Localized Frustration and Binding-Induced Conformational Change in Recognition of 5S RNA by TFIIIA Zinc Finger

2013 ◽  
Vol 117 (50) ◽  
pp. 15917-15925 ◽  
Author(s):  
Cheng Tan ◽  
Wenfei Li ◽  
Wei Wang
Keyword(s):  
Conformational Change ◽  
Zinc Finger ◽  
5S Rna

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 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 A Hydrophobic Segment within the 81-Amino-Acid Domain of TFIIIA from Saccharomyces cerevisiae Is Essential for Its Transcription Factor Activity

1998 ◽  
Vol 18 (1) ◽  
pp. 420-432 ◽  
Author(s):  
Owen Rowland ◽  
Jacqueline Segall
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Zinc Finger ◽  
Transcription Factor Activity ◽  
Factor Activity ◽  
5S Rna ◽  
Hydrophobic Residues ◽  
Acid Domain ◽  
Amino Acid Domain

ABSTRACT Transcription factor IIIA (TFIIIA) binds to the internal control region of the 5S RNA gene as the first step in the in vitro assembly of a TFIIIB-TFIIIC-TFIIIA-DNA transcription complex. An 81-amino-acid domain that is present between zinc fingers 8 and 9 of TFIIIA fromSaccharomyces cerevisiae is essential for the transcription factor activity of this protein (C. A. Milne and J. Segall, J. Biol. Chem. 268:11364–11371, 1993). We have monitored the effect of mutations within this domain on the ability of TFIIIA to support transcription of the 5S RNA gene in vitro and to maintain cell viability. TFIIIA with internal deletions that removed residues 282 to 315, 316 to 334, 328 to 341, or 342 to 351 of the 81-amino-acid domain retained activity, whereas TFIIIA with a deletion of the short leucine-rich segment 352NGLNLLLN359 at the carboxyl-terminal end of this domain was devoid of activity. Analysis of the effects of double and quadruple mutations in the region extending from residue 336 to 364 confirmed that hydrophobic residues in this portion of the 81-amino-acid domain, particularly L343, L347, L354, L356, L357, and L358, and to a lesser extent F336 and L337, contributed to the ability of TFIIIA to promote transcription. We propose that these hydrophobic residues play a role in mediating an interaction between TFIIIA and another component of the transcriptional machinery. We also found that TFIIIA remained active if either zinc finger 8 or zinc finger 9 was disrupted by mutation but that TFIIIA containing a disruption of both zinc finger 8 and zinc finger 9 was inactive.

An RNA Aptamer with High Affinity and High Specificity for the 5S RNA Binding Zinc Finger Proteins TFIIIA and p43

Biochemistry ◽  
2010 ◽  
Vol 49 (8) ◽  
pp. 1755-1765
Author(s):  
Tristen C. Weiss ◽  
Gary G. Zhai ◽  
Paul J. Romaniuk
Keyword(s):  
Zinc Finger ◽  
Rna Binding ◽  
High Specificity ◽  
Zinc Finger Proteins ◽  
Rna Aptamer ◽  
5S Rna ◽  
High Affinity

scholarly journals Role of TFIIIA zinc fingers in vivo: analysis of single-finger function in developing Xenopus embryos.

1993 ◽  
Vol 13 (8) ◽  
pp. 4776-4783 ◽  
Author(s):  
M B Rollins ◽  
S Del Rio ◽  
A L Galey ◽  
D R Setzer ◽  
M T Andrews
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Transcriptional Activation ◽  
Zinc Fingers ◽  
Wild Type ◽  
5S Rna ◽  
5S Rna Genes ◽  
Rna Genes

The Xenopus 5S RNA gene-specific transcription factor IIIA (TFIIIA) has nine consecutive Cys2His2 zinc finger motifs. Studies were conducted in vivo to determine the contribution of each of the nine zinc fingers to the activity of TFIIIA in living cells. Nine separate TFIIIA mutants were expressed in Xenopus embryos following microinjection of their respective in vitro-derived mRNAs. Each mutant contained a single histidine-to-asparagine substitution in the third zinc ligand position of an individual zinc finger. These mutations result in structural disruption of the mutated finger with little or no effect on the other fingers. The activity of mutant proteins in vivo was assessed by measuring transcriptional activation of the endogenous 5S RNA genes. Mutants containing a substitution in zinc finger 1, 2, or 3 activate 5S RNA genes at a level which is reduced relative to that in embryos injected with the message for wild-type TFIIIA. Proteins with a histidine-to-asparagine substitution in zinc finger 5 or 7 activate 5S RNA genes at a level that is roughly equivalent to that of the wild-type protein. Zinc fingers 8 and 9 appear to be critical for the normal function of TFIIIA, since mutations in these fingers result in little or no activation of the endogenous 5S RNA genes. Surprisingly, proteins with a mutation in zinc finger 4 or 6 stimulate 5S RNA transcription at a level that is significantly higher than that mediated by similar concentrations of wild-type TFIIIA. Differences in the amount of newly synthesized 5S RNA in embryos containing the various mutant forms of TFIIIA result from differences in the relative number and/or activity of transcription complexes assembled on the endogenous 5S RNA genes and, in the case of the finger 4 and finger 6 mutants, result from increased transcriptional activation of the normally inactive oocyte-type 5S RNA genes. The remarkably high activity of the finger 6 mutant can be reproduced in vitro when transcription is carried out in the presence of 5S RNA. Disruption of zinc finger 6 results in a form of TFIIIA that exhibits reduced susceptibility to feedback inhibition by 5S RNA and therefore increases the availability of the transcription factor for transcription complex formation.

scholarly journals Role of TFIIIA zinc fingers in vivo: analysis of single-finger function in developing Xenopus embryos

1993 ◽  
Vol 13 (8) ◽  
pp. 4776-4783
Author(s):  
M B Rollins ◽  
S Del Rio ◽  
A L Galey ◽  
D R Setzer ◽  
M T Andrews
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Transcriptional Activation ◽  
Zinc Fingers ◽  
Wild Type ◽  
5S Rna ◽  
5S Rna Genes ◽  
Rna Genes

The Xenopus 5S RNA gene-specific transcription factor IIIA (TFIIIA) has nine consecutive Cys2His2 zinc finger motifs. Studies were conducted in vivo to determine the contribution of each of the nine zinc fingers to the activity of TFIIIA in living cells. Nine separate TFIIIA mutants were expressed in Xenopus embryos following microinjection of their respective in vitro-derived mRNAs. Each mutant contained a single histidine-to-asparagine substitution in the third zinc ligand position of an individual zinc finger. These mutations result in structural disruption of the mutated finger with little or no effect on the other fingers. The activity of mutant proteins in vivo was assessed by measuring transcriptional activation of the endogenous 5S RNA genes. Mutants containing a substitution in zinc finger 1, 2, or 3 activate 5S RNA genes at a level which is reduced relative to that in embryos injected with the message for wild-type TFIIIA. Proteins with a histidine-to-asparagine substitution in zinc finger 5 or 7 activate 5S RNA genes at a level that is roughly equivalent to that of the wild-type protein. Zinc fingers 8 and 9 appear to be critical for the normal function of TFIIIA, since mutations in these fingers result in little or no activation of the endogenous 5S RNA genes. Surprisingly, proteins with a mutation in zinc finger 4 or 6 stimulate 5S RNA transcription at a level that is significantly higher than that mediated by similar concentrations of wild-type TFIIIA. Differences in the amount of newly synthesized 5S RNA in embryos containing the various mutant forms of TFIIIA result from differences in the relative number and/or activity of transcription complexes assembled on the endogenous 5S RNA genes and, in the case of the finger 4 and finger 6 mutants, result from increased transcriptional activation of the normally inactive oocyte-type 5S RNA genes. The remarkably high activity of the finger 6 mutant can be reproduced in vitro when transcription is carried out in the presence of 5S RNA. Disruption of zinc finger 6 results in a form of TFIIIA that exhibits reduced susceptibility to feedback inhibition by 5S RNA and therefore increases the availability of the transcription factor for transcription complex formation.

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