Molecular mechanisms of RNA polymerase--the F/E (RPB4/7) complex is required for high processivity in vitro

Abstract

Transcription elongation in vitro is affected by the interactions between RNA polymerase (RNAP) subunits and the nucleic acid scaffold of the ternary elongation complex (TEC, RNAP-DNA-RNA). We have investigated the role of the RNAP subunits F/E (homologous to eukaryotic RPB4/7) during transcription elongation and termination using a wholly recombinant archaeal RNAP and synthetic nucleic acid scaffolds. The F/E complex greatly stimulates the processivity of RNAP, it enhances the formation of full length products, reduces pausing, and increases transcription termination facilitated by weak termination signals. Mutant variants of F/E that are defective in RNA binding show that these activities correlate with the nucleic acid binding properties of F/E. However, a second RNA-binding independent component also contributes to the stimulatory activities of F/E. In summary, our results suggest that interactions between RNAP subunits F/E and the RNA transcript are pivotal to the molecular mechanisms of RNAP during transcription elongation and termination.

Figure 1.

Transcription elongation assay using synthetic nucleic acid scaffolds. Experimental setup (A). A 14 nt RNA oligonucleotide is pre-annealed to the DNA TS and subsequently incubated with recombinant RNAP and the NTS. Upon the addition of nucleotides RNAP extends the RNA primer completely independent of promoter elements and basal transcription factors. The templates used in this study are illustrated in (B). The RNA primer is highlighted in red and the DNA TS and NTS in black. The U₇ and U₅ terminator signals, and the A₇ and A₅ controls are highlighted in blue. Red arrows indicate the 3′ termini of terminated transcripts and blue arrows indicate the 3′ termini of transcripts generated by runoff. Structure and function of RNAP subunits F/E (C). ‘The S. shibatae RNAP structure (pdb 2WAQ) was manually superimposed on the DNA–RNA scaffold of the S. cerevisiae RNAPII elongation complex (DNA in yellow, RNA in red, pdb 1Y1W). The archaeal RNAP core is coloured grey, the two subunits F and E and highlighted in magenta and blue, and the RNAP clamp in green, respectively. Residues in subunit E that affect RNA binding are highlighted in red, and the RNA transcript that is not resolved in the crystal structures is sketched as red dotted line.

Figure 2.

The NTS stimulates the processivity. (A) The transcript pattern that was generated by in vitro transcription of the parental elongation scaffold (Figure 1B) under single-round conditions with RNAP (200 nM) in the presence (+NTS, grey bars) and absence of the NTS (−NTS, black bars). Samples were taken at the indicated time points (0.5, 1, 2, 5, 15, 30, 60 min). Histograms are based on quantization of at least three independent experiments (arbitrary units, AU). (B) The synthesis of the 71 nt-run off transcript normalized to the 60-min time point (−NTS). (C) The synthesis of total transcripts normalized to the 60-min time point (−NTS).

Figure 3.

The F/E complex enhances the processivity. (A) The transcript pattern generated by RNAPΔF/E (200 nM) using the parental elongation scaffold (Figure 1B) in the absence of the NTS either without (RNAPΔF/E) or following preincubation with 800 nM F/E (wt RNAP). (B) The transcript pattern generated by RNAPΔF/E (50 nM) in the presence of the NTS either without (RNAPΔF/E) or following preincubation with 200 nM F/E (wt RNAP). Samples were taken at the indicated time points. Note the difference in time scales (1–60 min −NTS and 0.5–20 min +NTS) and RNAP concentrations (200 nM −NTS and 50 nM +NTS) that are due to the higher activity level due to the presence of the NTS. Histograms are based on quantization of at least three independent experiments. (C) The synthesis of the 71 nt run off product wild type RNAP (grey bars) normalized to time point at 60 min (in arbitrary units, AU), and the total transcripts synthesized by wild type RNAP (grey bars) and RNAPΔF/E (black bars) normalized to the value at 60 min (−F/E). (D) The accumulation of the 71 nt transcript in the presence of the NTS by wild type RNAP (grey bars) and RNAPΔF/E (black bars), and the accumulation of total transcripts in the absence (black bars) or presence of F/E (grey bars). (E) RNAPΔF/E or RNAPΔK/F/E were preincubated with F/E and transcription reactions were carried out in the absence (−NTS) or presence of the NTS (+NTS).

