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LS > Eukaryotic Gene Regulation > Flashcards

Eukaryotic Gene Regulation Flashcards

0
Q

Different levels of regulation

A
  •   Chromatin remodeling
  •   RNA processing
  •   DNA Methylation
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1
Q

Are genes turned off permanently or temporarily?

A

In some cases genes are turned off permanently, can turn them into a permanent type of cell (permanently silenced). Others can be turned on and off

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2
Q

Do all cells have the same DNA?

A

the nuclei of cells contain a complete complement of genetic information, cells don’t lose chromosomes that have genes on them that certain cells don’t need

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3
Q

Dolly

A

Dolly the sheep in 1997 Wilmut and colleagues in Scotland
• Dolly died in 2003 (6 yrs old). She died from progressive lung disease, but also suffered from arthritis. Cloned animals tend to suffer maladies,including premature aging.

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4
Q

What is so amazing about cloning?

A

Nucleus of the first cell (that gave rise to dolly) came from a somatic cell. That cell had already differentiated and became a permanent udder/skin cell so they had to switch it back on to make different types of cells

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5
Q

Do all cells express the same genes?

A

•  Within an organism, each cell type has a unique protein expression profile.

Some things are the same, probably expressed at different levels. May express the same important/generic proteins used for cell maintenance, but there are many different ones that are cell specific

–  Some common genes such as rRNA genes are referred to as “housekeeping” genes because they are expressed at all times in all cell types

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6
Q

Gene Expression is highly regulated

A

All cells contain the same genetic material; however, different types of cells contain different kinds of proteins. Therefore, the expression of genes must be regulated/controlled in a cell specific manner

Expression of genes must also be controlled temporarily duringdevelopment and in response to certain environmental factors.

In addition, the level of gene expression is also regulated…certain proteins are present in higher levels than others in a
particular type of cell.

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7
Q

When is gene regulation most tightly controlled?

A

During embryonic development

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8
Q

Transcriptional control

A

Turning transcription on or off- binding of inhibitor, repressor protein to promoter sequence to turn off expression of the gene

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9
Q

RNA processing control

A

Alternative splicing

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10
Q

RNA transcript and localization control

A

Whether or not mRNA has appropriate signal peptide to take it where it needs to go

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11
Q

Translational control

A

Whether or not ribosome sits down on mRNA

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12
Q

mRNA degradation control

A

How quickly is the mRNA degraded, how long is it available to be translated? Seem can be translated multiple times to make multiple proteins

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13
Q

Protein activity control

A

Phosphorylation, dephosphorylation, proteolytic processing of insulin

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14
Q

How do hormones turn genes on?

A

Hormones (if steroid derivatives) can be lipid soluble and go through the cell membrane and bind to a receptor protein. typically dimerizes. the dimer of receptor and steroid hormone will bind to promoter region on DNA and turn on a gene. Response to hormone is turning on gene expression

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15
Q

A eukaryotic gene is regulated by many regulatory elements and proteins

A

Expression of genes are governed by the gene control region: promoter and regulatory elements

Regulatory or enhancer sequences

Specific DNA binding proteins bind to a gene’s regulatory sequences to determine where and when transcription will initiate; can either be activators or repressors

Regulatory elements can be found tens of thousands of base pairs either upstream or downstream from the promoter, or adjacent

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16
Q

Transcription Factors

A

Typically two domains (Structural or functional. Can have many motifs)

  1.   DNA-binding domain
  2.   Activation domain (Other proteins come and associate, activates polymerase and the rest of the proteins it is associated with to kick the polymerase off the promoter and transcribe the gene)

A stable framework so that those regions which recognize the DNA can be positioned to interact with the DNA double helix. Recognize promoter regions or enhancer regions

Stimulate transcription

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17
Q

How do transcription factors interact with the DNA?

A

These protein motifs fit around DNA so they recognize base pair patterns. Recognize promoter regions or enhancer sequence regions.

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18
Q

Examples of transcription factors

A

Beta helix loop helix

TFIIIA zinc finger motif Needed for transcription of 5s rRNA

Leucine zipper (beta zipper)

Hmg- high mobility group proteins bend DNA and bring enhancer sequences that are far away into close proximity to general transcription factor proteins or mediators

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19
Q

Why do transcription factors function as dimers?

A
  •   Heterodimerization expands the diversity of regulatory factors that can be generated
  •   3 bHLH proteins can make 6 different transcription factors

Can recognize many promoter sites, don’t need one transcription factor for each promoter sequences due to dimerization

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20
Q

Possible Mechanisms of Transcriptional Activation

A

Recruit transcription factors to vicinity of promoter (increase the local concentration)

Stabilize formation of pre-initiation complex

Disrupt local chromatin structure- How closely wrapped is the DNA around the histones

Acetylation/deacetylation of histones, modifying them

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21
Q

Model of all elements of transcriptional control

A

Regulatory transcription factors recruit chromatin-remodeling complex and HATs. Chromatin decondenses

When chromatin decondenses, a region of DNA is exposed, including the promoter

Regulatory transcription factors recruit proteins of the basal transcription complex to the promoter, looping DNA

RNA polymerase and the basal transcription complex join to form the initiation complex, transcription begins

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22
Q

Where are the enhancers and what do they do?

A

Enhancers upstream and downstream of coding region in this example. Proteins bind to these enhancers. Enhancers are cis, on dna adjacent to the gene. enhancer proteins recognize enhancer element and bind to transcription factors on the promotor. Gtf and enhancers bring in polymerase. Ca help stabilize pol on the DNA. many genes don’t have enhancer sequences, less efficient transcription

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23
Q

How do enhancers stimulate transcriptionfrom a distance?

A

Enhancers are composed of one or more binding sites for regulatory proteins, such as activators and repressors

Activators interact with other proteins (i.e. mediator complex) to bring in and stabilize transcription machinery, causing the intervening DNA to loop out

Mediator is the molecular bridge between activators and RNA Pol II

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24
Q

How do activators work?

A

Activator protein hits pol, RNA pol shoots off promoter. Also due to CTD phosphorylation. Looping of dna can interact with mediator. Mediator interacts with all tfs and pol, activator can hit that, change in its conformation and then mediator disassociates with pol and pol leaves its association with tfs and transcription begins. Increases efficiency

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25
Q

Mediators

A

Several DNA-bound activators can interact with a single mediator complex

The mediator complex is a type of co-activator—intermediary proteins that assist the transcription activators to stimulate initiation of transcription

Subunits of a mediator can bind to RNA pol II, activation domain of various activator proteins, and histone acetylation activity

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26
Q

How big are mediators?

A

Mediator proteins are very large, can interact with over 20 proteins at a time to increase efficiency of binding of pol 2 and transcription. Different regions have different functions

27
Q

Comparison of prokaryoticand eukaryotic activation

A
  •   Prokaryotes: activator binds immediately upstream of polymerase and directly contacts polymerase
  •   Eukaryotes: activator interacts with many proteins to recruit general transcription factors and RNA pol from a distance, leading to a loop in the DNA. Gene regulation before and after coding sequence
28
Q

Combinatorial Regulation

A

Factors can recognize many promoter regions through dimerization

If the expression of each transcription factor is unique, the expression pattern of each of the gene will be unique.

29
Q

Combinatorial control

A

Multiple transcription factors & multiple binding sites lead to complex regulatory systems

Many enhancer sites

30
Q

Regulation of the rat PEPCK gene

A

Transcription is controlled by a variety of transcription factorsthat interact with specific DNA sequences located in the
upstream regulatory region (can also be located downstream)

31
Q

How do we get such a variety of regulatory proteins?

A

When a cell divides regulatory proteins are inherited in one cell but not the other

Gene expression most important in embryo. Many patterns of gene expression, how you get different types of tissue cells. Change in combo of regulatory proteins that are inherited early on by the cells

32
Q

Histone acetylation and chromatin remodeling

A

DNA is packaged into nucleosomes

Deacetylated histones are “OFF”

Acetylation of histones prevents chromatin compaction and increases access to specific regions ofDNA

Histone acetyltransferases (HATs) acetylate residues on histone proteins

33
Q

How do acetyl groups prevent chromatin compaction

A

Acetyl groups bulky, allow loosening of DNA. Acetyl groups on histone tail are bulky and displaces DNA. Histone acetyl transferases add acetyl groups

34
Q

Chromodomains

A

promote protein binding to methylated lysines in the tail region of histone H3.

may
 also be associated with gene regulation

35
Q

Bromodomains

A

Bromodomains are generally found in proteins that regulate chromatin structure and gene expression, such as histone acetyltransferases and the ATPase component of certain nucleosome-remodeling complexes.

Bind to acetyl groups

Recognizes acetylated lysine residues (found at n terminus of histones)

36
Q

Transcription activators affect histone acetylation

A

Gcn4 is a yeast transcriptional activator

Its DNA-binding domain (DBD) interacts with the upstream activating sequence (UAS) of the genes it regulates.

Its activation domain (AD) interacts with a multiprotein histone acetylase complex that includes Gcn5 (histone acetylase activity)

Hyperacetylation of histone N-terminal tails around Gcn4 byGcn5 facilitate access of GTF required for initiation.

A subunit of TFIID has histone acetylase activity. Binds to promoter sequences and affects adjacent histones. Opens up DNA to start transcription

37
Q

Chromatin remodelingcomplexes: SWI/SNF

A

Is a co-activator that is required for activation of some yeast promoters

Use energy of ATP

Can transiently dissociate DNA from the surface of nucleosomes, promoting histone sliding

Induce formation of loop or bulge promote unfolding of condensed chromatin structures (conformational change)

Higher eukaryotes (i.e. Drosophila and mammals) also have multiprotein complexes with homology to yeast SWI/SNF complex

38
Q

Transcription of yeast HO gene by activators and co activators

A

HO gene is in condensed chromatin

SWI5 activator binds to upstream enhancer and interacts with SWI/SNF chromatin-remodeling complex

SWI/SNF decondenses chromatin by sliding, exposing histone tails

GCN5-containing histone acetylase complex associates with SWI5 & acetylates histone tails

SWI/SNF continues to decondense adjacent chromatin

Chromatin in HO control region is hyperacetylated

SWI5 is released

Activator SBF binds to the promoter-proximal elements

SBF binds to the mediator complex (which was recruited by binding proteins)

Pol II interacts with subunits of mediator complex and the GeneralTranscription Factors, resulting in the assembly of the pre-initiation complex

39
Q

How do genes get repressed?

A
  •   Different chemical nature of the DNA(i.e. methylation, chromatin structure, removing acetyl groups)
  •  The presence of repressor proteins
40
Q

Competitive DNA binding

A

Repressor covers enhancer/activator sequence

41
Q

Masking activation surface

A

Repressor interacts with activator so it can’t interact with mediator

42
Q

Direct interaction with gtfs

A

Repressor interacts with mediator or TFIID in a way which pol is not able to escape

43
Q

Recruitment of repressive chromatin remodeling complexes

A

Chromatin condensed

44
Q

Recruitment of histone deacetylases

A

When the promoter DNA is assembled onto a nucleosome with unacetylated histones, GTFs cannot bind the TATA box

45
Q

What specific repressors are active?

A

Repressors can direct histone deacetylation in nucleosomes that bind to the TATA box promoter-proximal region

UME6 is a repressor that binds to the upstream regulatory sequence (URS1) and interacts with Sin3 through the repression domain

Sin3 is a subunit of a multiprotein complex that also contains RPD3, a histone deacetylase.

RPD3 deacetylates histone N-terminal tails around UME6 binding site toinhibit GTF assembly at the TATA box, repressing gene expression

46
Q

Methylation of cytosine, CPG islands

A

Cytosine is methylated which turns off gene expression

Found in some but not all eukaryotes: can be stably inherited during replication, some permanently silenced

Cpg island found upstream of promoter region. 4 common CG sequences stop transcription, they are methylated

47
Q

Importance of DNA methylation: Arabidopsis thaliana

A

No DNA methylation (B & D) then gene inactivation is affected, so more leaves/stalks

Identified every methylated site in the genome, look to see phenotypes and its correlation with methylation of cpg sites. Methylome- what’s methylated

48
Q

What is the enzyme that methylated DNA?

A

DNA methyl-transferases

49
Q

Methylation and cancers

A

Active gene- cell growth regulated, DNA repaired, cell death

Cancer- Cpg island promoter is heavily methylated but it is Important to regulate cell cycle, when there’s no control mechanism cells grow and divide. Uncontrolled cell growth, accumulation of DNA damage, resistance to cell death

50
Q

Post-transcriptional Control

A
  •   Alternative splicing
  •   Tropomyosin from different muscle types- some exons are specific to tropomyosin in skeletal muscle or in smooth muscle, some exons are common to both
51
Q

Alternative splicing

A

Splicing proteins identify sites on introns, can identify different sites. During spicing we end up with different exons being included in final RNA. Seem exons tightly correlated and always included together

Exon splice enhancers

52
Q

EJC - NMD

A

Exon junction complexes put down where exons are joined. Still there when translation begins, ribosome knocks those proteins off. When nonsense mutation, ribosomes hit the stop codon then there is a release factor and everything else dissassociates, but there are still ejcs on it which sends up a flag for mRNA to be degraded. Ribosome is associated with mRNA almost immediately so other mRNAs with ejcs aren’t degraded right away

53
Q

Post-Transcriptional Control of Gene Expression

A

•  Regulates the stability of mRNA in the cell

54
Q

miRNAs

A

–  Represses translation of specific mRNA

–  Short single stranded RNA (21-22 nt) that bind to mRNA at the 3’UTR in a mismatch manner and represses translation of mRNA

–  Naturally occurring

Mark things for degradation. Controls whether mRNA is translated. When it combines with an mRNA it deactivates/ degrades it

55
Q

siRNAs

A

–  Represses translation of specific mRNA

–  Short single stranded RNA (21-22 nt) that binds to mRNAperfectly and results in cleavage of mRNA

–  Targets mRNA for destruction

Made to manipulate gene expression, can see what happens to organism if you shut off one gene

56
Q

What do mRNAs and siRNAs both interact with?

A

Both interact with a multiprotein complex, RNA-inducedsilencing complex (RISC)

57
Q

miRNA and siRNA

A

It is a powerful experimental tool to study gene function.

RNA interference is believed to be an ancient cellular defense against certain viruses and mobile genetic elements in both plants and animals- Thought to evolved to control viruses. Viruses have single stranded mRNA genomes, miRNAs can silence them

miRNAs important during development ~250 genes encoding miRNAs in humans

58
Q

miRNA and siRNA processes

A

MiRNA and siRNA precursors can be single stranded (usually forms a loop) or double stranded

Dicer- ribonuclease, cuts mi/siRNA to proper size

RISC (RNA-induced silencing complex) binds to and inactivates mRNA

Argonaute- Carries the RISC where it needs to go. mRNAs can be targeted for degradation after miRNAs bind

59
Q

Do miRNAs and siRNAs have perfect base pairing

A

miRNAs don’t have to have exact match

siRNAs are man made, so perfect match

60
Q

Translational control

A

Life span of mRNA

Temperature

61
Q

Life span of mRNA

A

–  mRNA for casein, when females breast feeding , make in their milk ducts mRNAs for casein, prolactin increases lifespan of mRNA that translates to casein. When not lactating, no prolactin, so mRNAs degraded easier

–  mRNA in oocyte, In the nucleus of egg, mRNAs in there that lasted, then mRNAs became translated when sperms comes

62
Q

Temperature

A

–  Phosphorylation of ribosomal proteins

–  Protein folding- denaturation and end of function

–  Viruses- proteins denature more readily in viruses because we have hsp, therefore fever would kill them

63
Q

Post translational control

A

STATs

64
Q

STAT protein

A

Activating or inactivating proteins already present in the cell- Phosphorylation

Growth signal activates STAT protein. Signaling molecule binds to cell surface receptor, interacts with inactive proteins, dimerizes. Activates STAT protein stimulates transcription

LS (17 decks)

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