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Foundations Week 7 > Gene Regulation I > Flashcards

Gene Regulation I Flashcards

1
Q

What experiments lead us to know that all cells have identical DNA content?

A

cloning experiments

  • the original experiments were carried out in the 1960s using frogs
  • cell nuclei isolated from skin cells could be used to regrow an entire animal
  • adult frog –> skin cells in culture dish –> nucleus in pipette –> inject nucleus into an unfertilized egg who’s nucleus was destroyed by UV light –> normal embryo formed which then became a tadpole!
  • most recently, process repeated for mammals; first mammal was Dolly the sheep from a mammary gland cell
  • underlying all of these studies is that the DNA present in a single cell contains the genetic information required to reconstitute the entire animal
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2
Q

Differences between cells depend on what?

A

the subset of genes that are active (transcribed)

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3
Q

Every cell must use the same set of genes to maintain basic cellular function; these include basic genes (examples?) and metabolic genes (examples?)

A

some examples of basic genes:
- ribosomal proteins, actin fiber proteins, etc.

some examples of metabolic genes:

  • ATP synthase
  • citric acid cycle enzymes
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4
Q

Cells with specialized functions must make _____ with specialized functions

A

proteins
ex: pancreatic cells make insulin
hair cells make keratins
eye cells make crystallin

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5
Q

What is the function of gene regulation?

A

transcription of different genes in different types of cells

  • after transcription, many additional levels of control are possible; these include alternative splicing, regulation of mRNA stability, translational control, etc
  • however, if a gene is not transcribed, all other forms of regulation are irrelevant
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6
Q

What do cells alter their pattern for?

A 
to respond to local cues for challenges
ex:
- respond to hypoxia (low oxygen)
- respond to heat stress
0 respond to increased BP
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7
Q

What are the components of RNA polymerase II?

A

it is a multi-subunit complex of 10-15 proteins

  • polymerase activity exists in a single component of the complex (RNA polymerase)
  • binding site for transcription regulator
  • repeating peptide sequence in the tail (with binding sites for RNA processing proteins)
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8
Q

What must RNA polymerase II interact with (bind to)?

A

the promoter region of protein coding genes

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9
Q

What is a promoter?

A

a region of DNA immediately surrounding the start site of transcription (where DNA copied into RNA)
- it contains gene regulatory sequences which are the binding sites for proteins called general transcription factors

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10
Q

What functions as the binding sites for proteins on a promoter?

A

gene regulatory sequences called general transcription factors

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11
Q

Promoters ______ contain gene speceific regulatory elements.

A

does not

*proteins are (experimentally) interchangeable between different genes)

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12
Q

Promoters are necessary, but not _____

A

sufficient

  • promoters do not contain gene specific regulatory elements
  • promoters do not control time or tissue of gene expression
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13
Q

Why does RNA polymerase need a promoter?

A

Because RNA polymerase II is not a DNA binding protein and has no particular affinity for dNA; it does not recognize a specific DNA sequence and bind it.

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14
Q

What is required to initiate efficient transcription?

A
  • other proteins are required to initiate efficient transcription; some, but not all, of these proteins are DNA binding proteins
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15
Q

How does RNA polymerase II locate itself at the correct transcription start site?

A
  • RNA polymerase II locates itself at the correct transcription start site by binding to the other proteins. The complex of RNA polymerase II with the other proteins is called the transcription initiation complex
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16
Q

What is the complex of RNA polymerase II with other proteins called?

A

the transcription initiation complex

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17
Q

What are the three typical general promoter sequences?

remember, these aren’t the only ones, not one of these is present in every promoter

A
  • TATA box: this is bound by TATA-binding protein (TBP), which is a component of the TFIID complex (A-T rich, meaning less stable)
  • the CCAAT box: bound by members of the CAT binding protein family
  • the GC box: binding site for the stimulatory protein family (Sp1-5
  • proteins that bind to the general promoter sequences are found in all cells at all times
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17
Q

Gene-specific positive regulatory elements are often called what?

A

enhancers

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18
Q

What is the significance of the A-T rich TATA box?

A

it is less stable due to A-T richness

  • note: TATA box is about 25 bases up from transcription start site
  • TATA box is the recognition sequence for a DNA binding protein complex called TFIID; TFIID contans TBP
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19
Q

What effects does the presence of gene-specific positive regulatory elements (enhancers) have?

A

the presence of these DNA sequences increases the level of transcription from an adjacent promotor

  • they control the time and tissue of expression
  • only work on promoter that is closest to them
20
Q

What are enhancers bound by?

A

positive regulatory proteins

21
Q

Enhancers are found at variable distances from the promoter, either upstream or downstream from a gene; as much as 200 kilobases away from the promoter. But if the promoter is 200 kilobases away, what does this suggest about other promoters?

A

any other promoters are further away; this promoter is the closest to the enhancer

22
Q

Enhancers appear to act at a distance, but what really happens to allow the protein bound to the enhancer to interact with the promoter?

A

DNA loops around

23
Q

Promotor sequences and enhancers are what?

A

regulatory sequences, to some extent

  • enhancers are specific (exactly with what cell, with what efficiency, and what time)
  • promoters are not
24
Q

Enhancers (upstream or downstream) are always on the same ____ of the scaffold as the ________.

A

loop

promotor (at beginning of gene)

26
Q

Negative regulatory elements are often also called what?

