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Mechanisms of Gene Regulation > Exam 3 > Flashcards

Exam 3 Flashcards

1
Q

Describe the CLIP technique

A
  • Cells are irradiated with UV light to cross-link RNA and protein molecules
  • Cells are lysed and RNA-protein complexes are immunoprecipitated using an antibody to the RNA-binding protein
  • RNA is digested with RNase, leaving only the short region of RNA to which the protein is bound
  • Digest with proteinase K, removing the RNA-binding protein
  • cDNA copies are made of the RNA molecules
  • Analysis via DNA sequencing
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2
Q

Describe microarrays

A
  • Can be used to catalog exons in mRNAs
  • Oligonucleotides are selected from each of the exons and hybridized with mRNA prepared from each tissue
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3
Q

Describe ribosome profiling

A
  • Often done after RNAseq, allows to check for what is being actively translated
  • Cells are treated with cycloheximide to inhibit protein synthesis, resulting in ribosomes stalling on the mRNA
  • Lyse cells and treat with RNase to leave only the short region of the RNA to which the ribosome is bound
  • Isolate ribosomes bound to the short RNA regions by sucrose density-gradient ultracentrifugation
  • Purify the bound RNA fragments
  • Make cDNA copies of the RNA fragments and analyze by DNA sequence analysis
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4
Q

Describe subtractive hybridization

A
  • Technique in which all mRNAs common to two samples are removed, leaving only those specifically expressed in one of the samples which can then be analyzed

Steps
- Make radioactive cDNA copy
- Subtractive hybridization to remove mRNAs expressed in undifferentiated 10T 1/2 cells
- Subtractive hybridization to remove mRNAs characteristic of terminal muscle differentiation or induced in all cells by 5-azacytidine
- Screen cDNA library from 10T 1/2 cells to isolate clones corresponding to remaining mRNA

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5
Q

Describe CstF

A
  • Cleavage stimulation factor
  • Preferentially binds to the poly (A) site of the mRNA encoding the membrane form compared to that of the secreted form
  • Early in B-cell development, when CstF levels are low, it binds to the poly (A) site for the membrane form. Later, when CstF levels are high, binding and cleavage will occur for the secreted form
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6
Q

Describe Rev

A
  • Regulator of expression of virion proteins
  • Rev is an RNA binding protein
  • Binds RRE (Rev response element) found in the 2nd intron of HIV transcripts
  • Acts at the level of RNA transport
  • Enhances transport of un-spliced or singly spliced RNA transcripts
  • Contains an NES
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7
Q

Describe Staufen

A

A protein that helps localize bicoid mRNA in fly embryos; also directs other mRNAs to dendrites (but not axons!) in mammalian cells

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8
Q

Describe Ste2

A
  • Binds to RNAPII while it is transcribing RNAs
  • Plays a key role in localizing specific mRNAs to particular locations within the cell
  • Ste2 moves from the RNAP to bind to the target sequence and directs localization of the RNA to the yeast bud
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9
Q

Describe IRES

A
  • Internal ribosome entry site
  • Amino acid starvation results in less translation by ribosomes that bind the 5’ cap
  • This is due to the inactivation of essential initiation factors for translation
  • Under starvation conditions, some proteins can still be made by ribosomes that load onto IRESs instead of cap
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10
Q

Describe the REST complex

A

Promotes epigenetic remodeling of core histones proteins at the promoter of target genes and inhibits the expression of neuronal genes in non-neuronal cells

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11
Q

Describe Notch/Delta

A
  • Notch-Delta is a system that mediates signaling between different cells, so that adjacent cells develop different phenotypes from one another
  • Notch, the transmembrane receptor of one cell, binds to Delta or Jagged, the transmembrane ligands belonging to the neighboring cell
  • Form of proteolytic processing
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12
Q

Describe Scp (SCP1, SCP2)

A
  • Small C-terminal phosphatase
  • SCP1 is a part of the REST complex, plays an integral role in REST’s ability to repress neuronal gene expression, can be inhibited by miR-124
  • SCP2 interacts with REST complex, represses transcription by dephosphorylating the RNAP II C-terminal domain on serine 5
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13
Q

What are the three ways TFs are phosphorylated by kinases?

A
  • Direct phosphorylation/association
  • Secondary messenger
  • Cascade
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13
Q

Explain with examples how proteolytic processing can regulate transcription? What proteins and triggering signals are involved?

A
  • Irreversible and ubiquitous post-translational modification, the breakdown of proteins into smaller polypeptides or amino acids

Example:
- Notch receptor processing forms a heterodimer of a membrane-associated chain (M) and extracellular space-associated chain (E)
- Notch receptor is activated by binding the ligand Delta on another cell
- Endocytosis of Delta stimulates cleavage events in the Notch M chain
- Notch tail released in cytoplasm can traffic to nucleus and be a TF

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14
Q

What are the two things required for activation of NFκB?

