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Mol. Cell Bio > Ch 12: Cell Nucleus and Gene Expression > Flashcards

Ch 12: Cell Nucleus and Gene Expression Flashcards

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1
Q

Inducible operon

A

the addition of a compound increases expression of the operon

example = the addition of lactose in minimal media upregulating the expresssion of beta galactose

1) inducer binds to the repressor protien
2) This prevents attachment to the operator
3) RNA polymerase transcribes the gene
4) genes transcribed and translated
5) lactose degraded by enzymes
6) fall in lactose concentration permits the repressor to bind and the operon is repressed

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2
Q

repressible operon

A

The addition of a compound represses the activity of the operon

example = when tryptophan is present, acts as a co-repressor and shuts off the operon

1) corepressor bind to the repressor protein
2) repressor binds to the operator
3) RNA pol is now unavalible to bind
4) Repressor inactive when tyrptophan levels fall
5) genes are transcribed
6) enzymes translated
7) tryptophan synthesized

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3
Q

4 key components of a bacterial operon

A

promoter

operator

repressor protien

regulatory gene

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4
Q

Positive control by cAMP

A

bacteria preferentially catavolize glucose over lactose since glucose suppresses the production of enzymes for other substrates

cAMP high when glucose levels are low

  • > cAMP binds to cAMP receptor protein (CRP)
  • > cAMP CRP complex binds to site on lac operon (positive regulator)
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5
Q

Import/ export across the nuclear envelope

A

proteins imported from the cytoplasm

mRNAs, tRNAs, ribosomes subunmits exported

snRNAs exported and snRNPs imported

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6
Q

Nuclear lamina

A

mechanical support and the site of chromatic attachment

assemnbly/ disassembly regulated by phosphorylation levels (cyclin dependent kinases)

-> this also applies the ht nuclear matrix proteins

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7
Q

Hutchinson-Gilford Progeria syndrome

A

HGPS

in fibroblast cells

synonymous mutation in a lamin A gene
-> causes splicing defect and truncated protein

causes premature aging, and leads to a lumpy nuclear envalope and asplicing defect leading to a truncated protein

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8
Q

Cytoplasmic face of the nuclear pore complex

A

composed of nucleoporin proteins

nuclear targeted protiens have nuclear localization signals (NLS)

NLS has short stretches of positively charged amino acids
(Pro-lys-lys-lys-arg-lys-val)

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9
Q

Process of importation of protiens into the nucleus

A

1) NLS containing cargo protien binds to a heterodimeric soluble NLS receptor called importin alpha/beta
2) protien escorted to outer surface of nucleus-docks with cytoplasmic filaments
3) filaments bend towards nucleus delivering complex to specific binding sites on NPC
4) Ran-GTP binds to importin/NLS and causes disassembly-imported cargo released

5) Part of NLS receptor (importin beta) shuttled back to cytoplasm with Ran-GTP
- > Ran-GTP hydrolyzed and released (Ran-GDP)

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10
Q

Chromosome structure

A

Average human cell conatins 6 billion base pairs of DNA
-> ~2 m of DNA

Chromosomes composed of DNA and protein (chromatin)

  • > histones-small basic proteins (+ve AA on histone bind to -ve charge on the DNA backbone)
  • > non-histone proteins-diverse size and function

proteins aid in packing of DNA

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11
Q

Nucleosomal Organization

A

Histone octamer comprised of 2 H2A, 2 H2B, 2 H3, 2 H4 proteins (8 in total)

beads on a string

Nucleosome core = 8 histone proteins + DNA

this is the first level of packing

histone proteins are HIGHLY conserved, and thus imply a functional role

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12
Q

Nucleosome core particle

A

H-terminal Tail H3

is flexible and can be modified

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13
Q

Higher levels of chromosome structure

A

30nm fiber-interaction between histone proteins of neighbouring nucleosomes

H1 (linker) histones are essential for formation of 30nm fiber

this is the second level of packing

Then, nucleosomes line up end to end in two stacks

alternating nucleosomes interact across helic via linker DNA
_> cohesion ring loops together the 30nm fibre