A

repressors (but proteins that bind to them are also sometimes called repressors)

*these may also be located at variable distances from the promoter

27
Q

What does the presence of negative regulatory elements (repressors) cause?

A

the presence of these sequences reduces levels of transcription from the adjacent promoter

28
Q

What are repressor sequences bound by?

A

negative regulatory proteins

29
Q

Assembly of the transcription initiation complex:

What does the initiation complex consist of?

A

a large number of proteins

  • these must all assemble at the promoter region
  • efficient binding of these protein factors appears to be facilitated by other proteins bound to the DNA in the promoter region, such as CAT binding proteins and Sp1
  • enhancer binding proteins can interact with this complex
30
Q

What is the first step of forming the transcription initiation complex?

A

binding of the TFIID complex to the TATA sequence

TATA-binding protein (TBP) is part of the TFIID complex. TBP causes bending (70 degrees) of the DNA duplex which opens up the helix (a minor groove) which allows entry of proteins that can unwind the DNA at the front (ultimately, by helicase)

31
Q

Assembly of the transcription initiation complex:

After TFIID complex has bound to the TATA sequence, what occurs?

A
  • soon after this, RNA polymerase II becomes part of the initiation complex
  • this association is quite unstable
  • despite the presence of RNA polymerase II, transcription does not commence at this time
32
Q

Assembly of the transcription initiation complex:

After TFIID complex has bound the TATA sequence and RNA polymerase II has become part of the initiation complex, what happens?

A
  • a protein with helicase activity joins the complex

- this is responsible for unwinding the DNA at the start site of trasncription

33
Q

Assembly of the transcription initiation complex:

After TFIID complex has bound the TATA sequence and RNA polymerase II has become part of the initiation complex, a protein with helicase activity joins and unwinds the DNA at the start site of transcription. Then what happens?

A

I believe this is when positive regulatory proteins bind

  • the major function of positive regulatory proteins is to stabilize the RNA polymerase II complex to the initiation complex
  • once assembly is complete and stable, transcription can commence
34
Q

CAT binding proteins and Sp proteins do what for the pre-initiation complex?

A

they stabilize the pre-initiation complex, together with TFIID and other proteins
*proteins that bind the general promoter are present in all cells at all times

34
Q

What is the overall structural result of the positive regulatory proteins?

A

they make a more stable environment for RNA polymerase II in the complex
*when the initiation complex is stable, transcription can commence

You have made this place that is so efficient that DNA can be transcribed almost as fast as an RNA polymerase II can come in and bind.

35
Q

fill in

A

fill in

36
Q

What is the effect of having multiple regulatory proteins (at the promoter site?)?

A

multiple positive regulatory proteins synergize to increase transcription from a promoter

38
Q

What forms the pre-initiation complex?

A

total repertoire of basal promoter proteins, plus transcription activator proteins

38
Q

Mutation of any bases in the TATA element results in what?

A

large reduction of transcriptional activity

39
Q

recruitment of repressive chromatin remodeling complexes

A
  • some activating proteins recruit chromatin remodeling factors
  • these can alter the conformation of the chromatin in the vicinity of the promoter and facilitate the binding of proteins of the preinitiation complex
  • one of the components of TFIID specifically binds to acetylated histones; therefore, acetylation of histones increases the affinity of TFIID for the promoter region and promotes binding to the TATA box
  • remodeling of some of the nucleosomes by the chromatin remodeling complex?
  • these alter conformation of chromatin near the gene, usually opening up he region of the chromosome so that it is more accessible –> high-level expression of genes in the loop
40
Q

Some inhibitory factors may recruit chromosome remodeling factors like histone deacetylases

What doe histone deacetylases do?

A

histone deacetylases reduce the affinity of TFIID for the promoter; therefore, a transcription complex is less likely to form

41
Q

Methylation can regulate gene activity

What are the two pathways for this?

A

human DNA can by METHYLATED SPECIFICALLY ON THE CYTOSINE RESIDUE OF CG BASE PAIRS; these modified residues are the targets of METHYLCYTOSINE BINDING PROTEINS, which in turn can do two things:

  • RECRUIT CHROMATIN REMODELING PROTEINS that CONDENSE THE CHROMATIN and make it less accessible to activating proteins
  • methylcytosine binding proteins can also RECRUIT HISTONE DEACETYLASES which reduce the affinity of chromatin for TFIID
42
Q

Methylation is believed to be important for what?

A

inactivating the very large stretches of DNA that are non-coding but which contain old viral sequences and transposable elements

43
Q

How are patterns of methylation preserved during DNA replication?

A

an enzyme preferentially modifies the new strand of DNA opposite the site of an existing methylcyotosine

44
Q

Overall, what is the goal of methylation?

A

to suppress gene activity

45
Q

Interesting fact:
at fertilization, the methylation of DNA is completely stripped away and reestablished about 5-7 days later, at the time of implantation. What can this cause?

A
  • during the first 5-7 days, the total number of transcripts from ancient retroviral genomes and transposable elements inserted into the human genome appears to outweigh normal cellular transcripts in the embryo
  • it is during this time that transposable elements may become mobile and insert into new locations in the genome
46
Q

Mutation of any bases in the TATA element results in what?

A

large reduction of transcriptional activity

47
Q

Binding of TBP to TATA sequences causes what?

A

extreme bending of the DNA and opens up the minor groove

*this facilitates initial separation of the two strands of DNA so that transcription can occur

Foundations Week 7 (10 decks)

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