A
  • Phosphorylation of IkB
  • Acetylation of NFκB
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15
Q

Describe the 4 general ways transcription factors can be activated.

A
  • Ligands
  • Protein : protein interactions
  • Kinase and kinase cascades
  • Proteolysis
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16
Q

Name three types of ligands that can activate transcription factors.

A
  • Extracellular ligands
  • Intracellular ligands/ligands that enter cells
  • Hormone ligands
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17
Q

Can you describe the details of signaling pathways triggered by extracellular ligands such as cytokines? For example, could you explain the JAK-STAT pathway starting with ligand binding and name the specific ligand that binds this receptor. What about the TGF beta pathway?

A

JAK-STAT
- Cytokine binding triggers a signal to JAK (a kinase).
- JAK phosphorylates STATs (signal transducers and
activators of transcription)
- STAT phosphorylation promotes dimerization and migration to nucleus
- STAT activates transcription

TGF beta
- TGFb binding leads to phosphorylation of R-Smad
- Phosphorylated R-Smad binds a Smad family protein, Smad4
- Heterodimer binds target genes to regulate transcription

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18
Q

What processes did we learn about that heat influences?

A

Heat promotes…

  • Dissociation of HSP90 chaperone
  • Trimerization
  • Phosphorylation is needed before HSF-1 can activate transcription
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19
Q

What is lateral inhibition? How do Delta and Notch interact to promote lateral inhibition pathways? What other proteins play a role in this process?

A
  • When one cell differentiates into another cell type but then prevents the neighboring cells from doing so
  • Activation of Delta stimulates Notch in the neighboring cells, while activation of Notch restricts Delta within the same cell
  • Other proteins: REST, SCP2, NeuroD, Id2, etc.
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20
Q

What mechanism regulates polyadenylation?

A

Inhibition of the poly A polymerase enzyme

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21
Q

Splicing efficiency and splice site competition can be influenced by what 3 things?

A
  • Ability to recruit splicing factors
  • Strength of SR binding sites
  • Presence or absence of ESEs
  • Position of splice site (is a proximal or distal downstream exon involved)
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22
Q

How does CBP know which activator to help? What regulates CBP interactions with various activators of transcription?

A
  • CREB* binding protein
  • CBP can be the target of phosphorylation and methylation, regulating its interactions with various activators of transcription
  • The methylation and phosphorylation help to direct the coactivator to its activator as well as cellular signaling pathways
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23
Q

What stages of translation can miRNAs regulate? How do miRNAs block translation?

A
  • Before processing
  • After processing
  • Bind to an mRNA target and induce its de-adenylation and subsequent rapid degradation
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24
Q

Please describe the role Rev plays in viral protein production. What does Rev rely on to do its job?

A
  • Rev acts post-transcriptionally to promote splicing/transport so the un-spliced and single spliced RNAs encoding the viral structural proteins and the reverse transcriptase accumulate
  • Requires a small amount of the fully spliced viral RNA which encodes Rev
  • Has also been shown to bind to a cellular export protein, mediating export of HIV RNA
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25
Q

What is the purpose of ribosome profiling?

A

It enables systematic monitoring of cellular translation processes and prediction of protein abundance

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26
Q

What does Heat Shock Factor bind to? What does HSF promote?

A
  • HSF binds to HSE
  • Promotes transcription even in the presence of protein synthesis inhibitors
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27
Q

What advantage does post-translational activation of TFs give a cell verses de novo synthesis of TFs?

A

It is much faster

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28
Q

What technique can be used to catalog exons in mRNAs? How does it work?

A
  • Microarrays
  • Oligonucleotides (representing genes or exons) on chips
  • Employ probes designed to detect each individual exon for known or predicted genes
  • Oligonucleotide array > hybridization > hybridization pattern
    ^ compare hybridization pattern between
    tissues
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29
Q

Explain how snRNPs are able to bind to the target sites in the RNA.

A
  • RNAs in snRNPs can base pair with the RNA that is being spliced
  • U1 snRNA binds 5` splice site (so does U6 snRNA)
    • U2 snRNA binds branch point
    • A at branch point doesn’t base pair
    • Branch point A’s 2OH can attack 5end
      of intron
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30
Q

What traits or abilities were predicted for a muscle-specific master regulator?

A
  • Expressed in skeletal muscle
  • Can induce expression of muscle-specific genes
  • Overexpression in non-muscle cells in culture induces muscle differentiation
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31
Q

What is the REST complex’s function? What are the various functions of REST complex components? What happens to REST during neuronal development?

A
  • Function: represses neuron-specific genes via a zinc finger domain
  • REST Repressor recruits SCP2 which can block transcriptional initiation
  • SCP helps REST block transcriptional initiation
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32
Q

How do hormones play a role in transcription by glucocorticoid receptor How are coactivators involved?