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14
Q

euchromatin

A

DNA that becomes dispersed during interphase

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15
Q

heterochromatin

A

DNA that remains compacted during interphase

constitutive = permanently compacted

factulative = transiently compacted
-> almost always related to gene expression

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16
Q

X chromosome inactivation

A

X chromosome randomly inactivated at early stage in development
-> done for dosage compensation reasons (females get two)

barr body

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17
Q

Lyon Hyopthesis

A

heterochromatization of female X chromosome occurs during development

heterochromatization is random (paternal or maternal X)

reactivation of x chromosome occurs before meiosis

XIST long non-coding RNA (lncRNA) initiates inactivaiton = guide sequence

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18
Q

Histone code

A

amino (NH2) terminal tails of histone protiens extend out past DNA

enzymatically modified by covalent addition of methyl (Me), acetyl (Ac), or phosphate (P) groups

=> affects the conformation fo the proteins epigenetics

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19
Q

protiens that bind selectively to modified H3 or H4 residues

A

proteins alter the structure and or function of chromatin

modification at one residue can influence events at other residues

=> subsequent recruitment, proteins bind more proteins

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20
Q

activation / deactivation of histones

A

typically:

acetylation = activation

methylation = inactivation

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21
Q

Steps of histone code and heterochromatic formation

A

epigenetics change the expression of DNA

1) RNAs transcribed from both strands of repeated DNA
2) forms dsRNAs
3) Dicer enzyme generates single stranded siRNA guide
4) Histone MTase guided to euchromatic DNA
5) methylation of K9 of H3
6) Binds to HP1 protein, HP1-HP1 interactions
7) Binds associated proteins with chromodomains, Binds to HP1

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22
Q

Mitotic chromosome preparation

A

transfer a drop of blood from a capillary pipette to culture medium
=> conatins substance which simulates mitosis in leukocytes

culture approximately 72 hours, then add colchine for 30min to 3 hours.
=> inductiohn of mitotic arrest

wash with fresh medium. Add hypotonic solution to cells. Let sit for 10 min.
Remove teh supernatent and add cold fixative.

disperse cells and observe karyotype?

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23
Q

Chromosomal aberrations

Inversions

A

Inversions

genetic information retained but can result in abnormal gametes

recombination can result in duplications or deletions of genes

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24
Q

Chromosomal aberrations

Translocations

A

all or one piece of a chromosome becomes attached to another chromosome

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25
Q

Telomeres

A

termial region of chromosomes

contain repeated sequences (TTAGGG) repeated up to 500 times

found in all vertebrate organisms

essential for telomere function

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26
Q

End replication process

A

DNA needs an RNA primer to be made

RNA is degraded Afterwards
-> leads to chromosome shortening

= senescence

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27
Q

Telomere structure

overhanging strands

A

3’ overhang invades DNA duplex and creates loop strucutre

binds telomere specific proteins (Protects telomere)

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28
Q

Telomerase function

A

RNA binds to telomere repeat

serves as template for DNA polymerization

Gap filled by polymerase enz alpha`-primase in germline cells

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29
Q

Hayflick limits

A

the shortest telomare length needed for replicative senescence, without this they die.

cells expressing telomerase are immorrtal and never reach this =- HeLa and cancer cells

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30
Q

Centromeres

A

contain a tandemly repeated 171 bp DNA sequence called alpha satellite DNA

binds to specific proteins
=> CENPA protein

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31
Q

Chromosomes are localized to specific regions of the nucleus

A

related to chromosome activity

more active chromosomes are centrally located, whereas periperhal chromosomes are less activee

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32
Q

interactions between distant genes

A

chromosome capture, transcriptionally active regions are coordinated together (like an operon= expression is corrdenated ) ,

genes are for a singular/ common biological process

33
Q

chromosome capture

A

chromatin is crosslinked with transcriptionally active regions are expressed together. OR formaldehyde is used to cross-link protein and bind these DNA fragments in place while they are being transcribed.

