Transcription factor purification

The general transcription factor TFllD is believed to be the key link between specific transcription factors and the general preinitiation complex. However, the purification and molecular characterization of TFllD from higher eucaryotes have been hampered by its instability and heterogeneity. All preparations of TFllD contain the TATA box-binding protein in combination with a variety of different proteins called TBP-associated factors, TAFs. When the preinitiation complex has been assembled, strand separation of the DNA duplex occurs at the transcription start site, and RNA polymerase II is released from the promoter to initiate transcription. However, TFIID can remain bound to the core promoter and support rapid reinitiation of transcription by recruiting another molecule of RNA polymerase. 

Gabrielsen, O. S., Homes, E., Komes, L., Ruet, A., and Oyen, T. B. (1989) Magnetic DNA affinity purification of yeast transcription factor T—a new purification principle for the ultrarapid isolation of near homogenous factor. Nucleic Acids Res. 17,6253-6267. 

The various transcription-control elements found in eukaryotic DNA are binding sites for regulatory proteins. In this section, we discuss the identification, purification, and structures of these transcription factors, which function to activate or repress expression of eukaryotic protein-coding genes. 

In vitro transcription by purified RNA polymerase II requires the addition of several Initiation factors that are separated from the polymerase during purification. These Initiation factors, which position polymerase molecules at transcription start sites and help to melt the DNA strands so that the template strand can enter the active site of the enzyme, are called general transcription factors. In contrast to the transcription factors discussed in the previous section, which bind to specific sites in a limited number of genes, general transcription factors are required for synthesis of RNA from most genes. 

Tang L, Grimm A, Zhang Y-X, Hutchinson CR (1996) Purification and characterization of the DNA-binding protein DnrI, a transcriptional factor of daunorubicin biosynthesis in Streptomyces peucetius. Mol Microbiol 22 801-813... 

Purification of general transcription factors, especiaUy TBP, has to be carried out at the maximum possible rate to prevent protein aggr ation over time. Concentration of GTFs can be performed only in a stirred cell to prevent formadon... 

The appearance of a distinct band corresponding to the accurate transcript of rat rDNA in an unfractionated hepatoma nuclear extract in response to exogenous HS-C is of considerable interest. This observation raises the possibility of using such a system to study rDNA transcription in response to a variety of physiological stimuli that are known to alter rRNA synthesis. Unfractionated samples have certain advantages over the fractionated samples since purification procedures could result in differential recovery of the transcription factors from control and test samples. 

Since then several transcription factors have been purified using DNA-affinity chromatography. The application of DNA-affinity chromatography is not limited to purification of transcription factors. All DNA binding proteins which show sequence specificity can be purified by this technique. 

Polynucleotide-ogarose. As discussed above, oligo-dT columns are used for purification of mRNA. Poly-U columns may also be used instead of oligo-dT. Similarly, specific polynucleotide sequences covalently linked to agarose may be used to purify transcription factors and other DNA-binding proteins. 

Austin R.J. and Biggin M.D. 1996. Purification of the Drosophila RNA polymerase II general transcription factors. Proc. Natl. Acad. Sci. 93 5788-5792. 

The purification and structural and functional characterization of the general initiation factors has proven extremely difficult. The specific function of the various factors, as well as their structural role in the entire complex, remains poorly resolved. According to the current model, the general transcription initiation factors, with which an exact start of transcription is possible in vitro, are required for the formation of a basal... 

K9. Kruse, B., Narasimhan, N., and Attardi, G., Termination of transcription in human mitochondria Identification and purification of a DNA binding protein factor that promotes termination. Cell 58, 391-397 (1989). 

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