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Genetics > M3 L14-15: Bacterial and Eukaryotic gene regulation > Flashcards

M3 L14-15: Bacterial and Eukaryotic gene regulation Flashcards

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1
Q

2 reasons eukaryotic gene expression is more complicated than bac

A

1) euk DNA is bound to histones –> can have dif chromatin states

2) reg sequences can be long range, not just in the promoter directly upstream of the gene

2.5) euk genomes have a lot more TFs (1400 vs 270 in E. coli)

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2
Q

pros and cons of unregulated gene expression (2 each)

A

pros: 1) all proteins present at all times, 2) don’t need to respond to enviro

cons: 1) metab costly, 2) can have antagonistic action of proteins involved in different metab pathways

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3
Q

is regulated expression evo favorable? why or why not? what operons were characterized and confirmed this?

A

yes, bc more energetically efficient - lac and trp operons

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4
Q

who experimented with the lac operon?

A

jaques manod, andre lwoff, francois jacob

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5
Q

what is constitutive expression? what kinds of genes are expressed this way?

A

when genes are expressed all the time

housekeeping genes that maintain basic cell function

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6
Q

what is regulated transcription? what are the two levels?

A

when genes are only expressed in certain conditions

1) regulate transcription initiation (on/off)

2) regulate amount of transcription (dimmer switch)

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7
Q

what are 3 other levels of gene regulation besides transcription?

A

1) mRNA stability
2) translation
3) post translational modiciations

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8
Q

what is negative control of transcription

A

binding a repressor to the DNA to prevent transcription

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9
Q

characteristics of repressors

A

allosteric domain (other protein binds and activates or inactivates repressor)

DNA binding domain (occupy space where RNA polymerase binds)

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10
Q

what is allostery

A

binding at one site of the protein alters the structure/function of another site

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11
Q

what 2 things can bind to repressor allosteric domains

A

1) inducer: protein binds to repressor, causes release of DNA, transcription proceeds

2) corepressor: protein binds to repressor, causes repressor to bind DNA, transcription blocked

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12
Q

what is positive control of transcription

A

binding an activator to facilitate transcription initiation

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13
Q

characteristics of activators

A

allosteric domain (can be activated or inactivated)

DNA binding domain (bind to facilitate transcription)

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14
Q

what 2 things can bind to activators

A

1) allosteric effector compounds: bind allosteric domain, allow activator to bind DNA, transcription proceeds

2) inhibitors: bind allosteric domain, prevent activator binding DNA, transcription not facilitated

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15
Q

what are operons

A

groups of genes and their shared regulatory regions (genes are usually involved in same pathways)

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16
Q

why are bacterial genomes so small and compact

A

most successful if they can reproduce fast under good conditions

large genomes are metab costly and slow to replicate (evo disadvantage)

operons are small and compact –> more efficient response when conditions are good

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17
Q

what is lactose? what can it be broken down into? changed into?

A

disaccharide of glucose and galactose

B-galactosidase can change a bond –> allolactose

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18
Q

what are the 3 proteins in the lac operon? how are they transcribed?

A

permease, lac Y (“permYase”)

b-gal, lac Z (“B-galactosidaZe”)

transacetylase, lac A (protects cell from damaging byproducts from lactose metab)

transcribed as polycistronic mRNA

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19
Q

in what conditions is the lac operon on/off? basal/leaky expression?

A

no glucose, no lactose: activator and repressor, leaky, OFF

glucose, no lactose: no activator, repressor, leaky, OFF

no glucose, lactose: activator, no repressor, normal expression, ON

glucose, lactose: no activator, no repressor, basal, OFF

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20
Q

what protein is used for lac operon neg regulation

A

lac repressor, homotetramer

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21
Q

what gene encodes lac repressor? where is it? how is it expressed?

