Pamela D. Popken-Harris, Research Associate
Michael R. Sullivan, Research Associate
a. A 50bp DNA sequence containing 434 bacteriophage operators OR1 and OR2 in linear conformation with two 434 cI repressor protein dimers docked at the operators.
b. Two glucocorticoid receptor protein (GR) dimers docked at 2 glucocorticoid response element (GRE) sites within a 50bp DNA sequence from mouse mammary tumor virus (MMTV) 5' long terminal repeat (LTR) in linear conformation (close-up of c).
c. Five GR dimers docked at five GRE sites within a 160bp DNA sequence from MMTV 5' LTR in linear conformation.
d. Five GR dimers docked at five GRE sites within a 160bp DNA sequence from MMTV 5' LTR in super-helical conformation.
e. Five GR dimers docked at five GRE sites within a 160bp DNA sequence from MMTV 5' LTR in super-helical conformation with histone protein core octamer shown.
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"Molecular Dynamics Simulation in Solvent of the Phage 434 cl Repressor Protein DNA Binding Domain Amino Acids (R1-69) in Complex with its Cognate Operator (OR1) DNA Sequence," L.F. Harris, M.R. Sullivan, and P.D. Popken-Harris, Journal of Biomolecular Structure & Dynamics, 17, p. 1 (1999). |
These researchers are conducting experiments investigating the mechanisms of a genetic switch controlled by DNA regulatory proteins. The group previously reported that a high degree of nucleotide similarity is conserved between nucleotide subsequences of the c-DNA that encode regulatory proteins, DNA recognition alpha helices, and their cognate operators or hormone response elements within the DNA to which they specifically bind. By model building, it was observed that amino acids located within the DNA recognition helices are spaced so that they align with trinucleotides identical to cognate codon-anticodon nucleotides within the DNA major groove halfsites and flanking regions of their DNA binding sites. These findings suggested that these amino acids may interact with their codon-anticodon nucleotides within their cognate DNA binding sites. These observations allowed the researchers to hypothesize a mechanism for a site specific DNA recognition code for DNA regulatory proteins. Molecular dynamics simulations are currently being conducted in solvent to investigate hydrogen bonding, van der Waals, and electrostatic interactions between amino acids of the DNA regulatory proteins and nucleotides of their specific DNA binding sites. Findings by this research group indicate that amino acids within the DNA recognition helices of both prokaryotic and eukaryotic regulatory proteins are interacting with their cognate codon-anticodon nucleotides within their specific DNA binding sites.
Preliminary findings suggest that the estrogen receptor (ER) and glucocorticoid receptor (GR) proteins might function at unique DNA binding sites in a manner similar to the classical genetic switch seen with the 434 bacteriophage repressor proteins interacting with their cognate DNA operator sites, see figure1a. This hypothesis is being investigated in both wetwork and computer experiments. It has been observed, using a gelshift assay, that the ER protein binds to non-consensus glucocorticoid response elements (GREs) located within the 5, LTR upstream of the MMTV proviral genes. A DNA sequence from the MMTV 5, LTR containing a consensus GRE and an adjacent non-consensus GRE site is shown in figure 1b. These two GRE sites, of the five GRE sites located within the MMTV 5, LTR sequence, see figure1c, are similar to the 434 bacteriophage operator sites comprising a genetic switch ,see figure 1a. These researchers are preparing to compute molecular dynamics simulations in solvent on the protein/DNA model of the putative GR/ER genetic switch as shown in figure 1b.
These researchers realize the importance of studying eukaryotic regulatory elements in their natural context to develop a better understanding of the requirements for controlling expression of transfected genes. Recently, a high-resolution crystal structure of a nucleosome core particle of chromatin was determined. This structure provided essential information for constructing an exact nucleosome model of the MMTV 5, LTR system that this group has studied for over a decade.
The chromatin structure of the MMTV LTR is highly ordered. In fact, one nucleosome encompasses the entire DNA sequence shown in figure 1c. These researchers are preparing a nucleosome model of the MMTV 5, LTR DNA sequence shown in figure 1c, with five GR dimers docked at the five GRE sites contained therein, see figure 1d and e.
Genetic engineering experiments designed to turn on or off gene transcription under the regulation of the steroid hormone receptor superfamily of DNA regulatory proteins are being continued, especially for the ER and GR proteins. These researchers are working interactively with both benchwork and computational methodology using the information gained to design laboratory experiments and to refine computer models. This back and forth experimentation using computers and benchwork is the basis for research in molecular medicine.
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URL: http://www.msi.umn.edu/about/publications/annualreport/ar2000/depts/MedSchool/Surgery_Abbott_Cancer/schultz.html |
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