Mechanisms regulating gene expression

Question 1

What is involved in the regulation of gene transcription?

Answer 1

Transcription- chromatin structure, histone modification (epigenetic), regulatory proteins (transcription factors)

DNA has non coding regions that regulate both transcription and translation.

Question 2

List the gene regulatory regions

Answer 2

Promoter = core promoter and proximal promotor region

1) Core promotor comprises DNA sequence within -40 to +40 of start of transcription (contains TATA box)
2) promotor proximal region -200 to +50 bp relative to RNa start site (contains the CCAAT) box
3) reguatory elements- enhancers (positive regulation) or repressors/ silencers (negative regulation). Bind transcription factor proteins (non covalently) that interact through DNA looping with basal transcription machinery. these elements act independently of orientation and distance to RNA transcription start site. So if you take a sequence and invert it, regulatory elements will still bind.
4) boundary elements are DNA segments at the ends of a gene region that function as insulators and bloack the influence of positive or negative DNA elements from affect adjacent genes. ex: prevent spread of heterochromatin

Question 3

What modifications can be made to histones and how do they affect transcription?

Answer 3

1) deacetylation - generates heterochromatin (condesnsed-inert chromatin) less transcription
2) acetylation - euchromatin (open active chromatin) more transcription
3) methylation- gene repression or expression depending on the residue methylated and the number of methy groups added.

Question 4

Explain how regulation of chromatin strucure acts cooperatively with transcription factor proteins to regulate gene expression.

Answer 4

Histone acetylation/ deacetylation, methylation/demethylation, and ATP dependent chromatin remodeling enzymes (SWI/SNF proteins) act to either make DNA more or less accessible to the protein complexes involved in transcription to eith up-regulate or down-regulate DNA transcription. facilitates the assembly of general transcription factors, mediator, and RNA polymerase at the promoter.

Question 5

Describe the order of events leading to transcription

Answer 5

Gene activator protein binds to chromatin → chromatin remodeling complex comes in and remodels chromatin → Histone modification enzymes covalently modify histones → Additional activator proteins bind to the gene regulatory region → mediator, general transcription factors, and RNA polymerase bind to assemble the pre-initiation complex at the promoter → binding of other activator proteins and rearrangement of proteins in the pre-initiation complex leads to transcription initiation.

Question 6

Regulatory elements (enhancer or repressor)

Answer 6

1) short sequences of DNA that bind to the transcription factors
2) can be located up to 50kB from the start site of transcription
3) regulatory elements can be placed upstream or downstream from start of transcription
4) Each regulatory element may bind multiple trancription factors

Question 7

histone acetylation

Answer 7

histone acetylation opens the chromatin. Lysines in the histone tails are acetylated, eliminating the side-chain positive charge (ammonium +) This breaks the charge interactions between adjacent nucelosomes allowing the chromatin structure to open.

done by histone acetyl transferases (HAT)

Question 8

Histone deacetylation

Answer 8

generates heterochromatin. catalyzed by histone deacetylases HDACs

Question 9

Histone tail modifications

Answer 9

Histone tails can be modified by histone methylases, adding a methyl group to lysines and arginines or histone kinases which ad phosphates to serine side chains

each modification type attracts proteins that specifically bind to the chemically modified site.

Question 10

Chromodomains

Answer 10

Domains on proteins that specifically bind to methylated lysines and arginines.

Question 11

bromodomains

Answer 11

domains of proteins that specifically bind to acetylated lysine.

Question 12

Properties of transcription factors

Answer 12

1) have modular designs with an activation domain, dimerization domain, and a DNA binding domain
2) The DNA binding domain often contains a structural morif with amino acis that interact with a unique DNA element exposed by the major and/or minor groove of the DNA helix.
3) Commonly (but not always) transcription factors are homo- or hetero-dimers. (i.e. HTH, HLH, and bZIP TFs)
4) Dimers bind to a palindrome in the DNA, which contains a symmetry appropriate for dimer binding to two succesive turns in the DNA helix

Question 13

Explain the modularity of TF proteins

Answer 13

Transcription factors can be divided into modules (domains). Usually have a dimerization module, have a DNA binding domain (module). Has an activation region (domain). Activation domain is the active part of the molecule can repress as well as activate. Never have observed the structure of an activation domain because they are dynamic and unobservable through crystallization technologies. These modules can be separated from one another. You can take a DNA binding domain and attach it to the domain of an activation domain.

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Question 14

Characteristics of helix-turn-helix

Answer 14

1) Each monomer contains a recognition alpha-helix joined by a short turn to a second alpha helix. The second helix supports the recognition helix by hydrophobis interactions.
2) The HTH is embedded in a domain that may be all alpha, an all beta, or an alpha-beta type of domain. The type of dimer interface interaction will depend on the transcription factor protein
3) In dimer HTH proteins, the recognition helices from each monomer bind to adjacent turns to the major groove. The DNA site is palindromic
4) the two copies of the recognition helix are are seperated by exactly one turn (3.4 nm)
5) Each half of the paindrome sequence binds a monomer

Question 15

Characteristics of the Helix loop helix proteins

Answer 15

1) Dimer structure. The two monomers are held together in a four helix bundle: each monomer contributes 2 alpha helices connected by a flexible loop of protein. A specific DNA sequence is bound by the two alpha helices that project from the frou-helix bundle.
2) each monomer contains a recognition helix joined by a loop to a second helix, which contins a leucine ziper motif.
3) The recognition helices from each monomer bind to adjacent turns of the major groove (usually a palindrome)
4) Myc, Max, Mad and MyoD

Question 16

Describe the binding of the leucine zipper motif

Answer 16

The leucines are in alignment at one edge of the helix (residues 1,8,15,22) forming hydrophobic interactions that keep the dimer together.

Question 17

Describe the structure of zinc finger motif

Answer 17

Zn atom is bound to teo Cys and two His or four Cys, each seperated by 2-3 amino acids. Each pair of Cys/His is seperated by 12-14 amino acids from the next pair of cys/his. This chain has an alpha helical segment and a beta sheet segment.

Typical TF has multiple Zn finger motifs that bind in tandem to adjacent turns of the major groove of the DNA.

Each recognition helix recognizes a specific piece of DNA, highly specific.

Question 18

Describe the structure of a protein containing the bZIP motif

Answer 18

Binds as a homo or hetero dimer to DNA

The dimer forms and apha helical scissor structure, each monomer is a long alpha-helix binding to a half-site of the palindromic element in the DNA.

DNA binding domain contains multiple basic amino acids.

dimerization is throug a leucine zipper (coiled-coil hydrophobic interaction)

Question 19

Describe the differences in binding of bZIP homo and heterodimers

Answer 19

homodimers bind to symetric DNA sequences and heterodimers bind to hybrid DNA sequences.

Question 20

What are the methods to analyze regulatory elements in gene expression

Answer 20

footprinting with DNAaseI (1) for transcription actor binding sites

Gel-mobility shift assay to show protein-DNA complexes

Chromatin immunoprecipitation (CHIP assay)

Reporter genes to assay regulatory elements in specific cells

Question 21

Describe DNA footprinting

Answer 21

Used to determine where an element might exist in a gene regulatory unit. Use PCR to generate copies of the DNA of the regulatory unit of the gene of interest where you think a regulatory protein may bind. Radioactively label the DNA, and mix it with cell lysate. If the lysate contains the protein that binds to the regulatory unit of the gene it will bind to it. Add a limiting amound of nuclease (1 phosphodiester bond per DNA oligo nucleotide unit). You will get DNA cut into successive lengths. The DNA bound to the protein will not be degraded. Use gel electrophoresis to seperate the DNA stands by weight. The region where the protein binds will show up as blank because it protected the DNA from the nuclease. so you now know the region of DNA that the TF binds to

Question 22

Gel mobility shift assay

Answer 22

Compare a radioactive fragment of DNA to a radio active fragment of DNA mixed with cell lysate containing the proteins that bind to the DNA. The protein(s) will bind to the DNA and it will move more slowly through the gel. Bands of differing intensity mean that different amount of protein is binding.

Question 23

CHIP assay (chromatin immunoprecipitation assay)

Answer 23

Used to isolate the DNA sequence that a regulatory protein binds to. Add formaldehyde to a cell to crosslink the proteins to DNA. Lyse the cell and use nuclease to break the DNA into small fragments. Add an antibody to the protein of interest, the antibody will cause the transcription factor bound to the DNA to precipitate (along with the DNA it is bound to). Make the solution acidic to reverse the crosslinks. This frees your DNA fragments to which you add primers, and amplify the DNA using PCR.

Question 24

Reporter Gene assay

Answer 24

Used to study an enhncer. The reporter gene is usually insect or bacterial genes (non mamalian). Insert this gene downstream from the enhancer elements. It is distance independent so it doesn’t have to be inserted right next to the enhancer. Transfect the reporter gene element into the cell. The level of gene expression will depend on the amount of transcription factors in the cell that will bind to the enhancer element in the reporter gene. The more transcription factors, the more product you will produce, the greater “report” you will see i.e. amount of light.