Quantitative analysis of transcription start site selection reveals control by DNA sequence, RNA polymerase II activity and NTP levels.

Transcription start site (TSS) selection is a key step in gene expression and occurs at many promoter positions over a wide range of efficiencies. Here we develop a massively parallel reporter assay to quantitatively dissect contributions of promoter sequence, nucleoside triphosphate substrate levels and RNA polymerase II (Pol II) activity to TSS selection by 'promoter scanning' in Saccharomyces cerevisiae (Pol II MAssively Systematic Transcript End Readout, 'Pol II MASTER'). Using Pol II MASTER, we measure the efficiency of Pol II initiation at 1,000,000 individual TSS sequences in a defined promoter context. Pol II MASTER confirms proposed critical qualities of S. cerevisiae TSS -8, -1 and +1 positions, quantitatively, in a controlled promoter context. Pol II MASTER extends quantitative analysis to surrounding sequences and determines that they tune initiation over a wide range of efficiencies. These results enabled the development of a predictive model for initiation efficiency based on sequence. We show that genetic perturbation of Pol II catalytic activity alters initiation efficiency mostly independently of TSS sequence, but selectively modulates preference for the initiating nucleotide. Intriguingly, we find that Pol II initiation efficiency is directly sensitive to guanosine-5'-triphosphate levels at the first five transcript positions and to cytosine-5'-triphosphate and uridine-5'-triphosphate levels at the second position genome wide. These results suggest individual nucleoside triphosphate levels can have transcript-specific effects on initiation, representing a cryptic layer of potential regulation at the level of Pol II biochemical properties. The results establish Pol II MASTER as a method for quantitative dissection of transcription initiation in eukaryotes.

In transcription antitermination by Qλ, NusA induces refolding of Qλ to form a nozzle that extends the RNA polymerase RNA-exit channel.

Lambdoid bacteriophage Q proteins are transcription antipausing and antitermination factors that enable RNA polymerase (RNAP) to read through pause and termination sites. Q proteins load onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and adjacent sigma-dependent pause element to yield a Q-loading complex, and they translocate with RNAP as a pausing-deficient, termination-deficient Q-loaded complex. In previous work, we showed that the Q protein of bacteriophage 21 (Q21) functions by forming a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of pause and termination RNA hairpins. Here, we report atomic structures of four states on the pathway of antitermination by the Q protein of bacteriophage λ (Qλ), a Q protein that shows no sequence similarity to Q21 and that, unlike Q21, requires the transcription elongation factor NusA for efficient antipausing and antitermination. We report structures of Qλ, the Qλ-QBE complex, the NusA-free pre-engaged Qλ-loading complex, and the NusA-containing engaged Qλ-loading complex. The results show that Qλ, like Q21, forms a nozzle that narrows and extends the RNAP RNA-exit channel, preventing formation of RNA hairpins. However, the results show that Qλ has no three-dimensional structural similarity to Q21, employs a different mechanism of QBE recognition than Q21, and employs a more complex process for loading onto RNAP than Q21, involving recruitment of Qλ to form a pre-engaged loading complex, followed by NusA-facilitated refolding of Qλ to form an engaged loading complex. The results establish that Qλ and Q21 are not structural homologs and are solely functional analogs.

Structural and mechanistic basis of σ-dependent transcriptional pausing.

In σ-dependent transcriptional pausing, the transcription initiation factor σ, translocating with RNA polymerase (RNAP), makes sequence-specific protein­DNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in σ-dependent pausing, the RNAP active center can access off-pathway "backtracked" states that are substrates for the transcript-cleavage factors of the Gre family and on-pathway "scrunched" states that mediate pause escape. Here, using site-specific protein­DNA photocrosslinking to define positions of the RNAP trailing and leading edges and of σ relative to DNA at the λPR' promoter, we show directly that σ-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 2­4 bp, and σ-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2­3 bp. Analogous experiments with a library of 47 (∼16,000) transcribed-region sequences show that the state scrunched by 2­3 bp­and only that state­is associated with the consensus sequence, T−3N−2Y−1G+1, (where −1 corresponds to the position of the RNA 3' end), which is identical to the consensus for pausing in initial transcription and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T−3 resides in the DNA nontemplate strand. A cryoelectron microscopy structure of a complex engaged in σ-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T−3 of the consensus sequence exerts its effects by facilitating scrunching.

