April 2 Lecture Outline

Developmental Cell Biology 220

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Lecture Outline for April 2, 1999

Need DNA-binding proteins to interact with RNA polymerase to initiate transcription. See Figure 10.8.

RNA polymerase II binds after TFIIB is in place. Both TFIIB and TFIID control the position of the polymerase on the DNA.
TFIIF - enzymatically unwinds the DNA helix.
TFIIE - ATPase associated with DNA
TFIIH - has protein kinase activity and adds phosphates to certain regions of RNA polymerase II. This makes it difficult for TFIID to bind and the RNA polymerase is released from the promoter to begin transcription.
TAFs (TATA-binding protein-associated factors) in Figure 10.9

Are important in regulating temporal-specific transcription (i.e., development)
Are important in regulating tissue-specific transcription of genes.
A given gene may have several different possible enhancer sites. Each enhancer may bind more than one protein factor to increase or decrease transcription.
Regulation of transcription is due to proteins bound at the enhancer sites interacting with proteins bound at the promoter.

Transcription factors (trans-regulators)

General structure contains an amino acid sequence which recognizes specific nucleotide sequences in the promoter and enhancer regions of the DNA.
General structure contains a trans-activating domain which may enhance the efficiency of building the transcription complex.
Homeodomain proteins (act to specify the anterior-posterior body axis)

Both the carboxy terminal and amino terminal interact with the DNA. There are 3a-helices in between.
Example: In Drosophila the bicoid protein is a transcription factor.

bicoid protein binds to promoter of the hunchback gene
the hunchback protein is a transcription factor which binds to enhancers of genes that are involved in forming the head and thorax.

contain a homeodomain, a POU domain, and a trans-activating domain.
Example: Pit-1 enhancer-binding protein. Pit-1 mRNA occurs in Rathke's pouch when it is histologically recognizable. 2-3 days later Pit-1 mRNA is translated. Growth hormone (GH) mRNA is translated when Pit-1 mRNA is translated. Pit-1 is a pituitary-specific factor which stimulates GH transcription and translation.
Example: Prolactin is synthesized in some pituitary cells. Prolactin is a hormone that stimulates milk production. Pit-1 protein binds to the enhancer and also binds with the estrogen/estrogen·receptor complex. The enhancer then can interact with the promoter (some 1300 base pairs away). This promotes transcription of the prolactin gene.
Pit-1 protein also binds to the enhancer for the Pit-1 gene. Why is this positive-feedback important in determining the fate of a cell's differentiation?

Example: E12, E47 are ubiquitous bHLH transcription factors which promote myogenesis in the presence of MyoD.

When Id transcription factor (inhibitor of differentiation) is present it binds E12 and E47. Id lacks a DNA-binding region.
FGF stimulates Id transcription
FGF activates protein kinase C, which phosphorylates myogenic bHLH proteins at the DNA binding sites, preventing binding to DNA.

basic leucine zipper transcription factors (bZip) are dimers with three different domains.

These have two or more 'fingers' which bind DNA.
The fingers are formed by interactions between cysteines and Zn and histidines.
Example: WT-1 is a transcription factor which binds to regulatory regions of genes active during kidney development.

Nuclear hormone-responsive elements are DNA sequences that bind hormone/hormone receptor complexes.
The hormone receptors contain 3 domains

Hormones acting in this way include: estrogen, progesterone, testosterone, cortisone, thyroxin, and also vitamin D and retinoic acid.
Sequence of events: hormone enters the cell, binds to the hormone-receptor protein, the hormone/hormone-receptor complex enters the nucleus and then dimerizes. Then the dimer binds to enhancer sequencesof the DNA.