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Bio 209 > Lecture 6 - Control of Gene Expression > Flashcards

Lecture 6 - Control of Gene Expression Flashcards

1
Q

Gene control examples in prokaryotes

A

Lac Operons
Trp Operons

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2
Q

Gene control examples in eukaryotes

A
  • Chromatin remodeling & position-effect variegation
  • Gene silencing: DNA methylation
  • Dosage compensation
  • Alternative splicing of mRNAs
  • mRNA stability
  • Regulatory RNAs (siRNA and miRNA)
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3
Q

Six steps at which eukaryotic gene expression can be controlled

A
  1. Transcriptional control
  2. RNA processing control
  3. RNA transport and localization control
  4. Translation control
  5. mRNA degradation control
  6. Protein activity control
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4
Q

Who developed the Operon Model?

A

Francois Jacob and Jacques Monod

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5
Q

Details of the Operon Model (controlling elements, components, expression of genes)

A

Controlling elements:
- Repressor -> regulator protein that represses transcription
- Operator -> site on DNA to which repressor binds

Components of an operon:
- Set of contiguous structural genes (shared function such as lactose catabolism or Trp anabolism)
- Promoter
- Operator -> situated b/t promoter and structural genes
- Multigenic mRNAs (polycistronic) -> multiple genes linked together on same operon

Offers efficient and coordinated expression of related genes

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6
Q

What is negative control of the operon model, and what are the two types?

A

Negative Control: repressors turn off transcription

Think repressors

Types: Inducible expression and repressible expression

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7
Q

What is inducible expression of the operon model?

A
  • Resting state: off (little to no transcription)
  • Free repressor bound to operator; transcription induced when inducer binds repressor => comes off
  • Involves catabolic operons like Lac Operon (degrade energy rich molecule to synthesize ATP) => cannot transcribe w/o energetic molecule
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8
Q

What is repressible expression of the operon model?

A
  • Resting state: on (active transcription)
  • Repressor/corepressor complex bind operator => inhibit transcription
  • Involves anabolic operons like Trp operon (biosynthesis operons; genes encode enzymes needed for synthesizing something) => keep transcribing until sufficient biosynthesis
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9
Q

Inducer vs co-repressor?

A

Both effector molecules

Inducer:
- Repressor already bound to operator
- Inducer binds to repressor => comes off of operator
- Induces transcription

CoRepressor:
- Repressor does not bind to operator until co-repressor binds to repressor
- Bind => corepressor-effector complex binds to operator
- Inhibits transcription

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10
Q

Lactose Operon (Lac Operon): what mechanism, what is the regulator gene, what does it encode, structure of product, what does it bind, what does that do

A
  • Inducible expression
  • lacl is the regulator gene, encodes Lacl repressor protein
  • Lacl is a tetramer
  • Lacl binds lac operator => operator has partial overlap w/ promoter => sterically prevents RNA polymerase from transcribing lacZ, lacY, and lacA
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11
Q

Function protein products of lacZ, lacY, and lacA genes

A

lacZ gene -> LacZ (beta-galactosidase) –> cleaves lactose into glucose and galactose; also forms allolactose inducer

lacY gene -> LacY (beta-galactosidase permease) -> pumps lactose into cells

lacA gene -> LacA transacetylase -> idk function

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12
Q

There is still low background of lacZ, lacY, and lacA, explain

True

False

A

True b/c needed to transport lactose into cell for metabolism

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13
Q

What is the inducer of lac operon? How is it made? Function?

A
  • Allolactose, a sensor of lactose
  • Made by LacZ beta-galactosidase from lactose
  • Function: binds Lacl repressor => releases from operator => transcription is induced
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14
Q

What is glucose induced repression of lac operon? Purpose? AKA?

A

Glucose prevents expression of lac operon

Purpose: glucose is the preferred carbon source, ensures glucose utilization over lactose when glucose is present i.e. ensures lac operon is only going to be induced when there is lactose and no glucose

AKA: Catabolite Repression

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15
Q

What does the promoter of lac operon contain?

A
  • CAP/cAMP binding site
  • RNA polymerase binds site
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16
Q

What is glucose induced repression of lac operon mediated by? How? Relation to glucose and transcription?

A

Mediated by:
- CAP (catabolite/cAMP activator protein) regulatory protein
- cAMP (cyclic AMP)

How mediated?
- Promoter contains CAP/cAMP binding site
- cAMP allosterically activates CAP
- CAP binds cAMP when cAMP present => CAP/cAMP complex binds to promoter => enhances transcription of lac operon

Relation to glucose and transcription?
- Low glucose => high cAMP => active CAP => CAP/cAMP complex binds promoter and exerts positive control of lac operon transcription
- High glucose => low cAMP => inactive CAP => no complex to bind promoter and no induction => ensures glucose is preferred energy source when present

17
Q

What are the conditions for MAX inducibility of lac operon?

A
  1. Lactose (allolactose)
  2. Relatively low glucose => high cAMP => high activated CAP protein
18
Q

Method of gene silencing in eukaryotes? What enzyme facilitates? Where does it occur? Does it happen in humans? Effects?

A
  • Methylation of cytosine
  • DNA Methyl Transferases
  • Methylation of cytosine typically occurs on CpG island clusters near transcription start site
  • Rarely methylated in humans
  • Altered nucleosome => less H1 and acetylated histones => hypersensitive to DNase I and transcriptionally inactive
19
Q

Methylated CpG associated with _____ transcription

A

Repressed

20
Q

Mechanism of methylated DNA, relation to cancer?