Structural and mechanistic basis of reiterative transcription initiation.

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.

XACT-seq: A photocrosslinking-based technique for detection of the RNA polymerase active-center position relative to DNA in Escherichia coli.

XACT-seq ("crosslink between active-center and template sequencing") is a technique for high-throughput, single-nucleotide resolution mapping of RNA polymerase (RNAP) active-center positions relative to the DNA template. XACT-seq overcomes limitations of approaches that rely on analysis of the RNA 3' end (e.g., native elongating transcript sequencing) or that report RNAP positions with low resolution (e.g., ChIP-seq and ChIP-exo). XACT-seq can be used to map RNAP active-center positions in transcription initiation complexes, initially transcribing complexes, and transcription elongation complexes. For complete details on the use and execution of this protocol, please refer to Winkelman et al. (2020).

Ssl2/TFIIH function in transcription start site scanning by RNA polymerase II in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, RNA polymerase II (Pol II) selects transcription start sites (TSSs) by a unidirectional scanning process. During scanning, a preinitiation complex (PIC) assembled at an upstream core promoter initiates at select positions within a window ~40-120 bp downstream. Several lines of evidence indicate that Ssl2, the yeast homolog of XPB and an essential and conserved subunit of the general transcription factor (GTF) TFIIH, drives scanning through its DNA-dependent ATPase activity, therefore potentially controlling both scanning rate and scanning extent (processivity). To address questions of how Ssl2 functions in promoter scanning and interacts with other initiation activities, we leveraged distinct initiation-sensitive reporters to identify novel ssl2 alleles. These ssl2 alleles, many of which alter residues conserved from yeast to human, confer either upstream or downstream TSS shifts at the model promoter ADH1 and genome-wide. Specifically, tested ssl2 alleles alter TSS selection by increasing or narrowing the distribution of TSSs used at individual promoters. Genetic interactions of ssl2 alleles with other initiation factors are consistent with ssl2 allele classes functioning through increasing or decreasing scanning processivity but not necessarily scanning rate. These alleles underpin a residue interaction network that likely modulates Ssl2 activity and TFIIH function in promoter scanning. We propose that the outcome of promoter scanning is determined by two functional networks, the first being Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow.

In eukaryotic organisms such as yeast, the process of converting genes into proteins begins with the transcription of DNA sequences into mRNA molecules. An enzyme called RNA Polymerase II (Pol II) is responsible for creating new strands of mRNA, but a variety of other so called transcription factors is also needed to kickstart the transcription process. These transcription factors are delivered to genes, where they attach to specific sequences, or promoters, which sit at the beginning of each gene. Once these transcription factors are in place, the double stranded DNA is unzipped to provide access to the DNA that will serve as the template for transcription. In budding yeast, Pol II and another specific transcription factor, known as TFIIH, work together to scan these promoter sequences to find the appropriate start sites of mRNA synthesis. However, several aspects of this process, such as how TFIIH works in promoter scanning, how far its scanning functions can extend, and how its activity is controlled, are currently poorly understood. Zhao et al. have investigated these questions in budding yeast. Using a range of genetic and genomic techniques, Zhao et al. found that certain sections of TFIIH were involved in choosing specific transcription start sites of mRNA synthesis during promoter scanning. These sections were identical in different eukaryotic organisms from yeast to humans, suggesting that these regions may be important for tuning or controlling the activity of TFIIH. Moreover, in yeast, the activity of TFIIH determines how far the scanning unit was able to move along the promoter DNA. Finally, Zhao et al. found that the initiation by promoter scanning was regulated by two distinct networks. The first network controlled how well mRNA synthesis could be initiated at individual transcription start sites; and the second network ­ driven by TFIIH ­ controlled which promoter sequences could be scanned to initiate transcription. This research provides an in-depth look into the early steps of the process of converting DNA into mRNA. The biological machinery used to initiate and control this action is highly conserved between yeast and humans, suggesting that the mechanisms for controlling the activity of these factors could be similar, even if their initiation processes may differ.

Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli.

Chemical modifications of RNA 5'-ends enable "epitranscriptomic" regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates for use as initiating entities, providing an ab initio mechanism for altering the RNA 5'-end. In Escherichia coli cells, RNAs with a 5'-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, "primer-dependent initiation." Here, we use massively systematic transcript end readout (MASTER) to detect and quantify RNA 5'-ends generated by primer-dependent initiation for ∼410 (∼1,000,000) promoter sequences in E. coli The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS-2NTSS-1NTSSWTSS+1, where TSS is the transcription start site, NTSS-1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS-2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS-2) through interchain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally encoded Ecoli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.

A spatiotemporal molecular switch governs plant asymmetric cell division.

Asymmetric cell division (ACD) requires protein polarization in the mother cell to produce daughter cells with distinct identities (cell-fate asymmetry). Here, we define a previously undocumented mechanism for establishing cell-fate asymmetry in Arabidopsis stomatal stem cells. In particular, we show that polarization of the protein phosphatase BSL1 promotes stomatal ACD by establishing kinase-based signalling asymmetry in the two daughter cells. BSL1 polarization in the stomatal ACD mother cell is triggered at the onset of mitosis. Polarized BSL1 is inherited by the differentiating daughter cell, where it suppresses cell division and promotes cell-fate determination. Plants lacking BSL proteins exhibit stomatal overproliferation, which demonstrates that the BSL family plays an essential role in stomatal development. Our findings establish that BSL1 polarization provides a spatiotemporal molecular switch that enables cell-fate asymmetry in stomatal ACD daughter cells. We propose that BSL1 polarization is triggered by an ACD checkpoint in the mother cell that monitors the establishment of division-plane asymmetry.

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

Pausing by RNA polymerase (RNAP) during transcription elongation, in which a translocating RNAP uses a "stepping" mechanism, has been studied extensively, but pausing by RNAP during initial transcription, in which a promoter-anchored RNAP uses a "scrunching" mechanism, has not. We report a method that directly defines the RNAP-active-center position relative to DNA with single-nucleotide resolution (XACT-seq; "crosslink-between-active-center-and-template sequencing"). We apply this method to detect and quantify pausing in initial transcription at 411 (∼4,000,000) promoter sequences in vivo in Escherichia coli. The results show initial-transcription pausing can occur in each nucleotide addition during initial transcription, particularly the first 4 to 5 nucleotide additions. The results further show initial-transcription pausing occurs at sequences that resemble the consensus sequence element for transcription-elongation pausing. Our findings define the positional and sequence determinants for initial-transcription pausing and establish initial-transcription pausing is hard coded by sequence elements similar to those for transcription-elongation pausing.

CapZyme-Seq: A 5'-RNA-Seq Method for Differential Detection and Quantitation of NAD-Capped and Uncapped 5'-Triphosphate RNA.

Nucleoside-containing metabolites such as the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD+ and NADH), 3'-desphospho-coenzyme A (dpCoA), and flavin adenine dinucleotide (FAD) can be incorporated as RNA 5' end caps by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase. We recently reported ″CapZyme-seq,″ a 5'-RNA-seq method that enables the differential detection and quantitation of relative yields of NCIN-capped RNA and uncapped 5'-triphosphate RNA. Here we provide the protocol for constructing cDNA libraries for CapZyme-seq. For complete information on the generation and use of this protocol, please refer to Vvedenskaya et al. (2018a).

Functional determinants of a small protein controlling a broadly conserved bacterial sensor kinase.

The PhoQ/PhoP two-component system plays a vital role in the regulation of Mg2+ homeostasis, resistance to acid and hyperosmotic stress, cationic antimicrobial peptides, and virulence in Escherichia coli, Salmonella and related bacteria. Previous studies have shown that MgrB, a 47 amino acid membrane protein that is part of the PhoQ/PhoP regulon, inhibits the histidine kinase PhoQ. MgrB is part of a negative feedback loop modulating this two-component system that prevents hyperactivation of PhoQ and may also provide an entry point for additional input signals for the PhoQ/PhoP pathway. To explore the mechanism of action of MgrB, we have analyzed the effects of point mutations, C-terminal truncations and transmembrane region swaps on MgrB activity. In contrast with two other known membrane protein regulators of histidine kinases in E. coli, we find that the MgrB TM region is necessary for PhoQ inhibition. Our results indicate that the TM region mediates interactions with PhoQ and that W20 is a key residue for PhoQ/MgrB complex formation. Additionally, mutations of the MgrB cytosolic region suggest that the two N-terminal lysines play an important role in regulating PhoQ activity. Alanine scanning mutagenesis of the periplasmic region of MgrB further indicates that, with the exception of a few highly conserved residues, most residues are not essential for MgrB's function as a PhoQ inhibitor. Our results indicate that the regulatory function of the small protein MgrB depends on distinct contributions from multiple residues spread across the protein. Interestingly, the TM region also appears to interact with other non-cognate histidine kinases in a bacterial two-hybrid assay, suggesting a potential route for evolving new small protein modulators of histidine kinases.

Universal promoter scanning by Pol II during transcription initiation in Saccharomyces cerevisiae.

BACKGROUND: The majority of eukaryotic promoters utilize multiple transcription start sites (TSSs). How multiple TSSs are specified at individual promoters across eukaryotes is not understood for most species. In Saccharomyces cerevisiae, a pre-initiation complex (PIC) comprised of Pol II and conserved general transcription factors (GTFs) assembles and opens DNA upstream of TSSs. Evidence from model promoters indicates that the PIC scans from upstream to downstream to identify TSSs. Prior results suggest that TSS distributions at promoters where scanning occurs shift in a polar fashion upon alteration in Pol II catalytic activity or GTF function. RESULTS: To determine the extent of promoter scanning across promoter classes in S. cerevisiae, we perturb Pol II catalytic activity and GTF function and analyze their effects on TSS usage genome-wide. We find that alterations to Pol II, TFIIB, or TFIIF function widely alter the initiation landscape consistent with promoter scanning operating at all yeast promoters, regardless of promoter class. Promoter architecture, however, can determine the extent of promoter sensitivity to altered Pol II activity in ways that are predicted by a scanning model. CONCLUSIONS: Our observations coupled with previous data validate key predictions of the scanning model for Pol II initiation in yeast, which we term the shooting gallery. In this model, Pol II catalytic activity and the rate and processivity of Pol II scanning together with promoter sequence determine the distribution of TSSs and their usage.

Detection of spacer precursors formed in vivo during primed CRISPR adaptation.

Type I CRISPR-Cas loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA. Immunity involves adaptation, the integration of ~30-bp DNA fragments, termed prespacers, into the CRISPR array as spacers, and interference, the targeted degradation of DNA containing a protospacer. Interference-driven DNA degradation can be coupled with primed adaptation, in which spacers are acquired from DNA surrounding the targeted protospacer. Here we develop a method for strand-specific, high-throughput sequencing of DNA fragments, FragSeq, and apply this method to identify DNA fragments accumulated in Escherichia coli cells undergoing robust primed adaptation by a type I-E or type I-F CRISPR-Cas system. The detected fragments have sequences matching spacers acquired during primed adaptation and function as spacer precursors when introduced exogenously into cells by transformation. The identified prespacers contain a characteristic asymmetrical structure that we propose is a key determinant of integration into the CRISPR array in an orientation that confers immunity.

