Identification of amino acid-base contacts in the Myc-DNA complex by site-specific bromouracil mediated photocrosslinking.

Dong, Qianping; Blatter, Erich E.; Ebright, Yon W.; Bister, Klaus

doi:10.1002/j.1460-2075.1994.tb06249.x

Cited by 26 publications

(19 citation statements)

References 26 publications

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“…Competitive gel shift analysis confirmed that sites lacking these groups were bound with lower affinity by Krox20 and NGFI-A VOL. 15,1995 DNA BINDING BY NGFI-A AND RELATED PROTEINS 2283…”

Section: Discussionmentioning

confidence: 99%

DNA-Binding Specificity of NGFI-A and Related Zinc Finger Transcription Factors

Swirnoff

Milbrandt

1995

Molecular and Cellular Biology

299

287

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NGFI-A is the prototypic member of a family of immediate-early gene-encoded transcription factors which includes NGFI-C, Egr3, and Krox20. These proteins possess highly homologous DNA-binding domains, composed of three Cys 2 -His 2 zinc fingers, and all bind to and activate transcription from the sequence GCGGGGGCG. We used a PCR-mediated random site selection protocol to determine whether other sites could be bound by these proteins and the extent to which their binding site preferences are similar or different. The high-affinity consensus sites generated from the selection data are similar, and the combined consensus sequence is T -G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (lowercase letters indicate bases selected less frequently).Using gel shift assays, we found that sequences that diverge from the consensus were bound by NGFI-A, confirming that there is greater variability in binding sites than has generally been acknowledged. We also provide evidence that protein-DNA interactions not noted, or whose importance was not apparent from the X-ray cocrystal structure of the NGFI-A zinc fingers complexed with DNA, contribute significantly to the binding energy of these proteins and confirm that an optimal site is at least 10 instead of 9 nucleotides in length. In contrast to the similarities in binding specificity among these proteins, we found that while NGFI-A, Egr3, and Krox20 have comparable DNA binding affinities and kinetics of dissociation, the affinity of NGFI-C is more than threefold lower. This could result in differential regulation of target genes in cells where NGFI-C and the other proteins are coexpressed. Furthermore, we show that this affinity difference is a property not of the zinc fingers themselves but rather of the protein context of the DNA-binding domain.

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“…Competitive gel shift analysis confirmed that sites lacking these groups were bound with lower affinity by Krox20 and NGFI-A VOL. 15,1995 DNA BINDING BY NGFI-A AND RELATED PROTEINS 2283…”

Section: Discussionmentioning

confidence: 99%

DNA-Binding Specificity of NGFI-A and Related Zinc Finger Transcription Factors

Swirnoff

Milbrandt

1995

Molecular and Cellular Biology

299

287

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“…Evidence from fusion proteins consisting of GAL4 and c-Myc suggested that the c-Myc transactivation domain is localized to its first 143 amino acids (93). Immediately N terminal to the dimerization domain is a domain rich in basic amino acids which directly contacts specific DNA sequences within the DNA major groove (41,45,56,57,60,143,144,185). c-Myc DNA binding sites (both canonical [5Ј-CAC GTG-3Ј] and noncanonical) have been identified by using a variety of in vitro protein-DNA binding assays (26,27,144).…”

Section: C-myc Transcription Factor Its Binding Partner Max and Madmentioning

confidence: 99%

c-Myc Target Genes Involved in Cell Growth, Apoptosis, and Metabolism

Dang

1999

Molecular and Cellular Biology

1,523

1,211

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“…The photoactivated cross-linking reagent BrU, an isosteric analog of thymine, has been widely used to identify positions in a recognition site that are in close contact with a DNA-binding protein (2,4,8). The formation of a BrU-mediated covalently linked protein-DNA complex indicates that the protein is in van der Waals contact with the methyl group of a substituted thymine.…”

Section: Mutations Of Base Pairs Outside Of the Carg Box Effect Transmentioning

confidence: 99%

“…The cross-linking efficiency is ϳ4% at position 15 but falls to Ͻ1% at position 14. The higher efficiency of cross-linking found at position 15 may indicate the close proximity of an aromatic amino acid (His, Phe, Trp, or Tyr) (2,8,49).…”

Section: Mutations Of Base Pairs Outside Of the Carg Box Effect Transmentioning

confidence: 99%