A

lacI (i), adjacent to operon, constitutive

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22
Q

what does the lac repressor bind (allosteric and DNA binding domains)

A

allosteric: allolactose (inducer), made from B-gal changing bond in lactose (no allolactose –> repressor binds DNA –> no transcription)

DNA binding domain: lacO, operator (includes TSS)

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23
Q

how do we get allolactose in the cell if the operon is off?

A

need permease to have lactose in the cell and need B-gal to have allolactose to induce the operon (but if the operon is off how do we have these proteins?)

expression is never truly zero (leaky and basal expression)

leaky: expression from repressor bound but activator not bound; effect of repression being reversible and not 100% efficient

basal: expression from no activator and no repressor

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24
Q

how is the lac operon positively regulated

A

when there’s low glucose, adenylate cyclase converts ATP to cAMP

cAMP binds CAP, CAP binds CAP binding site on operon –> facilitates RNA polymerase binding

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25
Q

what are constitutive mutants? what are the two types?

A

muts that cause operon to be expressed all the time

cis acting (only affect transcription of the chrom w/ mut)

trans acting (affect transcription of other chroms)

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26
Q

operator mutations - what happens? cis or trans? how do we know?

A

repressor can’t recognize operator –> constitutive expression

cis acting (know from studying partial diploids from F’ plasmids)

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27
Q

repressor coding sequence mutations - what happens? cis or trans?

A

I (i) - mutations alter DNA binding domain of repressor –> can’t bind operator, constitutive expression

trans acting because repressor from one chrom can bind to operon on dif chrom

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28
Q

super repressor muts - what happens? cis or trans?

A

change repressor allosteric domain –> allolactose can’t bind to induce operon (repressor stays bound)

Is muts act in trans (super repressors made from one chrom can bind to operator on other chroms)

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29
Q

catabolism vs anabolism

A

cat: break down, gain energy, operons usually inducible by presence of a molecule

anabolism: build, costs energy, operons usually repressible by end product (neg feedback)

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30
Q

what’s attenuation

A

some repressible operons can continuously control magnitude of expression based on concentration of end product (dimmer switch)

common where the end product must maintain near constant concentration

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31
Q

anatomy of trp operon

A

3 reg regions: promoter, operator, leader region (upstream of operon and transcribed)

5 structural genes: trpE, D< C, B, A

32
Q

what encodes trp repressor? where is it? how is the repressor activated?

A

trpR, outside operon, activate repressor w/ tryptophan (corepressor)

33
Q

in what conditions is the trp operon on/off

A

trp present –> off
trp absent –> on

*but also attenuation for medium level of transcription

34
Q

2 key properties in leader region (162 bp upstream operon) for attenuation in trp operon

A

1) 4 regions that can make self-comp stem loops

2) codes for separate 14AA peptide w/ 2 consecutive trp codons and own star/stop codons

35
Q

in what conditions do you form the different stem loops?

A

1-2: if ribosome doesn’t associate quickly (transcription/translation not coupled) –> slow transcription –> form 3-4 and terminate transcription if ribosome doesn’t bind

2-3: anti-termiantion loop, if trp codons translated slow –> ribosome does not occupy region 2

3-4: termination loop, if trp codons translated quickly –> ribosome occupies region 2 –> do not transcribe rest of trp operon

*each trpL mRNA chooses between 2-3 and 3-4, ratio of choices depends on trp concentration

36
Q

trp operon mutations

A

1) reduce complementarity in 3-4 stem loop –> reduce repression

2) mut in 2 consecutive trp codons –> operon controlled by other AAs (if code for stop codons instead, ribosome will fall off, not occupy region 2 –> form 2-3 loop –> constitutive expression)

37
Q

response to stress vs response to nutrients

A

nutrient changes are common, response requires a few genes

stress conditions less common, response req change in expression for many genes (change sigma subunit to recognize dif promoters)

38
Q

3 mechs for translation regulation in bacteria

A

1) main way translation repressor proteins bind mRNA: a pdt from each operon can bind own mRNA shine dalgarno –> prevent translation (neg feedback)

