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

Abstract

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.

Keywords: RNA polymerase; initial transcription; massively parallel reporter assay; photocrosslinking; promoter; sigma factor; transcription; transcription elongation; transcription pausing.

Figure 1.. Mechanisms of initial transcription and transcription elongation

(A) Initial transcription involves RNAP-active-center translocation through “DNA scrunching,” with RNAP in complex with initiation factor σ. Panel shows first six nucleotide-addition steps of initial transcription, starting from RNAP-promoter open complex (RPo), and yielding successive RNAP-promoter ITCs containing 2- to 6-nt RNA products (ITC,2-ITC,6). Following each nucleotide addition, the RNAP active center translocates forward through a DNA scrunching mechanism, from a pre-translocated state (pre; right column) to a post-translocated state (post; left column), and the RNAP-active-center A-site position advances by 1 bp. Grey, RNAP; yellow, σ; dark yellow, σ finger; blue, −10 element and TSS; P and A, RNAP-active-center P-site and A-site, respectively; black boxes, DNA nucleotides (nontemplate-strand nucleotides above template-strand nucleotides); red boxes, RNA nucleotides; positions numbered relative to the TSS position, +1. (B) Transcription elongation involves RNAP-active-center translocation through “DNA stepping,” with RNAP not containing σ. Panel shows two nucleotide-addition steps of transcription elongation, from TEC containing 11 nt of RNA (TEC,11) to TEC containing 13 nt of RNA (TEC,13). Following each nucleotide addition, RNAP translocates forward through a DNA stepping mechanism, between a pre-translocated state (pre; right column) and a post-translocated state (post; left column), and the RNAP-active-center A-site position advances by 1 bp. Symbols and colors as in A.

Figure 2.. Approaches to map the RNAP-active-center A-site position: NET-seq and XACT-seq

(A) Approach to map the RNAP-active-center A-site position through analysis of RNA 3’ ends. Procedure entails isolating nascent RNA and identifying and quantifying RNA 3’ ends by polyacrylamide gel electrophoresis (PAGE) or high-throughput sequencing (NET-seq). The RNAP-active-center A-site position (purple box; numbered relative to TSS position, +1) is approximated based on the RNA 3’ end (pink box); other symbols and colors as in Fig. 1. Different translocational states adopted by TEC containing single, defined 20-nt RNA product--e.g., reverse-translocated by 5 bp (TEC,20-reverse-5), reverse-translocated by 1 bp (TEC,20-reverse-1), pre-translocated (TEC, 20-pre), post-translocated (TEC,20-post), and hyper-translocated by 1 bp (TEC,20-hyper-1)--all yield the same RNA 3’ end and therefore cannot be distinguished. (B) Approach to map the RNAP-active-center A-site position through site-specific protein-DNA photocrosslinking. using RNAP derivative containing photoactivatable agent (green circle labeled Bpa) that crosslinks to DNA at defined distance from RNAP active center. Procedure entails UV-irradiating transcription complexes, isolating RNAP-crosslinked DNA, and identifying and quantifying crosslinking sites by analysis of primer extension products by PAGE or HTS (XACT-seq). The RNAP-active-center A-site position (purple box; numbered relative to TSS position, +1) is defined based on identity of crosslinking site (green box). Symbols as in A. Different translocational states adopted by a TEC containing single, defined RNA product yield different crosslinking sites and therefore can be distinguished. See also Fig. S1.

Figure 3.. Incorporation of Bpa at RNAP-β’ subunit residue R1148 does not affect pausing in vitro

(A) Transcription-elongation pausing. Top, sequence of DNA template containing 65-bp transcribed region with consensus sequence element for transcription-elongation pausing (red). Bottom, PAGE analysis of products of transcription reactions performed with RNAP-β’ ^R1148Bpa or RNAP-β’ ^wt. Gel shows RNA products at indicated times after addition of NTPs to complexes halted at position +29. +46, pause position. Pause-capture efficiencies (calculated as described in Landick et al., 1996) are means ± SD (n =3). (B) Initial-transcription pausing. Top, sequence of placCONS initial-transcribed region. Bottom left, products of reactions performed using ApA as initiating entity and NTP subsets that enable synthesis of RNA products of up to 7 nt (UTP/GTP) or up to 11 nt (UTP/GTP/ATP). Bottom right, products of reaction performed using ApA as initiating entity and NTP subset that enables synthesis of RNA products up to 11 nt, with RNAP-β’ ^R1148Bpa (β’ ^Bpa) or RNAP-β’ ^wt (β’ ^wt). Positions of 6-nt paused RNA product and 7- and 11-nt full-length RNA products are indicated. Percentage of 6-nt RNA products are means ± SD (n = 3). See also Fig. S2.

Figure 4.. RNAP-active-center A-site positions in initial-transcription pausing at the lacCONS promoter in vitro and in vivo

(A) In vitro. Top, placCONS. Middle, primer extension mapping of crosslinking sites. Bottom, position of RNAP-active-center A-site (purple) and nucleotide crosslinked to Bpa (green) defined relative to the TSS position. Markers, sequence ladder generated using placCONS. β’ ^Bpa, RNAP-β’ ^R1148Bpa; β’ ^Bpa; σ^{Δ finger}, RNAP-β’ ^R1148Bpa containing σ finger deletion; β’ ^Bpa; β^D446A, RNAP-β’ ^R1148Bpa containing β substitution D446A. (B) In vivo. Top, three-plasmid merodiploid system for co-production, in E. coli cells, of decahistidine-tagged, RNAP-β’ ^R1148Bpa, in the presence of untagged wild-type RNAP. First plasmid carries gene for RNAP βʹ subunit (grey rectangle) with nonsense codon (green) at residue βʹ R1148; second plasmid carries genes for engineered Bpa-specific nonsense-suppressor tRNA and aminoacyl-tRNA synthetase (white rectangles); third plasmid carries placCONS; and chromosome (brown oval) carries genes for wild-type RNAP subunits (light grey rectangle). Middle, primer-extension mapping of crosslinking sites. Bottom, position of RNAP-active-center A-site (purple) and nucleotide crosslinked to Bpa (green) defined relative to the TSS position, +1. Rif, rifampin; Markers, sequence ladder generated using placCONS. (C) Interpretation of results in A and B. Symbols and colors as in Figs. 1–2. See also Fig. S3.

Figure 5.

Promoter-position dependence of initial-transcription pausing for a library of 4¹¹ (~4,000,000) promoters in vivo (A) placCONS template library containing all possible sequences from promoter position +3 through +13 (placCONS-N11). These promoter positions encompass expected positions of crosslinking of RNAP-β’ ^R1148Bpa in initial transcription. (B) Left, PAGE analysis of RNAP-active-center A-site positions in vivo. Right, histogram showing signals detected in absence (black line) or presence (grey line) of Rif. Markers, sequence ladder generated using placCONS-N11. (C) Position of RNAP-active-center A-site (purple) and nucleotide crosslinked to Bpa (green) defined relative to the TSS position, +1. See also Fig. S4.

Figure 6.. Promoter-sequence determinants for initial-transcription pausing in a library of 4¹¹ (~4,000,000) promoters in vivo

(A) Position-independent, global, consensus sequence for initial-transcription pausing for RNAP-active-center A-site positions +5, +6, +7, +8, and +9. Consensus nucleotides are in black. (B) Percent occupancy at each initial-transcribed region tetranucleotide sequence for RNAP-active-center A-site positions +5, +6, +7, +8, and +9. Red, pink, cyan, and blue denote pause-site sequences with 3 of 3, 2 of 3, 1 of 3, and 0 of 3 matches to global consensus sequence, respectively. Mean ± SEM (n = 3). (C) Position-specific consensus sequences for initial-transcription pausing for RNAP-active-center A-site positions +3, +4, +5, +6, +7, +8, and +9. (D) Representative sequences yielding high RNAP occupancy at positions +5, +6, +7, +8, and +9. Colors as in Fig. 5C. (E) RNAP-active-center A-site positions and crosslinking positions in vitro for sequences of D. Lanes 1–4, sequence markers; lanes 5–9, data for RNAP-β’ ^R1148Bpa; lanes 10–14, data for RNAP-β’ ^R1148Bpa containing σ finger deletion. (F) RNAP-active-center A-site positions and crosslinking positions in vitro for sequences of D analyzed as pool. Histogram on right presents quantitation of RNAP occupancies at +5, +6, +7, +8 and +9, with data for RNAP-β’ ^R1148Bpa in red and data for RNAP-β’ ^R1148Bpa containing σ finger deletion in black. Ratios are means ± SD (n = 3). See also Figs. S5–S7.

Figure 7.. Relationship between sequence determinants for initial-transcription pausing and transcription-elongation pausing

(A) Consensus sequence for initial-transcription pausing. Positions are labeled relative to the RNAP-active-center A-site (purple box) and P-site. (B) Consensus sequence for transcription-elongation pausing. Top, positions numbered relative to RNA 3’ end (pink box). Bottom, positions numbered relative to RNAP-active-center A-site (purple box) and P-site. (C) Positions of consensus-sequence nucleotides in pre-translocated-state complexes in initial-transcription pausing (ITC-pre) and transcription-elongation pausing (TEC-pre). Grey boxes with white lettering, high-information-content DNA nucleotides of consensus sequence; grey boxes without white lettering; low-information-content nucleotides of consensus-sequence; pink boxes with white lettering, high-information-content RNA nucleotides of consensus sequence. Dotted rectangle, region enlarged in D. Other symbols and colors as in Fig. 1. (D) Enlarged view, showing unfavorable interaction of RNAP β residue D446 with nontemplate-strand pyrimidine (Y) at A-site (crossed-out bracket labeled βD446). (E) Positions of consensus-sequence nucleotides in post-translocated-state complexes in initial transcription pausing (ITC-post) and transcription-elongation pausing (TEC-post). Dotted rectangle, region enlarged in F. (F) Enlarged view showing favorable interaction of RNAP β residue D446 with nontemplate-strand purine (R) at A-site (bracket labeled βD446).