Interactions between RNA polymerase and the "core recognition element" counteract pausing

Abstract

Transcription elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native elongating transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G₋₁₀Y₋₁G(+1) (where -1 corresponds to the position of the RNA 3' end). We demonstrate that sequence-specific interactions between RNAP core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a posttranslocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.

Fig. 1. Identification of consensus PE

A. Sequence logo for consensus PE from NET-seq. Red, bases with ≥0.2 bit sequence-information content. B. In vitro transcription assays with consensus PEs and mutant PEs. Top, templates. Bottom, results. +29, RNA before addition of UTP; +46, RNA in TEC at PE; RT, read-through RNA; red, consensus PE bases.

Fig. 2. Contributions of individual base pairs of consensus PE

A. In vitro transcription assays with yrbL PE derivatives. Red, consensus PE bases. B. Schematic representation of TEC in pre-translocated state at consensus PE (top) and TEC in post-translocated state at consensus PE (bottom). White boxes, DNA; gray boxes, RNA; gray shading, RNAP; red, consensus PE bases; i and i+1, RNAP active-center i and i+1 sites.

Fig. 3. Sequence-specific RNAP-G_CRE interactions modulate pausing

A. Structural organization of TEC in post-translocated state at consensus PE (left) and RPo in transcription initiation at promoter containing consensus CRE (right; 21). The presence in each case of an unpaired G at downstream end of nontemplate strand of transcription bubble suggests the possibility of equivalent sequence-specific interactions between RNAP core and the G. Red at left, bases of the consensus PE; red at right, G_CRE. B. Results of fluorescence-detected equilibrium assays (top) and kinetic (bottom) assays of interactions of RNAP derivatives with nucleic-acid scaffolds containing G, A, T, or an abasic site (X) at position corresponding to G_CRE. ntDNA, nontemplate-strand DNA; tDNA template-strand DNA. C. Sequence logos for consensus PE with RNAP-β^WT (top; 0.4 bit sequence-information content at G_CRE) and RNAP-β^D446A (bottom; 1 bit of sequence-information content at G_CRE), as defined by mNET-seq. Red, bases with ≥0.2 bit sequence information content. D. Pause-capture efficiencies of RNAP β^WT (left panels) and RNAP β^D446A (right panels) at yrbL PE and gltP PE in vitro. +29, RNA before addition of UTP; +46, RNA in TEC at PE; asterisks, RNA in TECs at additional sites where RNAP β^D446A exhibits higher pause-capture efficiency than RNAP β^WT; RT, read-through RNA; red, consensus PE bases.

Fig. 4. Sequence-specific RNAP-G_CRE interactions modulate translocation bias

A. Structural organization of TEC in pre-translocated state at consensus PE (left; unfavorable RNAP-CRE interaction) and TEC in post-translocated state at consensus PE (right; favorable RNAP-CRE interaction). PPi, pyrophosphate. B. Nucleic-acid scaffolds used for translocation-bias assays. Asterisk, radiolabel on RNA 5′ end; box, position corresponding to G_CRE; red, consensus PE base. C. Translocational bias for RNAP β^WT and RNAP β^D446A on nucleic-acid scaffolds containing G or T at position corresponding to G_CRE. Gel images show pyrophosphorolysis reaction progress from 0-30 min. 9-nt, scaffold; 8-nt, product of pyrophosphorolysis; 10-nt, product of “chase” reaction with GTP (+1G template) or UTP (+1T template). Graph shows fraction of unaltered scaffold (mean±SEM; 3 measurements) as function of time.