2) antisense RNA: binds mRNA and prevents translation

3) riboswitches: bind a segment of mRNA to a regulatory molecule

39
Q

example of antisense RNA translation regulation in bac

A

IS10 encodes a transposase

bac can tolerate low expression but high expression disrupts normal genes

IS10 has antiparallel promoters
1) P-in: weak, express transposase
2) P-out: strong, express overlapping antisense RNA

when transposase mRNA and antisense RNA bind, sequesters shine dalgarno, transposase not translated

*imperfect, occasional transposase translation

40
Q

example of a riboswitch

A

glmS protein catalyzes GlcN6P (sugar) formation

low GlcN6P –> riboswitch inactive, translate mRNA

high GlcN6P –> riboswitch active, part of 5’ UTR acts as ribozyme –> cleaves part of mRNA –> RNase J1 degrades 3’ cleavage pdt (also neg feedback)

41
Q

Compare and contrast tryptophan’s role as a co-repressor in the trp operon and GlcN6P’s role in regulating glmS. How are these mechanisms similar? How are they different?

A

They are both examples of negative feedback (end pdt represses expression)

trp regulates at the transcriptional level (prevent or allow transcription of 5 trp operon structural genes)

GlcN6P regulates at the translational level (riboswitch): Glcn6P bound to mRNA –> part of 5’ UTR acts as ribozyme –> cleaves part of mRNA –> RNase J1 degrades 3’ cleavage pdt (also neg feedback)

42
Q

4 types of gene expression in eukaryotes

A

1) constitutive
2) inducible/repressible
3) temporal (timing in development)
4) spatial (region of the body/tissue type)

43
Q

3 types euk cis-acting regulatory seqs

A

1) core promoter
2)enhancers
3) silencers

44
Q

char of core promoter

A

sim to bacteria located directly upstream gene

binds general TFs and RNA pol II

contains TATA box

45
Q

char of enhancers and silencers

A

interact with other ptoteins and core promoter –> change amt transcription

can be anywhere relative to gene

may have many binding sites for many TFs to bind –> dif outcomes

qualitative (on/off) and quantitative (dimmer) regulation

*enhancers and silencers are cis-acting but the TFs that bind are trans-acting

46
Q

example of a gene with tissue specific expression

A

tissue specific expression driven by presence/absence of certain TFs

sonic hedgehog –> limb and digit dev, brain organization (limbs/brain require dif amounts expression)

long range enhancer in limbs with limb-spec TFs, short range enhancer in brain with brain-spec TFs –> dif pattern expression

mut in SHH regulatory seqs –> polydactyly; use CRISPR to insert cobra SHH reg seq in mouse –> serpentize

47
Q

how do we get morphological evolution? how do we know?

A

mutations in master regulatory genes (tooklit genes) not new genes/changes in protein seqs

human and chip protein seqs 99% same but dif timing, tissues, and amounts expression

48
Q

what is modularity

A

idea that you can change some components of enhancers without affecting others

49
Q

what are locus control regions

A

highly specialized enhancer elements that regulate expression of many genes with related functions (similar to operons but no polycistronic mRNA)

50
Q

Explain modularity in the context of thalassemia

A

anemia from imbalance of a and B globins (usually 1:1)

fetal O2 reqs change over development –> need to change expression of dif globins

regulatory muts in a or B globin LCR –> mess up timing/amt of expression –> thalassemia: anemia from imbalance of a and B globins (usually 1:1)

51
Q

what is binding site turnover

A

when enhancer binding sites remain and genes reg in similar way but with dif combos of TFs (modularity bc changing TFs but not outcome)

52
Q

are enhancers highly conserved? any exceptions?

