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Fluorescence Kinetic Studies of DNA Unwrapping in Transcription Initiation with NTP addition and in Open Complex Dissociation by high salt
Author(s) -
Callies Katelyn,
McNerney Christina,
Cimperman Clare Kai,
Xue Andrew,
Kan Hertina,
Sreenivasan Raashi,
Chhabra Munish,
Record Tom
Publication year - 2018
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.2018.32.1_supplement.652.26
Subject(s) - förster resonance energy transfer , dna , biophysics , transcription (linguistics) , dissociation (chemistry) , fluorescence , chemistry , transcription bubble , promoter , rna , microbiology and biotechnology , biology , polymerase , biochemistry , gene , physics , gene expression , linguistics , philosophy , rna dependent rna polymerase , quantum mechanics
λP R promoter DNA is bent and wrapped around E. coli RNA polymerase (RNAP) in the stable open complex (RP o ), as demonstrated by Cy3‐Cy5 fluorescent energy transfer (FRET) between dyes attached 115 bp apart on the far‐upstream (−100) and downstream (+14) position of DNA. Here we report the use of FRET and PIFE (single‐dye fluorescence enhancements that indicate close contacts between RNAP and the far‐upstream or downstream DNA) to investigate unwrapping of upstream DNA after NTP addition to initiate transcription, form a short RNA‐DNA hybrid and escape from the promoter. We observe two kinetic phases of roughly equal amplitude in the decrease in FRET in transcription initiation. The first kinetic phase (0.1 s to 10 s) of DNA unbending/unwrapping appears slower than the rate of NTP binding and dinucleotide synthesis, indicating that this first phase of unbending/unwrapping occurs in the processes of translocation and hybrid extension. The second kinetic phase (at times > 10 s) corresponds to that of production of full‐length (14 bp) RNA transcripts, as determined in parallel single‐round transcription assays. These results indicate that the second phase of DNA unbending and unwrapping occurs in promoter escape. In addition, we report the kinetics of unwrapping in dissociation by salt‐upshifts. These experiments reveal that salt‐induced unwrapping occurs during the conversion of the stable open complex to the unstable open intermediate I 2 , prior to DNA closing. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .