Structural and mechanistic basis of reiterative transcription initiation

Abstract

Reiterative transcription initiation, observed at promoters that contain homopolymeric sequences at the transcription start site, generates RNA products having 5′ sequences noncomplementary to the DNA template. Here, using crystallography and cryoelectron microscopy to define structures, protein–DNA photocrosslinking to map positions of RNAP leading and trailing edges relative to DNA, and single-molecule DNA nanomanipulation to assess RNA polymerase (RNAP)–dependent DNA unwinding, we show that RNA extension in reiterative transcription initiation 1) occurs without DNA scrunching; 2) involves a short, 2- to 3-bp, RNA–DNA hybrid; and 3) generates RNA that exits RNAP through the portal by which scrunched nontemplate-strand DNA exits RNAP in 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 slipping of RNA relative to DNA within a short RNA–DNA hybrid, and with extrusion of RNA from RNAP through an alternative RNA exit.

Structural visualization of transcription initiation in action

“…Our proposed mechanism of transcription initiation in mammals is generally similar to the well-characterized transcription initiation by bacterial RNAP, which forms CC, OC, ITC, and EC complexes and undergoes promoter opening, bubble expansion, bubble collapse, and promoter escape through a DNA-scrunching mechanism (88)(89)(90)(91). The bacterial s factor, which is functionally equivalent to eukaryotic GTFs, associates with RNAP and stabilizes the upstream promoter in the ITC.…”

Purification of synchronized Escherichia coli transcription elongation complexes by reversible immobilization on magnetic beads

“…Second, a G +5 G +6 dinucleotide was included because it favors productive initiation from the λP R promoter ( 29 ). Third, homopolymeric sequences were limited to a maximum of two nucleotides to minimize the potential for reiterative transcription initiation ( 30 , 31 , 32 , 33 ). Fourth, the C3 RNA sequence and its DNA complement are predicted to be unstructured by the ‘fold’ algorithm of the RNAStructure RNA secondary structure prediction software ( 34 ).…”