Highly efficient 5' capping of mitochondrial RNA with NAD+ and NADH by yeast and human mitochondrial RNA polymerase.

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~15% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial transcriptional outputs, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

Analysis of Bacterial Transcription by "Massively Systematic Transcript End Readout," MASTER.

A systems-level view of cellular gene expression requires understanding the mechanistic principles governing each step of transcription. In this chapter, we describe a massively multiplexed method for the analysis of the relationship between nucleic acid sequence and transcription termed "MASTER," for massively systematic transcript end readout. MASTER enables parallel measurements of transcription output from at least 410 (~1,000,000) individual template sequences in vitro and in vivo. MASTER involves constructing a DNA template library of barcoded sequences, generating RNA transcripts from the library during transcription in vitro or in vivo, and analyzing the relative abundance and 5'-end sequences of the RNA transcripts by high-throughput sequencing. MASTER provides a powerful, rapid, and versatile method to identify sequence determinants of each step of transcription and to define the mechanistic basis by which these sequence determinants dictate transcription output.

Role for GrgA in Regulation of σ28-Dependent Transcription in the Obligate Intracellular Bacterial Pathogen Chlamydia trachomatis.

The obligate intracellular bacterial pathogen Chlamydia trachomatis has a unique developmental cycle consisting of two contrasting cellular forms. Whereas the primary Chlamydia sigma factor, σ66, is involved in the expression of the majority of chlamydial genes throughout the developmental cycle, expression of several late genes requires the alternative sigma factor, σ28 In prior work, we identified GrgA as a Chlamydia-specific transcription factor that activates σ66-dependent transcription by binding DNA and interacting with a nonconserved region (NCR) of σ66 Here, we extend these findings by showing GrgA can also activate σ28-dependent transcription through direct interaction with σ28 We measure the binding affinity of GrgA for both σ66 and σ28, and we identify regions of GrgA important for σ28-dependent transcription. Similar to results obtained with σ66, we find that GrgA's interaction with σ28 involves an NCR located upstream of conserved region 2 of σ28 Our findings suggest that GrgA is an important regulator of both σ66- and σ28-dependent transcription in C. trachomatis and further highlight NCRs of bacterial RNA polymerase as targets for regulatory factors unique to particular organisms.IMPORTANCEChlamydia trachomatis is the number one sexually transmitted bacterial pathogen worldwide. A substantial proportion of C. trachomatis-infected women develop infertility, pelvic inflammatory syndrome, and other serious complications. C. trachomatis is also a leading infectious cause of blindness in underdeveloped countries. The pathogen has a unique developmental cycle that is transcriptionally regulated. The discovery of an expanded role for the Chlamydia-specific transcription factor GrgA helps us understand the progression of the chlamydial developmental cycle.

"NAD-capQ" detection and quantitation of NAD caps.

RNA 5' cap structures comprising the metabolic effector nicotinamide adenine dinucleotide (NAD) have been identified in diverse organisms. Here we report a simple, two-step procedure to detect and quantitate NAD-capped RNA, termed "NAD-capQ." By use of NAD-capQ we quantitate NAD-capped RNA levels in Escherichia coli, Saccharomyces cerevisiae, and human cells, and we measure increases in NAD-capped RNA levels in cells from all three organisms harboring disruptions in their respective "deNADding" enzymes. We further show that NAD-capped RNA levels in human cells respond to changes in cellular NAD concentrations, indicating that NAD capping provides a mechanism for human cells to directly sense and respond to alterations in NAD metabolism. Our findings establish NAD-capQ as a versatile, rapid, and accessible methodology to detect and quantitate 5'-NAD caps on endogenous RNA in any organism.

CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5' Capping with NAD.

Nucleoside-containing metabolites such as NAD+ can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

The mechanism of variability in transcription start site selection.

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.