Digest cross linked DNA, and add biotin Tags and ligated together. the proteins binding the fragments together at the trascription regions is removed, biotin pulls down is used to to pull oyut DNA and is then sequenced for bioinformatics

34
Q

Compartmentalization of mRNA processing

A

localized areas of mRNA transcription within the nucleus

corrdenated by nuclear matrix protein.

nuceuls is isolated in detergents and hihg salt conditions. All that is left is the nuclear matrix protiens; how they were discovered. Play an importnat role in transcription corrdenation in the cell

35
Q

Characteristics of nuclear matrix proteins

A

insolube in high salt and high detergent conditions

composed of netword of crisscrossing protien fibrils

anchor for machinery for transcription,m RNA processing

protein structure scaffold

36
Q

Control of gene expression in eukaryotes

A

higher eukaryotes have specialized cells, and specific expression of genes within specilized cells

cells become differentiated

still retain complete geneic information

demonstrated by nuclear transplantation experiments (dolly the sheep)

37
Q

dolly cloneing

A

enucleated egg cells fused with cells derived from mammary gland (epithelial cells)

Reduced serum levels caused epithelial cells to enter quiescent state (G0) the unmasked unexpressed genes

fused cells activated by brief electric pulse

egg implanted in surrogate sheep

38
Q

Levels of control of gene expression

A

Trascriptional: control over whether a specific gene is transcribed and how often

Processing level: How a primary transcript is spliced

Translational level: control over whether a transcript is translated or not and if so, at what levels

Post translational levels: mechanisms that regulate the activity and stability of proteins

39
Q

DNA microarrays

A

yeast cells are grown in two different growth media. then you do a bulk RNA extraction, and synthesize cDNAs with an oligo dt primer (Thiamine nuclotides) when with total RNA they only stick to mRNA because they have poly-A tails. cDNAs are polymerized with fluorescent green dye Cy3 or red Cy5.

Then take the mixed populations of cDNA and analyzed on a microarray = microscope grid with little cells, each cell has a gene, and the samples is hydridized to the cells and will either fluoresce red, green or yellow or nothing dictating which genes are being expressed

can also tell by fluorecense intensity the level of expression

40
Q

Role of transcription factors

A

general transcription factors bind at the core promoter sites

specific transcripiton factors bind to specific regulatory sites in gene
=> also interact with other transcription factors in what is known as cominatorial control

DNA binding and protein-protein binding domains

41
Q

Common TR motifs

A

ZInc finger motif

Helix-loop-helix

Leucine zipper motif

42
Q

Zinc finger motif

A

zinc ion coordinated between tow cysteine and two histidine residues

makes afinger-like structure that fits intothe DNA groove

multiple fingers allow specificity

FTIIIA is a TF with 9 zinc fingers
bound to DNA of 5S rRNA gene

43
Q

Helix-loop-helix motif

A

dimeric MyoD complex

basic aa region binds to negative DNA backbone

dimerization increases diversity of regulatory factors

homo or hetero dimers

Myogenin promoter fused to beta-galacrosidase gene

myoD(HLH TF) is and activator of myogenin

44
Q

Leucine zipper motif

A

Helix with a leucine every 7th amino acid, hydrophobic interactions of leucine zip the two helixes together

basic aa to bind to the backbone

AP-1 is and example heterodimer

45
Q

regulation of PEPCK

A

phosphoenolpyruvate carboxykinase

gluconeogenic gene

converts pyruvate to glucose (sort of ) = oxaloacetate to phosphoenol pyruvate

enzyme synthesized when glucose is low

regulation includees:
=DNA regulatory sequences
=Specific TF
=Signal transductionpathway that activates expressison (hormones)

46
Q

PEPCK regulatory region

A

GRE
TRE
IRE

47
Q

PEPCK regulatory region GRE

A

glucocorticoid response element

binds to glucocorticoid receptor

48
Q

PEPCK regulatory region TRE

A

thyryoid receptor element

binds to thyroid hormone receptor

49
Q

PEPCK regulatory region IRE

A

insulin recepotr element

binds insulin hormone

50
Q

Deletion analysis

A

for analyzing promoters for regulatory elements

Use a reporter plasmid (like GFP) to monitor effect

promoter construsts = fused promoter to a reporter and disect the promoter by removing parts of the promoter = delection constructs

measured by the intensity of GFP fluorescence

51
Q

DNA footprinting assay

A

reveals sights within a promoter that are protein binding sights

Take DNA fragment (thought to be a promoter) and end lable it (P-32)

take nuclear extract (including TF), and mix with labled DNA. TFs will bind to the DNA. Then we treat with DNase I, will digest the DNA but not where the protein is bound.
=> TF will procect from DNA digestion