A

typically yes highly conserved, NS doesn’t tolerate mutations bc they’re highly organized and muts are more likely to decrease function

exception is binding site turnover; some enhancers can have dif TFs bind but produce same protein (modular)

53
Q

example of euk transcription activation

A

yeast gal pathway uses enhancer

req 4 genes for galactose import and use: gal1, 2, 7, 10 (all have own promoters and all reg by gal4 TF)

gal4 constitutively expressed and always bound to upstream activator seq (UAS) enhancer element (but not always activated)

no galactose –> gal3 in cytoplasm, gal80 bound to gal4, blocks activation domain –> gal4 can’t activate transcription

galactose present –> gal3 moves to nucleus –> gal3 binds gal80 –> gal80 releases gal4 and opens activation domain –> can activate gal1, 2, 7, 10

54
Q

example of euk transcription repression

A

yeast gal pathway also uses silencer (interferes with enhancer, no binding of operator/promoter)

glucose present: make Mig1 –> binds silencer between UAS and gal1, 2, 7, 10 genes –> recruits Tup1 –> complex interferes iwth gal4 enhancer element

glucose absent: phosphorylate Mig1 –> send it out of nucleus

55
Q

how to direct long range enhancers

A

insulator seqs: bind proteins, direct enhancers to interact with specific promoter, bloc comm btwn enhancer and wrong promoters, direct more preference to one enhancer over others

56
Q

consequences of insulator seq muts

A

inappropriate enhancer activation –> genetic defects in humans

57
Q

what are the 2 types of heterochromatin

A

1) constitutive: always closed transcriptionally inactive

2) facultative: can be euchrom or heterochrom dep on timing and tissue type

58
Q

what is position effect variegation

A

structural mutations can put seq that are normally in euchrom regions into heterochrom regions

partially in euchrom region and partially in hetero region –> variegation (red and white drosophila eyes)

59
Q

4 epigenetic histone marks

A

1) H3K27me3: repressive mark in facultative hetero (“3 Me added on histone H3 @ lysine 27”; facultative region will not be transcribed)

2) H3K9me3: repressive mark in constitutive heterochrom (constitutive region; never transcribed)

3) H3K9ac: euchrom mark - activation (“add acetyl group @ lysine 9 on histone H3”)

4) H3K4me: euchrom mark - activation

60
Q

4 key char of histone marks

A

1) alter chromatin structure
2) transmissible during cell div
3) reversible
4) don’t change DNA seq (epigenetic)

61
Q

cast of characters for histone modification

A

1) chromatin readers: proteins that rec and bind certain histone marks (maintain activity or inactivity)

2) chromatin writers: catalyze chem mod of histones (add activating or repressive marks)

3) chromatin erasers: catalyze removal of chem mods on histones

62
Q

what is chromatin remodeling? what are the 2 ways to displace a histone?

A

move histones rather than modifying them

1) nucleosome sliding: nucleosome stays bound but displaced to expose reg seqs

2) nucleosome ejection/repositioning: remove nucleosome and put it somewhere else (via swi/snf complex)

63
Q

example of chromatin remodeling

A

pho5 in yeast: encodes phosphatase (removes phosphates from things if cell doesn’t have enough -repressed if excess phosphate, activated if low phosphate)

pho4: encodes protein that facilitates pho5 regulation

high phosphate: pho4 in cytoplasm; TATA blocked by -1 nucleosome, UASp2 blocked by -2 nucleosome, UASp1 (enhancer) bound to pho2 (transcription activator) which is bound to NuA4 (acetylase), promoter histones not acetylated

low phosphate: pho4 in nucleus: pho4 binds pho2, NuA4 acetylates nearby histones and opens chromatin, pho2/4 complex displaces -2 nucleosome, another pho4 binds and recruits swi/snf complex to remove nucleosomes -1, -3, -4, generat TFs bind TATA –> activate transcription

64
Q

what are lncRNAs? example of how they regulate gene expression?

A

long noncoding RNAs

Xist is a gene that is transcribed from inactivated X chroms –> encodes lncRNA that covers the chrom it came from –> facilitates HDACs (acetylases) and methylases binding the X chrom –> condense into barr body

65
Q

what is allele specific expression? is it common? exceptions?

