The mechanism of variability in transcription start site selection

Abstract

During transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded ‘transcription bubble,’ and selects a transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA ‘scrunching’ and ‘anti-scrunching,’ the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the transcription bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.

XACT-Seq Comprehensively Defines the Promoter-Position and Promoter-Sequence Determinants for Initial-Transcription Pausing

“…We first used RNAP-b' R1148Bpa to detect initial-transcription pausing at placCONS in vitro (Figure 3A). The results in Figure 3A, lanes 5, 8, and 11, confirm the previously reported ability of the RNAP-b' R1148Bpa to detect the RNAP-active-center A-site position with single-nucleotide resolution in static initial-transcribing complexes in the absence of NTP substrates in vitro (Winkelman et al, 2015;Winkelman et al, 2016a;Winkelman et al, 2016b;Yu et al, 2017). The results in Figure 3A, lanes 6, 9, and 12, demonstrate that the detected position of the RNAP-active-center A-site position does not change upon addition of a 2-nt RNA (ApA), indicating that the resulting ITC,2 is in a pre-translocated state (ITC,2-pre).…”

“…XACT-seq takes advantage of an RNAP derivative that has a photo-activatable crosslinking amino acid p-benzoyl-L-phenylalanine (Bpa) incorporated at RNAP-b' subunit residue R1148 (RNAP-b' R1148Bpa ), which, upon photo-activation in vitro or in vivo, forms covalent crosslinks with DNA at a position exactly 5-nt downstream of the RNAP-active-center A-site (Figure 2B; Yu et al, 2017). In previous work, this reagent was used for structural analysis of static, trapped transcription complexes (Winkelman et al, 2015;Winkelman et al, 2016a;Yu et al, 2017). In this work, we apply the reagent for analysis of actively-transcribing complexes.…”

XACT-seq comprehensively defines the promoter-position and promoter-sequence determinants for initial-transcription pausing

“…In vitro photo-crosslinking and crosslink mapping experiments were done using procedures described in (Yu et al, 2017). For the experiments in Figure 3A Figure 5C, 50 µl reactions containing 20 nM RNAP holoenzyme, 4 nM template, and 1 X RB were incubated for 5 min at 25°C, 5 uL of 1 mM ATP, 1 mM CTP, 1 mM GTP, and 1 mM UTP were added, reactions were incubated 2 min at 25°C, and subjected to UV irradiation for 2 min at 25°C.…”

“…30 units of DpnI was added, the reaction was incubated at 37°C for 16 h, and DNA was recovered using a PCR purification kit (Qiagen). The recovered DNA was treated with 10 units of T4 polynucleotide kinase (PNK) (New England Biolabs) in 1 X T4 PNK buffer containing 20 µM ATP for 30 min at 37°C, 400 units of T4 DNA ligase (New England Biolabs) was added, reactions were incubated at 16°C for 16 h, 1 µl of the reaction was introduced into electrocompetent DH10B cells, and cells plated on LB agar plates containing 50 µg/ml kanamycin, and recombinant plasmid DNA was isolated from individual transformants.Plasmid pCDF-CP(Yu et al, 2017) contains a CloDF13 replication origin, a selectable marker conferring resistance to spectinomycin, and two BglI recognition sites that are used to introduce DNA fragments upstream of transcription terminator tR2. Plasmid pCDF-lacCONS(Yu et al, 2017) is a derivative of pCDF-CP containing sequences from positions -88 to +70 of placCONS inserted into BglI-digested pCDF-CP.…”

Understanding transcription across scales: From base pairs to chromosomes

“…Most genes are transcribed from multiple, distinct TSSs (Forrest et al, 2014;Mejía-Almonte et al, 2020). TSS selection is tissue dependent and influenced by sequence context and splicing (Forrest et al, 2014;Fiszbein et al, 2019;Yu et al, 2017). TSS conservation is variable among mammals, indicating that speciesspecific gene expression regulation may be influenced by differential start site use (Forrest et al, 2014;Fiszbein et al, 2019).…”