PT  - JOURNAL ARTICLE
AU  - Yu Liu
AU  - Jared T. Winkelman
AU  - Libing Yu
AU  - Chirangini Pukhrambam
AU  - Yu Zhang
AU  - Bryce E. Nickels
AU  - Richard H. Ebright
TI  - Structural and mechanistic basis of RNA extension in reiterative transcription initiation: RNA slipping without DNA scrunching
AID  - 10.1101/2021.05.08.443277
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.05.08.443277
4099  - http://biorxiv.org/content/early/2021/05/09/2021.05.08.443277.short
4100  - http://biorxiv.org/content/early/2021/05/09/2021.05.08.443277.full
AB  - In standard transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA, selects a transcription start site, and--using a “scrunching” mechanism, in which RNAP remains bound to the promoter, unwinds additional DNA, and pulls the additional unwound DNA past its active center, synthesizing an RNA product having a 5’ sequence complementary to the DNA template. In an alternative pathway of transcription initiation, termed “reiterative transcription initiation,” primarily observed at promoters containing homopolymeric sequences at or near the transcription start site, RNAP binds to promoter DNA, unwinds promoter DNA, selects a transcription start site, and--using a mechanism that has not previously been defined--generates an RNA product having a 5’ sequence that contains a variable number of nucleotides not complementary to the DNA template. Here, using x-ray crystallography to define structures, using protein-DNA-photocrosslinking to map positions of RNAP leading and trailing edges relative to DNA, and using single-molecule DNA nanomanipulation to assess RNAP-dependent DNA unwinding, we show that RNA extension in reiterative transcription initiation (1) occurs without DNA scrunching, (2) involves a short, 2 bp (post-translocated state) to 3 bp (pre-translocated state) RNA-DNA hybrid, (3) and can involve an RNA product positioned as in standard transcription initiation and a DNA template strand positioned differently from standard transcription initiation. The results establish that, whereas RNA extension in standard transcription initiation proceeds through a scrunching mechanism, RNA extension in reiterative transcription initiation proceeds through a slippage mechanism, with sliding of RNA relative to DNA within a short, 2-3 bp, RNA-DNA hybrid.Competing Interest StatementThe authors have declared no competing interest.