A

different amounts of expression of the maternal and paternal alleles - most genes do not show this (express mat and pat alleles equally)

some genes exhibit genomic imprinting: amt or pat allele declared transcriptionally inactive in gamete formation (via methylating one parent’s allele)

DNA methylation (repressive mark) not same as histone methylation

66
Q

example of genomic imprinting

A

IGF2 in mammals: insulin-like growth factor 2

H19: nearby gene

maternal chrom: ICR (imprinting ctrl region) not methylated, insulator can bind –> distant enhancer promotes H19 transcription, not IFG2

paternal chrom: ICR and H19 both methylated –> insulator does no bind –> enhancer acts on IGF2 promoter –> express IGF2 not H19

67
Q

explain genomic imprinting for IGF2 from evo perspective

A

F does not want to express high amts IFG2 bc that would cause offspring to grow more –> uses more maternal resources –> want all offspring to grow equally

M wants his offspring to grow more than offspring from other males (increase own fitness) - achieve by having offspring produce more IGF2

68
Q

what is RNA interference

A

silence genes via double stranded RNA

dicer/molec ruler cuts dsRNA into 21-25 bp frags –> si/miRNA associates w/ RISC complex –> degrades passenger RNA and look for complementary mRNA –> 3 poss outcomes (frst 2 dep on amt of identity btwn RNA bound to RISC and mRNA)

1) destroy complementary mRNA via argonaut protein

2) bind complementary mRNA and prevent translation

3) RNAi machinery alters chromatin state via RNA induced transcrptional silencing

69
Q

where to get dsRNA for RNA interference (3 sources)

A

1) bidirectional promoters: producve si (small interfering RNA)

2) miRNA (micro RNA) genes fold on themselves

3) dsRNA from virus

70
Q

explain RNA induced transcriptional silencing

A

RITS complex: also has argonaut, carries siRNA to nucleus –> bind complementary nascent RNA –> RITS complex recruits histone mod enzymes to spread heterochrom marks

important for shutting down non-genic repeats

71
Q

how did RNAi evolve? why down regulate is already transcribed? how do we know?

A

defense against transposable elements

mutations in RNAi machinery activates normally silent transposable elements

72
Q

Compare and contrast the characteristics of the core promoter with that of an enhancer sequence.

A

Similar:
Sequences that regulate transcription of certain genes

Different:
1) Core promoter usually upstream of the gene, contains the TATA box and binds general TFs and RNA pol II

2) Enhancer sequence can be anywhere with respect to the gene it’s regulating, binds proteins that facilitate transcription

73
Q

How can the property of modularity give rise to different species using different binding sites and different trans-acting proteins to regulate a gene, but the pattern of gene expression remains similar between these species?

A

With modularity, you can change one aspect of the regulatory module (like the specific proteins and binding sites), but as long as the proteins have similar functions, they can still produce the same output (same pattern of expression).

74
Q

compare and contrast gal pathway and lac operon

A

similar:
1) can both be induced and repressed
2) expression regulated by proteins binding DNA

different:
1) gal pathway is in yeast (euk), lac operon is in bac
2) gal genes regulated via enhancer and silencer, lac operon regulated via activator and repressor
3) lac operon makes polycistronic mRNA

75
Q

Histone deacetylases remove acetyl groups from histones. Would we expect greater activity of these enzymes in a particular genomic region to increase or decrease transcriptional activity of that region and why?

A

Decrease transcription of that region because acetyl groups are typically euchromatin markers → usually increase transcription so removing them would decrease transcription.

76
Q

Identify each of the following histone marks as either generally heterochromatic or euchromatic: H3K27me3, H3K9ac, H3K9me3, H3K4me

A

H3K27me3 and H3K9me → hetero
H3K9ac and H3K4me → euchromatic

Genetics (19 decks)

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