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Chapter: Genetics and Molecular Biology: Xenopus 5S RNA Synthesis

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Structure and Function of TFIIIA

One of the motivations for studying regulation of the 5S genes was the hope that phenomena totally unheard of in prokaryotes would be found in eukaryotes, and the first well-studied eukaryotic system seemed likely to yield a rich harvest.

Structure and Function of TFIIIA

One of the motivations for studying regulation of the 5S genes was the hope that phenomena totally unheard of in prokaryotes would be found in eukaryotes, and the first well-studied eukaryotic system seemed likely to yield a rich harvest. For the most part, what has been found has turned out to be phenomena that are not unique either to the 5S genes or to eukaryotes. For example, the stable state that is determined by a protein or complex of proteins that remain bound for long periods of time has proven to be a valuable concept, but it is not unique either to eukaryotes or to the 5S system. Even the startling discovery of an internal control region turns out not to be unique, either to eukaryotes or to genes transcribed by RNA polymerase III. The zinc fingers, which were first found in TFIIIA, are a common structure in eukaryotes, but they also exist in prokaryotes.

The discovery of the zinc fingers is illustrative of the unexpected source for many discoveries. While exploring the cause for substantial losses during the purification of TFIIIA from 7S particles, workers noticed that steps like gel filtration that could separate the protein from small molecules led to substantial loss of TFIIIA activity. Also, addition of metal ion chelators increased losses. Prior addition of Zn++, but not other ions, prevented the losses. With this strong clue that zinc ion was involved, they performed atomic adsorption spectroscopy and found indeed that each molecule of the protein contained five to ten zinc ions.

 

The presence of multiple ions in the protein suggested that an ion-binding substructure or domain might be repeated within the pro-tein. This suspicion was reinforced by the finding that upon extended proteolytic digestion of the protein there remained multiple species of a 3,000-4,000 dalton peptide. Upon examining the sequence of the protein for a repeated sequence of about 30 amino acids, one was found. The marvelous property of these repeating units was that they possessed two cysteines and two histidines spaced just as required for these four amino acids to chelate zinc (Fig. 15.8), as was discussed.


Figure 15.8 Locations of cysteines and histidines in zinc fingers.


 

Since TFIIIA possesses nine zinc fingers, and each can contact three bases of of the DNA, the protein can contact part of the 40 bases of the essential internal control region in the 5S gene. The portion of the protein that does not contact DNA likely contacts one or both of the other proteins required for most active transcription of 5S genes, TFIIIB or IIIC. There is a 10,000 dalton portion of the protein at the N-terminal end that probably is involved in this function since it does not possess zinc fingers and its removal eliminates transcriptional activation by TFIIIA.

 

The structure of a zinc finger was predicted by comparison with other proteins containing liganded metal ions and close examination of the sequences of zinc fingers. Subsequent determination of the structure of artificial zinc fingers by nuclear magnetic resonance methods and X-ray crystallography confirmed the predicted structure.


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