answer 220

Intrinsic Death Pathway activated by proteins that reside in the mitochondria (Bak, Bax, Bcl-2, Cytochrome C (CytC) activated in response to DNA damage, cell stress, lack of adhesion, lack of trophic signals Extrinsic Death Pathway activated through cell surface death receptor activated by direct contact with other cells that direct them to self-destruct by apoptosis

Answer 221

DNA damage or ultraviolet irradiation stimulates apoptosis: - Induces synthesis of Puma BH3-only protein - Puma binds to Bak, Bax, and Bcl-2. - Bak and Bax form oligomeric pores that release Cyt c.

Answer 222

Cell lack of adhesion to substrate – apoptosis in adhesion-dependent cells: - Disruption of integrin signaling - Release of the Bim BH3-only protein from the cytoskeleton - Bim binds to Bak and Bax to promote pore formation and Cyt c release.

Answer 223

Pro-survival proteins (Bcl-2, Bcl-x) are abundant. They bind to pro-apoptotic proteins (Bak or Bax, less abundant), prevent oligomerization and close pore

Answer 224

Bad, Bim, Puma, when activated, displace Bcl-2 from Bak or Bax and allow to open the pore

Answer 225

Cyt C is released, binds to Apaf-1 and activates Caspase 9, which activates the executioner caspases (in intrinsic pathway)

Answer 226

The progression of cancer from a normal somatic cell to a full-grown tumor can be thought to take place in four stages. Step 1: Initiation. Except in rare cases in which cancer is caused by a singular genetic event, such as infection by a tumor virus, we usually cannot pinpoint the initiating event but a somatic mutation is usually suspected. Step 2: Cancer progression. Precancerous cells acquire mutations that dysregulate growth-control pathways, causing inappropriate cell proliferation. Most cancers progress by a process of natural selection, sequentially acquiring multiple such mutations in different pathways. Step 3: Evasion of cancer cell elimination. Precancerous cells are normally removed from the body by either apoptosis or immune-surveillance processes. To progress beyond this stage, tumor cells must acquire additional somatic mutations that allow them to evade these systems. Step 4: Tumor growth and dispersal. Continued growth of a tumor requires a blood supply and tumor cells must acquire yet more changes that promote angiogenesis. To spread throughout the body by metastasis, cells from a solid tumor must acquire the ability to migrate from their original site and adhere at a new location in the body.

Answer 227

Genome destabilization P53 Inhibitors of CDKs Sensors of DNA damage (ATM, ATR) DNA repair genes

Answer 228

- G1/S Cyclins or CDKs - Regulators of G1/S (Rb protein) - Growth Factor Receptors and Signal transduction proteins

Answer 229

Degrade the basement membrane Migrate on extracellular matrix (ECM) fibers away from the primary tumor to reach the blood vessels Can be attracted by signals such as epidermal growth factor (EGF), which can be secreted by macrophages Penetrate the blood vessel endothelial cell layer that forms the vessel walls and enter the bloodstream

Answer 230

Carcinoma cells penetrate the ECM and blood vessel wall: Use the actin cytoskeleton to extend invadopodia Produce matrix metalloproteases and other proteases that degrade basement membranes to open a path for metastasis

Answer 231

circulating tumor cell adheres to the blood vessel lining in a new location and migrates through it to colonize a new tumor in the underlying tissue. Fewer than 1 in 10,000 cells that escape the primary tumor survive to form a secondary metastatic tumor

Answer 232

Individual chromosome number is altered Composite chromosomes composed of pieces from different chromosomes

Answer 233

- causes the mutated cell to divide to form a localized polyp of benign tumour cells

Answer 234

- Loss of APC tumor-suppressor gene (chromosome 12) - activation of K-Ras oncogene (chromosome 12) - Loss of p53 tumor-suppressor gene (chromosome 17) - Other changes that in combination can lead to tumor cells invading blood vessels, allowing metastasis to occur

Answer 235

1) The active receptor phosphorylates inactive SMAD in the cytosol, causing it to oligomerize and activate 2) Active Smad translocates into nucleus where it binds to the following genes to induce their transcription

Answer 236

Smad stimulates expression of: 1) p15 (which inhibids CDK4) - this causes cells to arrest in G1 2) An inhibitor of a protease that degrades extracellular matrix - cells do not migrate.

Answer 237

changes in the body not a part of reproduction (ex. growth of pubic hair, muscle in boys and breasts in girls)

Answer 238

onset of menstruation (usually 12)

Answer 239

strategy of dealing with emotions where we learn to look at our experience through a different frame - like turning something negative into a learning experience

Answer 240

putting off immediate temptations in order to focus on long term goals

Answer 241

abstract reasoning and logic and higher cognitive abilities (starts to go up after age 12)

Answer 242

moral development learning right from wrong

Answer 243

preconventional conventional postconventional

Answer 244

- people reason based on self-interest, avoiding punishment

Answer 245

people reason based on social conventions (traditions), can involve authority figures "Because mom said so"

Answer 246

Level of reasoning that considers justice and fairness

Answer 247

windows of time during which exposure to a specific type of environmental stimulation is needed for normal development of a specific ability ex. becoming fluent in a language --> should be introduced to that language in really early years

Answer 248

fitting new information into the belief system you already posses ex. all girls have long hair - that person has short hair but is a girl! (azlan)

Answer 249

creative process where people modify their belief structures based on experience ex. that girl has short hair, so girls can have short hair

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