Figure 4.

The stimulatory effect of RNAP subunits F/E on transcription elongation correlates with the RNA binding activity. (A) The binding of wild type F/E and three mutant variants of the F/E complex (5 and 10 µM) to a 54 nt ³²P-labelled riboprobe in EMSAs. (B) Transcription pattern generated by RNAPΔF/E (200 nM) preincubated with wild type F/E, F/E^K33E, F/E^R37E or F/E^loop in a dose-response experiment (8, 80 or 800 nM) using the parental elongation scaffold (Figure 1B) in the absence of the NTS (reaction time is 20 min). (C) The activity of the F/E variants in the presence of the NTS in a time course experiment. Transcript patterns generated are by RNAPΔF/E in the absence of F/E or preincubated with 80 nM F/E, F/E^K33E, F/E^R37E or F/E^loop. Samples were taken at the indicated time points (40, 90 and 300 s). (D) Quantization of the 71 nt transcript of panel (B) normalized to RNAPΔF/E. (E) Quantization of the 71 nt transcript of panel (C) normalized to RNAPΔF/E at 300 s (in arbitrary units).

Figure 5.

Transcription termination of archaeal RNAP is facilitated by poly-U signals. (A) A stretch of seven U-residues (U₇-signal) but not A-residues (A₇-signals) in the template facilitate efficient transcription termination. The parental template generates a 71 nt runoff transcript, whereas the +53 and +66 U₇ terminator templates generate 53 or 66 nt transcripts, respectively. The +53 and +66 A₇ templates lead to the synthesis of runoff transcripts. All reactions were carried out for 5 min, include the NTS and contain 50 nM wild type RNAP. (B) Reducing the number of U-residues from seven (U₇-signal) to five (U₅-signal) increases transcription read through. Reactions were carried out for 5 min using 50 nM RNAP. (C) Both U₇- and U₅-signals can terminate transcription without the NTS. Reactions were carried out for 20 min using 50 nM RNAP. Panels (D) and (E) show that both run off- and terminated-, but not stalled-transcripts are released from the elongation complex. Products of transcription elongation reactions using the +66 A₇-, +66 U₇- and +51 U₅-templates were separated electrophoretically under native (D) and denaturating conditions (E). As a negative control for transcript release stalled transcription elongation complexes were generated by omitting GTP from a transcription reaction (‘no GTP') using the +66 A₇-template. Under these conditions a 41 nt transcript is synthesized which remains associated with RNAP. Adding GTP to this reaction after 10 min (‘GTP at 10’) and letting the reaction proceed for another 10 min generates the 66 nt run off transcript (E) that is released (D). Transcripts synthesized from U₇- and U₅-templates are released (D). In addition a proportion of transcripts can be found in low mobility region of the native gel that corresponds to paused complexes (D and E). For unclear reasons the U7 (+66) template leads to more far upstream pausing (at <30 nt) than the U7 (+66) and U5 (+51) templates.

Figure 6.

RNAP subunits F/E enhance transcription termination from weak U₅-terminator signals. (A) Comparison of transcript patterns generated by wild type RNAP and RNAPΔF/E using parental-(‘run off’), +53 U₇- and +51 U₅-templates. Reactions were carried out for 90 s using 50 nM RNAP in the presence of the NTS. RNAP subunits F/E stimulate the formation of RNA transcripts, but have no significant influence on pausing at position +41 (‘no GTP’) or on transcription termination directed by the strong +53 U₇-signal. (B) The transcript pattern generated by wild type RNAP, RNAPΔF/E and RNAP F/E^loop using the +51 U₅- and +26 U₅-terminator templates. Reactions were carried out for 20 min including the NTS and using 50 nM RNAP. (C) Quantization of the ratio between terminated and read through transcripts in panel (B).