A
  • GR Binding of hormone glucocorticoid promotes DNA binding activity in vivo
  • Co-activators activate transcription by interacting with the basal transcription complex and/or by modifying chromatin structure
33
Q

You might be asked to diagram or list the steps for transcription factor activation by extracellular ligands, secondary messengers or via a kinase cascade

A

TF activation by extracellular ligands:
- Steroid/thyroid hormone can diffuse directly through the membrane and bind to its receptor
- Protein will bind integral membrane receptor and the signal will propagate inwards
- Example: JAK-STAT pathway

TF activation via a kinase cascade:

34
Q

Is Glucocorticoid receptor an activator or a repressor? How does it decide what it will be?

A

Can be activated by hormone ligands to become an activator and/or a repressor

35
Q

How does HSP90 influence entry of glucocorticoid receptors into the nucleus?

A
  • If HSP90 is bound, glucocorticoid receptors cannot enter into the nucleus
  • Upon the loss of HSP90, the NLS is exposed and the GRs can enter the nucleus
36
Q

Define/describe alternative RNA splicing

A
  • A cellular process in which exons from the same gene are joined in different combinations
  • Tissue specific splicing differences are common, which yields different mRNAs and possibly different proteins
37
Q

Define/describe RNA processing

A
  • The sequence of events through which the primary transcript from a gene acquires its mature form
  • Includes: capping at the 5’ end, addition of a poly(A) tail at the 3’ end and splicing to remove introns
38
Q

What is CLIP used for?

A

Used for characterizing RNA sequences bound by RNA binding proteins

39
Q

Define/describe PARP1

A
  • poly (ADP ribose) polymerase I
  • Following heat shock, PARP1 becomes activated and modifies the poly A polymerase by the addition of polyADP-ribose molecules, resulting in the inhibition of the poly A polymerase enzyme so that no polyadenylation occurs following the 3’ cleavage of the mRNA
40
Q

Define/describe alternative polyadenylation

A
  • When different cleavage sites are used in different cell types, producing polyadenylated mRNAs of different lengths
  • The RNAs of different lengths can be with or without binding sites for specific RNA-binding proteins or miRNAs and are therefore subject to different patterns of regulation
41
Q

Define/describe 3’ untranslated region (UTR)

A
  • Regulate mRNA-based processes, such as mRNA localization, mRNA stability, and translation
  • Length of 3’ UTR is altered by alternative polyadenylation
42
Q

Define/describe nuclear export signal (NES)

A

Rich in leucine residues, which promotes transport of the RNA to which it has bound from the nucleus to the cytoplasm

43
Q

Define/describe protein kinase

A

A protein kinase is a kinase which selectively modifies other proteins by covalently adding phosphates to them

44
Q

Define/describe heat-shock element (HSE)

A
  • What HSF binds to
  • Type of signaling molecule
45
Q

Define/describe heat-shock proteins. How does HSP90 work?

A
  • HSPs are a family of proteins produced by cells in response to exposure to stressful conditions
  • Is bound to the GR until steroid binding, which then causes it to dissociate and reveal the NLS
46
Q

Define/describe signaling molecule

A

If a signaling molecule enters a cell, it can potentially bind to a TF and directly regulate its activity

47
Q

Define/describe ligand

A

A molecule that can bind to a receptor. This causes a change in cell signaling, and ultimately, cell behavior or structure

48
Q

Define/describe nGRE-negative glucocorticoid response element

A
  • The presence of an nGRE results in the receptor binding as a trimer of three receptor molecules rather than the dimeric form which binds to the standard GRE
  • Transcription repression via 4 different possible mechanisms
48
Q

Define/describe nuclear localization signal

A
  • An amino acid sequence that ‘tags’ a protein for import into the cell nucleus by nuclear transport
  • Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface
49
Q

Define/describe chaperone

A
  • A family of proteins that play a vital role in the stabilization of unfolded proteins
  • This stabilization aids in many processes such as translocation, degradation, and folding
50
Q

Define/describe extracellular signaling molecules

A

Cues, such as growth factors, hormones, cytokines, extracellular matrix components and neurotransmitters, designed to transmit specific information to target cells

51
Q

Define/describe STAT (signal transducers and activators of transcription)

A
  • Family of TFs
  • intracellular transcription factors that mediate many aspects of cellular immunity, proliferation, apoptosis and differentiation. They are primarily activated by membrane receptor-associated Janus kinases (JAK)
52
Q

Define/describe the JAK-STAT pathway

A
  • Cytokine binding triggers a signal to JAK (a kinase).
  • JAK phosphorylates STATs (signal transducers and activators of transcription)
  • STAT phosphorylation promotes dimerization and migration to nucleus
  • STAT activates transcription
53
Q

