Architectural arrangement of the small nuclear RNA (snRNA)-activating protein complex 190 subunit (SNAP190) on U1 snRNA gene promoter DNA.

Matthew T Doherty,William E Stumph,Yoon Soon Kang,Cheryn Lee

doi:10.1074/jbc.m112.407775

Matthew T Doherty, William E Stumph + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m112.407775

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Abstract

Myb repeats ∼52 amino acid residues in length were first characterized in the oncogenic Myb transcription factor, which contains three tandem Myb repeats in its DNA-binding domain. Proteins of this family normally contain either one, two, or three tandem Myb repeats that are involved in protein-DNA interactions. The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is a heterotrimeric transcription factor that is required for expression of small nuclear RNA genes. This complex binds to an essential promoter element, the proximal sequence element, centered ∼50 base pairs upstream of the transcription start site of snRNA genes. SNAP190, the largest subunit of SNAPc, uncharacteristically contains 4.5 tandem Myb repeats. Little is known about the arrangement of the Myb repeats in the SNAPc-DNA complex, and it has not been clear whether all 4.5 Myb repeats contact the DNA. By using a site-specific protein-DNA photo-cross-linking assay, we have now mapped specific nucleotides where each of the Myb repeats of Drosophila melanogaster SNAP190 interacts with a U1 snRNA gene proximal sequence element. The results reveal the topological arrangement of the 4.5 SNAP190 Myb repeats relative to the DNA and to each other when SNAP190 is bound to a U1 promoter as a subunit of SNAPc.

Highlights

SNAP190, the largest subunit of the small nuclear RNA (snRNA)-activating protein complex (SNAPc), interacts with DNA via 4.5 Myb repeats
The small nuclear RNA-activating protein complex (SNAPc) is a heterotrimeric transcription factor that is required for expression of small nuclear RNA genes
By carrying out hydroxylamine digestion of the protein at NG peptide bonds subsequent to the site-specific protein-DNA photo-cross-linking, it is possible to map or localize the region in the protein to which the cross-linking occurs. By employing such a strategy, we recently determined that phosphate position 1 of the U1 proximal sequence element A (PSEA) cross-linked to a region of DmSNAP190 located between amino acid residues 359 and 483 [18]

Summary

Background

SNAP190, the largest subunit of the snRNA-activating protein complex (SNAPc), interacts with DNA via 4.5 Myb repeats. By using a sitespecific protein-DNA photo-cross-linking assay, we have mapped specific nucleotides where each of the Myb repeats of Drosophila melanogaster SNAP190 interacts with a U1 snRNA gene proximal sequence element. Arrangement of SNAP190 Myb Repeats on U1 Gene Promoter DNA amino acids residues in length), that constitute the DNA-binding domain of the c-Myb transcription factor [21, 22]. The studies reported here reveal that all 4.5 Myb repeats of DmSNAP190 are located in close proximity to the DNA Based upon these data and the solved crystal structures of the repeats of the Myb protein itself, it became possible to accurately model the three-dimensional spatial arrangement of the 4.5 DmSNAP190 Myb repeats on the DNA when DmSNAPc binds to the U1 PSEA sequence. We discovered that several of the nucleotides in the 3Ј half of the PSEA did not cross-link to any of the Myb repeats but rather cross-linked to the N-terminal domain of DmSNAP190 that precedes the Myb repeats

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION