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Roles of the Initial Transcribed Sequence on Productive and Non‐Productive Initiation
Author(s) -
Dyke Sarah,
Tang Rebecca,
Felth Lindsey C.,
Henderson Kate L.,
Record M. Thomas
Publication year - 2019
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.2019.33.1_supplement.458.10
Subject(s) - rna polymerase , transcription (linguistics) , abortive initiation , dna , biology , promoter , transcription bubble , rna , microbiology and biotechnology , genetics , biophysics , gene , gene expression , linguistics , philosophy
Transcription initiation by E. coli RNA polymerase (RNAP) begins with specific binding of RNAP to promoter DNA, the ~100 bp region from the UP‐element (−40 to −60) and farther‐upstream DNA to the initial transcribed region (ITR), extending ~20 bp downstream from the transcription start site (+1). The sequence and length of upstream promoter DNA elements (including −10, −35 and UP elements) are major determinants of the rate of formation of the open promoter complex [1], while the sequence and length of the discriminator element (between the −10 element and the transcription start site (+1) and the length of the ITR are major determinants of the lifetime of the open complex [2,3]. Recently, we investigated the effects of the discriminator on initiation of productive and abortive transcription, as well as on escape of RNAP from promoter contacts in the open complex during productive initiation [2]. For the four T7A1 and λPR promoter/discriminator combinations, we found that the discriminator determined the lifetime and stability of the open complex and the escape point of RNAP in initiation. Here we compare initiation kinetics and RNA product distributions for the previously‐characterized ITR of the λPR promoter with two variant ITR: a repeating trinucleotide sequence (AUG), and a scrambled (“random”) sequence of A, U, and G. Initiation experiments are performed by adding mixtures of ribonucleotides (NTP, one 32P‐labeled) to preformed RNAP‐promoter open complexes, either by conventional mixing (10 seconds), or with a rapid quench flow (RQF) methods (1 millisecond). RNA products are separated by gel electrophoresis and visualized with a phosphorimager. Preliminary experiments comparing these ITR show similar kinetics of escape from the promoter and productive initiation of long RNA, but very different patterns of synthesis of short (abortive) RNAs by nonproductive complexes which cannot efficiently escape. In addition, a post‐escape transient pause at a RNA length of 13 nucleotides, at which length the nascent RNA has entered the RNA exit channel of RNAP, shows very different kinetics for the random ITR as compared to the repeating and original λPR ITR. Measurements of lifetimes and stabilities of these open complexes are planned to determine if they are affected by the ITR. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .