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GN 311 > Lec 27: Regulation of Eukaryotes > Flashcards

Lec 27: Regulation of Eukaryotes Flashcards

1
Q

Transcriptional controls

A 
1. Transcription
2 splicing and processing
3 transport to the cytoplasm
4 degradation of mRNA
5 translational regulation
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2
Q

Chromatin structure with transcription

A
  • is very important in regulation-affects gene expression
  • genes must unwind from the histones before transcription can take place
  • transcriptionally active DNA is loosely coiled
  • PUFFS and balbani rings in active areas
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3
Q

DNase I Hypersensitivity Sites

A
  • as regions become transcriptionally active, regions around the genes become highly sensitive to the action of DNase I. These regions are called DNase I HYPERSENSITIVITY SITES
  • develop about 1000 nucleotides upstream from the start of transcription
  • allows for more open configuration of chromatin this allows for relaxation of chromatin therefore regulatory proteins can gain access to binding sites on the DNA
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4
Q

What are the 3 processes that affect gene regulation?

A

DNA methylation
Histone Methylation
Chromatin Remodeling

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5
Q

Histone structure

A

2 domains:

  • globular domain interns with other histones and the DNA
  • Positively charges tail interacts with negatively charge phosphate groups on DNA
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6
Q

Methylation of Histones

A
  • can either increase or decrease transcription depending on which amino acid are methylated
  • can activate or repress gene expression (usually reprises)
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7
Q

DNA methylation

A
  • by DNA methyl Transferase tends to cause genes to be TURNED OFF (silenced).
  • 5mc, common at CpG island near the start of transcription
  • heavily methylated causes silence
  • Methylated CpG sequences “attract” deacetylases further repressing transcription
  • In genreal an increase in methylation results in decreased transcription of DNA
  • 40%of genome is GC pairs
  • 27% of GC paris have 5-methylated cytosines
  • High methylation in BARR BODIES- female mammals have inactive X chromosome that have extensive methylation
  • Repetitive sequences tend to be heavily methylated
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8
Q

Acetylation of Histones

A
  • weakens interactions with DNA and may allow transcription factors to bind to DNA
  • acetylation usually stimulates transcription
  • the addition of Lysine prevents formation of the 30 nm fiver, causing chromatin to vein open configuration and available for transcription
  • enzyme histone acetyl transferase
  • Loosens DNA-histone association by neutralizing the positive charge on the histones
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9
Q

Deacetylaton of HIstones

A
  • restores chromatin structure, which represses transcription
  • enzyme Histone deacytlases
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10
Q

Acetylation in Arabidopsis

A
  • controls flowering
  • FLC gene-supressing flowering (until after an extended period of coldness) when it is turned on
  • FLC gene encodes a regulatory protein that represses the activity of other genes that affect flowering. Acetylation of the FLC gene turns the gene on preventing flowering
  • The activity of the FLC is controlled by FLD-stimulates flowering by repressing the action of FLC
  • FLD encodes a deactylase enzyme which removes acetyl groups from histones
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11
Q

Chromatin Remodeling

A

SLIDING THE NUCLEOSOME ALONG THE DNA so that DNA can change from part of the nucleosome to part of the area between nucleosome

CONFORMATIONAL CHANGES IN THE DNA OR NUCLEOSOMES to allow the DNA bound to the nucleosome to be more exposed and more able to bind transcriptional activators

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12
Q

Promoter

A

TATA box: deletions alters site of transcription

CCAAT and GC boxes: required for efficient transcription

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13
Q

Enhancer

A
  • cis acting, but not always next to gene
  • non-specific- increases transcription of nearby genes
  • Act at a distance and are orientation independent
  • Promote transcription from nearby promoters
  • may enhance more than one gene
  • may work at a distance from a promoter
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14
Q

Insulators

A
  • Blocks (insulates) genes from the effects of the other genes
  • Cis-element but can have different effects
  • Boundary element that function to insulate the effects of enhancers
  • position dependent manner to block enhancers from genes that they should not affect
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15
Q

Silencers

A

cause repression (inhibit transcription) by binding to elements in the regulatory region or distant to the promoter.

