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Footprinting analysis of the interactions between E. coli RNA polymerase and promoter DNA that produce very long abortive transcripts (VLATs)
Author(s) -
Thandar Mya,
Lee Ahri,
Hsu Lilian M
Publication year - 2012
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.26.1_supplement.738.1
Subject(s) - rna polymerase , transcription (linguistics) , polymerase , biology , footprinting , promoter , dna , microbiology and biotechnology , genetics , rna , gene , base sequence , gene expression , linguistics , philosophy
T5 phage N25 is a prototypical strong Eσ 70 promoter whose transcription initiation is rate‐limited at the promoter escape step, leading to extensive abortive initiation. The position of promoter escape depends not only on the promoter‐polymerase affinity but also on the initial transcribed sequence (ITS). Thus, N25 aborts to +11 and escapes at +12 whereas its variants with ITS changes— N25 anti (+3 to +20) and DG203 (+3 to +10)—aborts to +15 and +19 and escapes at +16 and +20, respectively. Studies with GreB showed that while all abortive transcripts longer than 4 nt can be rescued (via cleavage‐reelongation)—suggesting they arose by RNAP backtracking, a fraction of the VLATs (16–19 nt) is resistant—indicating that they are formed by a mechanism different from RNAP backtracking (Chander et al. 2007 Biochemistry 46 , 12687). We proposed that GreB‐resistant VLATs are the products of RNAP hyper‐forward translocation during escape. We tested this mechanism by placing Eco RI roadblock at various locations in DG203 downstream. The results indicate that GreB‐resistant VLATs are produced only when RNAP can forward translocate (via scrunching) to the 16 th –19 th positions and, during subsequent escape, hyper translocate when a spatial allowance of 2 bp (optimally 4–5 bp) downstream exists. Additional proofs are derived from KMnO 4 and Exo III footprinting analyses. Supported by NSF‐RUI Grant (MCB‐ 0841452) to L. M. Hsu