The Function of Individual Zinc Fingers in Sequence-specific DNA Recognition by Transcription Factor IIIA

Transcription factor IIIA (TFIIIA) employs an array of nine N-terminal zinc fingers to bind specifically to both 5S RNA and 5S DNA. The binding of TFIIIA to 5S RNA and 5S DNA was studied by using a protease footprinting technique. Brief treatment of free or bound TFIIA with trypsin or chymotrypsin generated fragments which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fragments retaining the N terminus of TFIIA were identified by immunoblotting with an antibody directed against the N terminus of TFIIIA. Proteolytic footprinting of TFIIIA complexed with 5S DNA derivatives reinforced other evidence that the three N-terminal zinc fingers of TFIIIA bind most tightly to 5S DNA. Proteolytic footprinting of TFIIIA in reconstituted 7S ribonucleoprotein particles revealed different patterns of trypsin sensitivity for TFIIIA bound to oocyte versus somatic 5S RNA. Trypsin cleaved TFIIIA between zinc fingers 3 and 4 more readily when the protein was bound to somatic 5S RNA than when it was bound to oocyte 5S RNA. A tryptic fragment of TFIIIA containing zinc fingers 4 through 7 remained tightly associated with somatic 5S RNA. Zinc fingers 4 through 7 may represent a tightly binding site for 5S RNA in the same sense that fingers 1 through 3 represent a tightly binding site for 5S DNA.

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The role of zinc fingers in transcriptional activation by transcription factor IIIA.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.1.168 ◽

1993 ◽

Vol 90 (1) ◽

pp. 168-172 ◽

Cited By ~ 19

Author(s):

S. Del Rio ◽

D. R. Setzer

Keyword(s):

Transcription Factor ◽

Transcriptional Activation ◽

Zinc Fingers ◽

Transcription Factor Iiia

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Proteolytic footprinting of transcription factor TFIIIA reveals different tightly binding sites for 5S RNA and 5S DNA.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5149 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5149-5158 ◽

Cited By ~ 14

Author(s):

D F Bogenhagen

Keyword(s):

Transcription Factor ◽

Binding Site ◽

Zinc Fingers ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

5S Rna ◽

Transcription Factor Iiia ◽

Tryptic Fragment ◽

N Terminus ◽

5S Dna

Transcription factor IIIA (TFIIIA) employs an array of nine N-terminal zinc fingers to bind specifically to both 5S RNA and 5S DNA. The binding of TFIIIA to 5S RNA and 5S DNA was studied by using a protease footprinting technique. Brief treatment of free or bound TFIIA with trypsin or chymotrypsin generated fragments which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fragments retaining the N terminus of TFIIA were identified by immunoblotting with an antibody directed against the N terminus of TFIIIA. Proteolytic footprinting of TFIIIA complexed with 5S DNA derivatives reinforced other evidence that the three N-terminal zinc fingers of TFIIIA bind most tightly to 5S DNA. Proteolytic footprinting of TFIIIA in reconstituted 7S ribonucleoprotein particles revealed different patterns of trypsin sensitivity for TFIIIA bound to oocyte versus somatic 5S RNA. Trypsin cleaved TFIIIA between zinc fingers 3 and 4 more readily when the protein was bound to somatic 5S RNA than when it was bound to oocyte 5S RNA. A tryptic fragment of TFIIIA containing zinc fingers 4 through 7 remained tightly associated with somatic 5S RNA. Zinc fingers 4 through 7 may represent a tightly binding site for 5S RNA in the same sense that fingers 1 through 3 represent a tightly binding site for 5S DNA.

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Induced Fit and “Lock and Key” Recognition of 5S RNA by Zinc Fingers of Transcription Factor IIIA

Journal of Molecular Biology ◽

10.1016/j.jmb.2005.12.010 ◽

2006 ◽

Vol 357 (1) ◽

pp. 275-291 ◽

Cited By ~ 52

Author(s):

Brian M. Lee ◽

Jing Xu ◽

Bryan K. Clarkson ◽

Maria A. Martinez-Yamout ◽

H. Jane Dyson ◽

...

Keyword(s):

Transcription Factor ◽

Zinc Fingers ◽

5S Rna ◽

Induced Fit ◽

Transcription Factor Iiia

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Structure of WT1 zinc fingers bound to its cognate DNA: Implications of the KTS insert

10.1101/271577 ◽

2018 ◽

Author(s):

Raymond K. Yengo ◽

Elmar Nurmemmedov ◽

Marjolein M.G.M. Thunnissen

Keyword(s):

Crystal Structure ◽

Transcription Factor ◽

Amino Acid ◽

Dna Binding ◽

Rna Splicing ◽

Zinc Fingers ◽

Dna Recognition ◽

Life Forms ◽

Target Dna ◽

Partner Proteins

ABSTRACTWT1 is a transcription factor with a DNA binding N-terminal domain containing four C2H2-type zinc fingers. In order to perform its role as a transcription factor, WT1 needs to specifically recognize and properly bind to its target DNA. How this is done is still not completely clear. Two of WT1’s major isoforms are distinguished by the presence or absence of a 3 amino acid insert, Lysine-Threonine-Serine (KTS) in the linker between zinc-fingers 3 and 4. This KTS insert is conserved throughout all life forms expressing WT1. The –KTS isoform, which acts as a transcription factor, binds DNA with higher affinity than the +KTS isoform, which is thought to participate in RNA splicing and interaction with partner proteins. This study was aims at elucidating the effect of the KTS insert on DNA binding. Here we present the crystal structure of WT1 zinc fingers 2-4, with and without the KTS insert, bound to the WT1 9-base pair cognate DNA sequence, refined to 1.9 Å and 2.5 Å respectively. The structures show that the +KTS isoform of WT1 recognizes DNA with the same specificity as the –KTS isoform. The only differences in the DNA bound conformation of the two isoforms are found within the linker containing the KTS, and these mainly involve the loss of the C-capping interactions thought to stabilize the complex. These structures provide the molecular detail necessary for the interpretation of the WT1 transcriptional DNA recognition and validation of its transcriptional targets.

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