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Open complex contacts: an analysis of the interaction between T5 N25 discriminator region and E. coli RNA polymerase
Author(s) -
White Cassandra L,
Hsu Lilian M
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.679.8
Subject(s) - rna polymerase , promoter , polymerase , physics , microbiology and biotechnology , dna , protein subunit , nucleotide , biophysics , discriminator , rna , chemistry , biology , biochemistry , gene , gene expression , optics , detector
The discriminator (DIS) region referring to the 6–8 bp between the +1 start site and the −10 box of the promoter has been shown to make direct contact with RNA polymerase (RNAP) to stabilize the open complex. Previous research showed that the second nucleotide on the non‐template strand downstream of the −10 box, when it is a G, in the rrnB P1 C −7 G and λ P R promoter contacts the σ1.2 region (Haugen et al . 2006 Cell 125 , 1069; 2008 PNAS 105 , 3292), but in the T5 N25 promoter contacts the β’ subunit. These promoters differ in the three nucleotides immediately downstream of the −10 box: GGG in rrnB P1 C −7 G, GGT in λ P R , and AGA in N25 . To determine if the G‐content in these three bases affect the contact of the G −5 residue with RNAP, we constructed three promoters – the wild type N25 (where DIS is A −6 GATTC −1 ), A − 6 G N25 , and A − 6 G/A − 4 G N25 – and subjected them to UV‐induced crosslinking to RNAP via their −5 S 6 G residue. Preliminary results showed that all three promoters transcribe very similarly, indicating that the stability of the open complex is minimally affected by the DIS mutations. In crosslinking, while both the wild type N25 and the A − 6 G N25 were found to contact the β’ subunit, the A − 6 G/A − 4 G N25 promoter failed to show any crosslinking. Our results suggest that the path of the DIS DNA may differ from promoter to promoter, thus lending different degree of stabilization to the open complex. Sponsored by an NSF grant (RUI‐0841452) to L. M. Hsu