Fragments are separated by gel electrophoresis

the only DNA fragments that will show up on the gel are those with the end lables

52
Q

Genome wide location analysis

A

treat cells with formaldehyde to kill cells and cross-link transcription factors to DNA

Isolate chromatic and shear it into fragments

incubate TF bound DNA with antibodies that bind the TF of interct

Chromnatic immunoprecipitation (ChIP)

reverse cross-links and puriify DNA

amplify DNA fragments with flurorescent probes and hybridize DNA to a microarray (this time to intergenic// non-coding regions) to find response elements = promoter sequences

53
Q

Activation of GRE by streoid hormone

A

glucocorticoids (chrtisol) secreated during stress

glucocorticoid receptors (GR) binds hormone

Translocated to nuclues

hormone/ receptors dimer binds to GRE palindrome

54
Q

Two classes of transcriptional activators

A

1) interact with casal transcription machinery (RNA pol)

2) act on chromatin to change stas
=> Remodeling complex, histone modificaiton complex

55
Q

Histone modifications

A

acetylation localized primarily int he promoter region of active genes

H3K36 methylation occurs moslty in the transcribed regions

Methylated HK36 recruits Rpd3 enzyme that deactylates lysine residues

Deactylated lysine resides prevents initiation of transcription within the coding region

56
Q

Transcriptional activator (via acetylation of histones)

A

DNA is respressed state = deactylated

GR binds and recruits CPB coactivators (histone acetyltransferase)

lysine redsides acetylates

acetylated histones recrutie SW1/SNF remodeling complex

TFIID binds to open region of DNA-=TAF 250 and RBP acetylate additoinal nucleosomes

RNA pol II binds to promoter

57
Q

Chromatin remodeling complex SW1/ SNF

4 ways it works

A

sliding exposes TATA site

Reorginization of histone octamer provides access to promoter

histone variants exchanged for H2A/ H2B

Histone octamer disassembled

58
Q

Nucleosomal Landscape of Yeast Genes

A

5’ end of gene has highly defined chromatin structure

Necleosome free region (NFR) at transcription start site

-1 nucleosome undergoes most extensive modifications upon transcriptional activation

modificaitons at the beginning and the ending of the gene

59
Q

Paused polymerases

A

RNA pol II held downstream of promoter

held in paused state by inhibitory factors DSIF and NELF

inhibiton relived by phosphorylation and elonation factors ELL

to quickly recruit RNA pol II

transcription factors may act at level of transcritipns elongations

permits rapid activation of genes

60
Q

Transcriptional repression

A

HDAC activitiy deacetylates histones

Histone methytransferase methylates histones
=> Lys 9 of histone H3

figure 12-48

deacytelated and methylated

Greater activity of acetylase (KAT) turns promoter on

Balance between acetylation and deacetylation can cause promoter to be responsive to activation

Greater activity of deacetylase (KDAC) turns promoter off

promoters ina responsive state= balance between on and off (not black and white)

61
Q

Long noncoding RNAs as transcription repressors

A

lncRNAs guide protein complexes to specific sites on chromatin
HOTair lncRNA transcribed form HOXC locus

two main roles

3’ end interactes with the coREST complex to demethyalltes H3K4 LSD1 resides

5’ ends of HOT air interactes with PRC2 complex
and methylates H3 K27 resides

Results in Transcriptional respression of HOXD locus

HOXC gene turns off the HOXD gene

62
Q

DNA methylation

A

epigenetic control of gene exprsssion by methylation of promoters

maintains inactive state

methylation at CG rich islands (Dnmt1 enzyme) (Symmerical recognition site)

63
Q

DNA methylation levels

A

methylation levels change during development

Blastocyst has low level of methylation

adult somatic cells highly methylated

in Dolly they reversed these methylation paterns to get back to the primordial germ cell stage through starvation

64
Q

Genomic imprinting

A

selective methylation of alleles depending on maternal or paternal origin

genes remain imprinted in developing embryo

associated with rare geneic disorders

preder-willi syndrome-deletion of portion of chromosome-15

indiciduals only affected when mutated chromosome inherited from father
=> maternal chromose is methyalted (not active)

65
Q

Processing level control

alternative spicing [fibronectin gene]

