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Testing the mechanism of very long abortive transcript (VLAT) formation via hyper forward translocation by E. coli RNA polymerase
Author(s) -
Vallery Tenaya Kristine,
Jiang Yunnan,
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.679.7
Subject(s) - rna polymerase , transcription (linguistics) , chromosomal translocation , polymerase , biology , rna , dna , microbiology and biotechnology , genetics , gene , linguistics , philosophy
Transcription initiation at the T5 phage N25 – an E coli Eσ 70 – promoter is rate‐limited at the promoter escape step; this leads to the generation of an abortive RNA ladder of 11 nt with escape at +12. The length of the abortive ladder depends on the initial transcribed sequence (ITS). Thus N25 anti , with ITS changes from +3 to +20, and DG203 , with ITS changes from +3 to +10, abort to +15 and +19, respectively (Hsu et al. 2006 Biochemistry 45 , 8841). Abortive RNAs ≤15 nt were shown to result from RNA polymerase (RNAP) backtracking, since their levels are ameliorated by GreB. In contrast, VLATs, referring to abortive RNAs 16–19 nt in length, are resistant to GreB and were deemed to result from RNAP hyper translocation (Chander et al. 2007 Biochemistry 46 , 12687), during which RNAP forward translocates by >1 nt, thus overshooting the 3′‐end of the transcript. VLATs cannot be elongated further and are subsequently released from the arrested complex. To investigate the proposed VLAT mechanism, we placed an E 111 Q EcoR I roadblock at several locations in the DG203 downstream DNA, which bracket the promoter escape transition, to prevent forward translocation. Preliminary results reveal that E 111 Q EcoR I binds downstream DNA tightly to prevent VLAT formation in a predicted manner, thereby supporting the hyper forward translocation model. Supported by NSF Grant (RUI‐0841452) to L. M. Hsu