Eukaryotic Promotors and Gene Structures

In prokaryotic cells the focus of the regulation of gene expression is almost entirely at the level of the initiation of transcription. However, in eukaryotic cells, regulation of gene expression can occur at all stages of transcription and translation, and even at protein stability.

Opportunities for Gene Regulation in Gene Expression in Eukaryotes

Those common to prokaryotes and eukaryotes

1. Initiation of transcription
2. mRNA stability
3. Initiation of translation
4. Protein stability

Those unique to eukaryotes

1. Chromatin structure
2. RNA processing
3. Transport to cytoplasm

RNA Polymerase

Prokaryotes need only one type of RNA polymerase for transcription. However, in eukaryotes, three are involved. These include RNA polymerase I, RNA polymerase II, and RNA polymerase III.

RNA pol I

• Transcribes ribosomal RNAs
• Promotor is located at -45 to +20 position
• Located in nucleolus
• Has 14 subunits

RNA pol II

• Transcribes what will become mRNAs
• Promotor is located way upstream (up to -70,000) to -25
• Located in the nucleoplasm
• Has 12 subunits

RNA pol III      

• Transcribes snRNAs and tRNA
• Promotor located +50 to +100. (Note that this position is downstream. Therefore, RNA polymerase first binds to the promotor and then backs up to the transcriptional start site and then transcribes through the promotor)
• Located in the nucleoplasm
• Has 17 subunits

Modularity of Eukaryotic Promotors

RNA polymerases do not independently cause the initiation of transcription. For them to work, several proteins called transcriptional factors and mediator proteins, must come into play, resulting in binding the RNA polymerase at the promotor site. Other proteins such as those causing bending of the DNA may also be necessary. Having all the right transcriptional factors together ensures that the right genes are expressed at the right time.

Steps in Transcriptional Complex Assembly

1. TFIID binds to the TATAAA box with the help of a protein called the TATA-binding protein (TBP) which is a part of TFIID
2. TFIIA and TFIIB binds to TFIID
3. TFIIF binds to TFIID and TFIIB while recruiting RNA polymerase II
4. Other proteins including TFIIE and TFIIH bind to the promotor, completing the initiation complex

The specific protein factors used in the transcriptional complex may be ubiquitous or specific to the function required (e.g. response to heat shock or growth factors), the location of the cell, or the phase of development of the organism (e.g. embryo, infant, or adult).

Without the appropriate proteins to assist an RNA pol II, no expression of a gene will occur. At the same time, inappropriate expression of genes is possible by introducing gene-activating proteins where they are not needed. This has been demonstrated by experiments with fruit flies conducted by Walter Jakob Gehring (1939-2014). In his experiment, Gehring and his team genetically engineered fly larvae to produce a gene-activating protein called GAL4 in many different body parts including wings, legs, and antennae. The result was the production of flies with eyes in a variety of unusual anatomical positions.

How Proteins are Able to Bind DNA

Proteins are able to interact with the DNA via interactions with its minor and major grooves. These grooves allow proteins to access elements along the edges of up to 6 base pairs that protrude into these grooves. Three of the main mechanisms by which they are able to accomplish this include the zinc finger motif, the helix-turn-helix motif, and the leucine zipper.

1. Zinc finger motif. This motif is characterized by stabilizing zinc ions which create finger-like folds in the protein. These folds are able to fit into the major groove of the DNA. The transcription factor TFIIIH, is a good example of this motif.
2. Helix-turn-helix motif. This motif consists of two alpha helixes, a long and a short one separated by a proline amino acid. The unique structure is able to fit into the major groove.
3. Leucine zipper. This motif is characterized by a series of leucine amino acids connecting two alpha-helix peptide chains resulting in a crisscrossed protein structure. The “unzipped” ends of the cross can fit into the major groove.

Reference: Krane, D. 2021. Bio 2110 Molecular Biology Video Lecture. Wright State University – Lake Campus.