A

In fibroblasts both exons EIIB and EIIA are present but not in lever mRNA where they are both missing

alternative splice varients

trans acting factors in different cell types vary

66
Q

Mechanisms of Alternative Splicing

A

a) changes in 5’ splice site can affect pairing with U1 snRNA
=> two potential 5’ splice sites

b) SR proteins bind to Exon or Intorn splicing enhancer (ESE, ISE) sites
=> hnRNP proteins bind to wxon or intron suppressor sites (ESS, ISS)
=> Reguatles whether U2AF, U1snRNP, and U2snRNP bind to pre-mRNA +> influence if cryptic splicing ro exclusion of the exon

67
Q

Splicing and Human diseaae

A

15% of point mutations linked to human disease cause splicing defects

mutations often occur in exons
=> not in splice sites
=>dont create cryptic splice sites

Often cause exon exclusion or inclusion
=> mutations in purine rish ESE sites (exon splicing enchancer sites)
=> also occur in ESS sites (exon splicing supressor sites)

68
Q

Tau isoforms

A

three main transcripts

2kbp in nucleus
6kbp in neurons
9kbp in retina nad peripheral necrvous system

in adult human brain exons 2, 3, and 10 are alternatively spiced

5 isoforms of tau protein

iclusion of exon 10 produces 4R tau isoform
exclusion of exon 10 produces 3R tau isoform

ratio of 3R tau to 4R tau is crucial for microtubule assembly

1:1 ratio required

69
Q

control of mRNA translation

A

translations control
=> mRNA localization (fly embryos)
=> mRNA stability
==> mRNA degredation

posttranslational control
=> protein degradation
=> proteasome structure/ function

70
Q

Cytoplasmic localization of mNA

A

3’ UTR sequence determines location of mRNA

RNA binding proteins bind recognition sequence

Bicoid at anterior end

oskar at the posterior end

swap UTRs and the chromosomes will move in opposite directions

71
Q

look at

A

figure 12-56b

does not matter that you are inhibitng the production of transcripts with actinomycin becuase they are already present

72
Q

Translational activaiotnof mRNAs in Sea Urchin eggs

A

CPEB protein phosphorylated (after fertilizaton)
=> maskin displaced
=> recruites CPSF protein

CPSF protein recruits poly A polymerase
=> dissociation of maskin from eIF4E
=> recruitment of eIF4G (required for translation)\

calcium activated protein kinase = CPEB

masked to unmasked transcripts

73
Q

Gene regulation and enviroment

A

changing cellular environment
=> stress (heat) activates protein kinases
==> Phosphorylates eIF2 (blocks furhter translation)

specific kinases for different stressors
=> heatshock
=> viral infection
=> unfolded proteins
=> amino acid starvation
74
Q

Control of Ferritin mRNA translation

A

ferritin protien binds and blocks toxic effect of iron (iron buffer)

expression increases in response to high iron levels

irons binds the response elements IRP in the inactive state such that translation is active (IRP is repressed//in inactive state)

75
Q

enzymes that degrade mRNA

A

deadenylase degrades poly A tail

decapping enzyme degrades G cap

after poly A tail and methyl guanosine cap are removed, exonuclease degrades mRNA in a 5’ to 3’ direction

3’ tail fold mRNA to accosiated wtih 5’ cap to protect mRNA from degredation

exosome degrades from the 3’ end

76
Q

where does mRNA degredation occur

A

in cytoplasmic granules called P-bodies

77
Q

role of miRNA in translational control

A

miRNA bind to the 3’ UTR to inhibit translation

early embryonic development requires miRNA translation control

Experiments conducted in whihc specific miRNAs are deleted

example
=deletion of miR-1 class of miRNA results in defects in heart development

-> may be due to overexpression of specific mRNAs

78
Q

mechanisms of mRNA reguations by miRNAs

A

deadenylation followed by decaping and degredation

proteolysis: degradaation of nascent peptide

initiation block: repressed cap recognition or 60s joining

elongation block: slowed elongation or ribosome ‘drop-off’

79
Q

Posttranslational control of mRNA

A

proteaseomes digest abnormal proteins
=> misfolded, incorrectly associated

Proteins marked by ubiquitin protein
=> directs protein to proteasome

Mol. Cell Bio (6 decks)

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