Mass Spectrometric Analysis of a Native Zinc-Finger Structure: The Glucocorticoid Receptor DNA Binding Domain

1995 ◽  
Vol 117 (12) ◽  
pp. 3319-3324 ◽  
Author(s):  
H. Ewa Witkowska ◽  
Cedric H. L. Shackleton ◽  
Karin Dahlman-Wright ◽  
John Y. Kim ◽  
Jan-Aake Gustafsson
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Zinc Finger ◽  
Mass Spectrometric ◽  
Binding Domain ◽  
Mass Spectrometric Analysis ◽  
Spectrometric Analysis ◽  
Dna Binding Domain

scholarly journals The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

1990 ◽  
Vol 10 (10) ◽  
pp. 5128-5137 ◽  
Author(s):  
M M Witte ◽  
R C Dickson
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dna Binding ◽  
Zinc Finger ◽  
Binding Specificity ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Transcription Activator ◽  
Activator Proteins ◽  
Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

A position-dependent transcription-activating domain in TFIIIA

1993 ◽  
Vol 13 (12) ◽  
pp. 7496-7506
Author(s):  
X Mao ◽  
M K Darby
Keyword(s):  
Amino Acids ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Zinc Finger ◽  
Transcriptional Activation ◽  
Transcriptional Activity ◽  
Rna Polymerase Iii ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
5S Rna

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

scholarly journals Structure refinement of the glucocorticoid receptor-DNA binding domain from NMR data by relaxation matrix calculations

1995 ◽  
Vol 247 (4) ◽  
pp. 689-700 ◽  
Author(s):  
Marc A.A. van Tilborg ◽  
Alexandre M.J.J. Bonvin ◽  
Karl Hård ◽  
Adrian L. Davis ◽  
Bonnie Maler ◽  
...  
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Structure Refinement ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Relaxation Matrix ◽  
Nmr Data

Structure and Dynamics of the Glucocorticoid Receptor DNA-Binding Domain:  Comparison of Wild Type and a Mutant with Altered Specificity†

Biochemistry ◽  
1997 ◽  
Vol 36 (37) ◽  
pp. 11188-11197 ◽  
Author(s):  
Helena Berglund ◽  
Magnus Wolf-Watz ◽  
Thomas Lundbäck ◽  
Susanne van den Berg ◽  
Torleif Härd
Keyword(s):  
Dna Binding ◽  
Glucocorticoid Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Wild Type ◽  
Structure And Dynamics
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