A

Not fully understood

  • Methylated CpG bind protein complex that represses nearby genes
  • MeCP2 changes chromatin structure

Cancer: Increased hypermethylation of CpG islands are seen in various cancers

21
Q

What is imprinting? How is methylation involved?

A

Imprinting: gene expression is controlled by its parental origin, gene remembers

Methylation involvement:
- Methylation is established in parental germ line
- Somatic cells develop w/ same methylation pattern
- Germ line development methylation is erased and reestablished based if oogenesis (ovum/female creation) occurs

22
Q

What is the reason behind dosage compensation in eukaryotes? What are the three methods of dosage compensation?

A

Typically, expression of genes on X chromosome will be twice as much in females as males => need dosage compensation

3 methods:
1. Inactivation (mammals)
2. Hyperactivation (fruit flies)
3. Hypoactivation (worms like C. elegans)

23
Q

How does inactivation dosage compensation work in mammals?

A
  • Begins at site called X inactivation center (XIC), which spreads in opposite directions towards ends of chromosome
  • XIST (X inactive specific transcript, a long noncoding RNA) gene is transcribed (maintained) as 17 kb RNA only from inactive X chromosome=> binds and coats inactive X chromosome specifically => inactivates entire X chromosome (all genes)
24
Q

Do both chromosomes express XIST gene? What happens later in development?

A

Yes

Later in development:
- Random X chosen to be inactive
- XIST transcript from one chromosome stabilizes and eventually envelops whole X chromosome
- XIST transcript from other chromosome disintegrates and transcription is repressed by methylation of promoter

25
Q

What does the inactive X chromosome form during interphase? What happens during S phase? What is the acetylation level of histone H4?

A

A darkly staining mass called the Barr Body

Specifically in S phase, it decondenses and is replicated after all other chromosomes (maintained but remains transcriptionally silent)

Low levels of acetylated histone H4 => heterochromatin => repressed transcription/expression

26
Q

How does hyperactivation in fruit fly/drosophila males work?

A
  • Elusive mechanism
  • Complex of proteins/RNA (expressed only in males) bind to specific sites on male X chromosome => bind 30-40 sites => likely to be involved in chromatin remodeling (helicase and histone acetyl transferase)
27
Q

How does hypoactivation of X chromosomes in worms/C. elegans females work?

A
  • Elusive mechanism
  • Protein binds specifically to both x chromosomes in females, not the X in males => hypoexpression/repression of expression on both female X chromosomes
28
Q

Relationship between hyperactivation of drosophila and hypoactivation of C. elegans?

A

Opposite mechanisms

29
Q

What is alternative splicing of mRNA in eukaryotes?

A
  • Spliceosomes in nucleus remove introns and can also modify coding sequence by removing exons
  • Dependent on RNA/spliceosome interaciton
  • Economizes on genetic information => create numerous related yet different proteins
30
Q

Alternative splicing of transcripts from the sex-lethal gene i male and female drosophila determine ____. How?

A

Fruit fly sex; females exclude internal stop codon, males do not

31
Q

How does mRNA stability act as method of gene expression in eukaryotes? How is lifespan of mRNA controlled

A
  • mRNA in the cytoplasm can be translated by several ribosomes simultaneously and continues until mRNA is degraded
  • Control lifespan of mRNA => control expression of genes

Lifespan is controlled by:
1. PolyA tail length
2. 3’ UTR sequence (AUUUA promotes rapid degradation)
3. Metabolic state of cell
4. Regulatory mechanisms

32
Q

What are the two related mechanisms for RNA dependent regulation of gene expression?

A
  1. Directed degradation of mRNA (RNAi)
  2. Translational inhibition of specific mRNAs
33
Q

What is RNAi - directed degradation of mRNA? What organisms observed? What experiment? Why does this occur?

A
  • Observed in C. elegans and Drosophila
  • First observed while performing negative control in anti-sense experiment => sense strand silences expression
  • Concluded that introduction of double stranded RNA homologous to gene of choice => more efficient silencing than either sense or antisense alone
  • Why: introducing anti-sense sequence hybridizes/bp w/ sense strand => dsRNA sets off RNA degrading enzymes => eliminate mRNA sequence
34
Q

What is the mechanism of Action-RNAi?

A
  1. Initiation
    - Long dsRNA => 21-23 nucleotide siRNA duplexes via Dicer nuclease cleavage, each siRNA strand has a 2-nucleotide 3’ overhang
  2. Effector Step
    - siRNA duplex bound to RNA-induced silencing complex (RISC)
    - Duplex unwound via ATP
    - Antisense strand bound to RISC, sense strand diffuses away
    - RISC/antisense strand complex binds homologous mRNA transcript w/ 100% complementarity
    - Target mRAN is cleaved in the middle of the duplex by non-Dicer RNase
    - Cleaved mRNA fragments recognized as unproductive by cell and thus degraded
35
Q

What is the mechanism translational inhibition of specific mRNAs?

A
  1. Initiation
    - Mediated by miRNAs (micro RNAs),
    - miRNAs are abundant in human genome and transcribed as part of longer RNA molecules
    - miRNAs processed in nucleus into 70-100 nucleotide hairpins by dsRNA specific ribonuclease (Drosha)
    - Hairpins transported out of nucleus by transportin/exportin-5- dependent mechanism
    - Digested by Dicer in cytoplasm => 21-23 nucleotide duplex w/ 2 nucleotide 3’ overhang
  2. Effector step
    - Single strand of mature miRNA bound to complex similar to RISC
    - Complex binds to mRNAs that are significantly but not 100% complementary
    - Bound mRNAs not translated

Bio 209 (10 decks)

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