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16
Q

Transcriptionally active DNA characteristics

A

Loosely coiled

-Puffs and Balbiani rings

17
Q

ROLE OF nonhistones in gene regulation

A

nuclear scaffold

18
Q

Where do Transcription factors bind

A

bind to DNA (H bonds), other transcription factors, and polymerase to regulate transcription

19
Q

DNA Binding Domains

A

Leucine Zipper
Helix-Turn-Helix (homeodomain)
Zinc Fingers

20
Q

Induction of Transcription

A

Coordinated Gene Regulation can occur
– Response elements: common regulatory elements upstream from the start sites of a collective group of genes that respond to a common environmental stimulus

Environmental and Nutritional Factors may induce transcription
– Temperature (Heat-Shock Proteins)
– Light (RBC = ribulose 1, 5, Bisphosphate Carboxylase)
– Hormones:
1) steroids: progesterone, estrogen, testosterone
2) Peptide: insulin, prolactin

21
Q

Heat Shock Genes

A

Sudden heat causes increased expression of some genes

-regulated at transcription level

22
Q

Heat shock genes in Drosophilia

A

HSTF is
phosphorylated when animal is heat stressed and binds to specific sequences to make the gene more accessible to RNA polymerase II.

23
Q

Light Induces RBC

A

an enzyme critical for photosynthesis

  • transcription is stimulated by light
  • se increase in RBC transcript in leaves after exposure to light
24
Q

Northern Blot

A

Shows relative amounts of RNA present

25
Q

Hormonal Regulation

A

Some hormones bind to cytoplasmic receptor and then complex binds to DNA to regulate gene expression – eg. steroids
•
Some hormones act at cell surface and appear to signal transcription factors - eg. insulin

26
Q

Transcription factors can:

A

-Interact with other proteins
• Be modified (resulting in altered action)
• Be localized (different ones present in different places)
• Be degraded

27
Q

Alternative Splice Sites in mRNA processing

A
  • Can result in different proteins in different tissues or different times in development
  • Regulation of splicing probably is a very important mechanism for controlling eukaryotic gene expression
28
Q

Alternative Splicing (intron removal) in Drosophila Sex Determination

A

-Sxl (sex lethal) is the master regulator of sex determination in Drosophila
-Ratio of X chromosomes to autosomes determines whether or not Sxl will produce a protein
– If the X:A ratio is 1.0. the Sxl protein is produced. Which sex is this? Female
– If the X:A ratio is 0.5, the Sxl protein is not produced. Which sex is this? Male

  • Processing occurs to make a functional polypeptide in females and a nonfunctional polypeptide in males
  • Different Exons put into proteins depending on sex chromosome composition
  • Cascade-Gene product at one locus directs splicing of pre-mRNA at next locus to cause development to go along one path or the other. Similar decisions at each developmental step.
29
Q

Evidence for Control at Translation Level

A
  • Big increase in protein synthesis after egg fertilization without RNA synthesis
  • Activation of T-cells includes a sudden increase in translation prior to T cell proliferation and seems to be due to an increase in translation initiation factors that cause an increase in protein synthesis which in turn causes a proliferation of T cells.
30
Q

Transport Control

A

Additional Levels of Gene Regulation

  • 1⁄2 of primary transcripts degraded in nucleus
  • Spliceosome retention model
31
Q

Degradation of mRNA

A
  • Amount of protein synthesized depends on availability of mRNA for translation
  • Amount of mRNA available depends on rates of synthesis and rates of degradation
32
Q

mRNA Degradation by Ribonucleases

A

10 or more types of ribonucleases
• Interaction between nucleases and specific
mRNA internal structure is important
• Typical mechanism:
– Poly A tail is shortened by RNases
– Once tail is at critical length, 5’ cap is removed – RNA is then degraded from the 5’ end
– other mechanisms are possible such as cleavage at internal sites.

33
Q

Mechanisms of Gene Regulation by RNAi

A

1mRNA cleavage
– siRNA combines with proteins and binds mRNA and then cleaves the mRNA (slicer activity). mRNA then degrades.
2Inhibition of translation
– pairing of miRNA with mRNA can decrease translation of the mRNA
3Transcriptional silencing
– siRNAs alter chromatin structure by binding to the chromatin, attracting enzymes that methylate the tails of histones. This causes DNA to bind the histone more tightly and decreases transcription.
– Direct methylation of DNA by miRNAs also decreases transcription
4Slicer independent degradation of mRNA
– mechanism not clear, but involves miRNA binding to mRNA

34
Q

RNAi

A

in eukaryotes shuts off gene expression using double stranded RNAs
dsRNA is cleaved by the enzyme dicer to form miRNA or siRNA that is about 24 nts long

35
Q

miRNA

A

is transcribed from a distinct gene and targets other genes for regulation

36
Q

siRNA

A

comes from mRNA, transposons or viral RNA and targets the genes that it comes from.

37
Q

what happen when miRNA binds with siRNA

A

bind with proteins to form a complex that base pairs with mRNA and either
• inhibits translation (miRNA) or
• degrades mRNA (siRNA)

GN 311 (17 decks)

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