Define/describe Smads

A
  • Family of TFs
  • Main signal transducers for receptors of the transforming growth factor beta (TGF-B) superfamily, which are critically important for regulating cell development and growth

TGF-B Pathway:
- TGFb binding leads to phosphorylation of R-Smad
- Phosphorylated R-Smad binds a Smad family protein, Smad4
- Heterodimer binds target genes or regulate transcription

54
Q

Define/describe cyclic AMP

A
  • Secondary messenger
  • Made from ATP by activated G-protein activating adenylyl cyclase
55
Q

Define/describe G-proteins

A
  • Activated by ligand binding
  • A family of proteins that act as molecular switches inside cells, and are involved in transmitting signals from a variety of stimuli outside a cell to its interior
56
Q

Define/describe adenylyl cyclase

A

Enzymes that synthesize the intracellular second messenger, cyclic adenosine monophosphate (cAMP), which in turn triggers a cascade of biochemical changes that regulate a number of important cellular processes

57
Q

Define/describe G protein coupled receptors

A
  • Integral membrane proteins
  • Cellular receptor is NOT associated with kinases, instead receptors are coupled to G proteins (GPCR)
58
Q

Define/describe ATP

A
  • Adenosine triphosphate
  • Energy currency of the cell
59
Q

Define/describe second messenger

A

Small molecules and ions that relay signals received by cell-surface receptors to effector proteins

60
Q

Define/describe receptor tyrosine kinase

A
  • Family of TF, integral membrane proteins
  • Bind to a variety of growth factors that regulate the growth and differentiation of specific cell types
  • Binding of ligand induces autophosphorylation of RTK
  • Part of MKKK cascade
61
Q

Define/describe Ras proteins

A
  • Act as molecular switches which are activated by binding to GTP. They play a role in the cascade of cell process control
  • Ligand binding induces RTK autophosphorylation
  • Phosphorylation recruits adaptor proteins that allow interaction with Ras proteins.
  • Ras:adaptor interactions promote Ras GTP binding (active Ras)
  • Ras-GTP binds Raf kinase which starts kinase cascade
62
Q

Define/describe signaling cascade

A

A sequence of successive activation reactions involving enzymes (enzyme cascade) or hormones (hormone cascade) characterized by a series of amplifications of an initial stimulus

63
Q

Define/describe tumor necrosis factor

A
  • A multifunctional cytokine
  • Proinflammatory, involved in autoimmune disease
64
Q

Define/describe notch

A

Notch receptor is a heterodimer composed of a membrane-spanning chain (M) and an extracellular chain (E)

65
Q

Define/describe delta

A

A protein in an adjacent cell that notch’s extracellular chain binds to

66
Q

Define/describe Akt

A
  • Akt signaling
  • Targets SF2
  • Effects on splicing
  • Recruitment of U1snRNP
67
Q

Define/describe master regulatory transcription factor

A
  • Regulatory proteins that are predominantly responsible for regulating the expression of multiple genes
  • Master regulatory genes for skeletal muscle: Myf5, MyoD, Mrf4, myogenin
68
Q

Define/describe nucleoside 5-azacytidine

A
  • Activates master regulator
  • Induces MyoD expression
69
Q

Define/describe basal transcription complex

A

MyoD achieves its affects by interacting with co-activator molecules which can then interact with components of the BTC (most basic set of proteins necessary for RNA polymerase to function at a site of transcription in eukaryotes)

70
Q

Define/describe Id

A
  • Inhibitory protein
  • MyoD forms an inactive heterodimer with the Id factor
  • Id levels decline when cells are induced to form differentiated myotubules by removal of growth factors
71
Q

Tissue specific splicing factors can play a role in alternative splicing by…

A

interacting with cis-acting sequences in mRNA

72
Q

Alternative splicing can be regulated by…

A
  • Tissue specific splicing factors
  • Constitutively expressed splicing factors
73
Q

What can constitutively expressed splicing factors by regulated by?

A
  • Post-translational modifications: methylation of CARM1 promotes omission of certain exons
  • Quantitative variations: the concentration of splicing factors can influence which sites they bind (high affinity sites will be occupied first)
74
Q

The proteins produced by alternative splicing can differ in what two ways?

A
  • Functions
  • Interactions with other proteins
75
Q

How is nonsense mediated decay utilized as a form of regulation?

A

Exons with stop codons can be spliced in to promote degradation

76
Q

In what two ways can polyadenylation be regulated?

A
  • Amount of polyadenylation
  • Site of polyadenylation
77
Q

Enhancement of polyadenylation can promote _______ while induction of de-adenylation can lead to _______

A

stability, degradation

78
Q

How can alternative polyadenylation lead to mRNAs being produced that encode different proteins?

A

If the alternative polyadenylation leads to alternative splicing

Mechanisms of Gene Regulation (4 decks)

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