Real-Time Observation of Backtracking by Bacterial RNA Polymerase

Abstract

RNA polymerase (RNAP) backtracking is a backward sliding of the enzyme along DNA and RNA. It plays important roles in many essential processes in bacteria and in eukaryotes. We describe here a fluorescence-based approach that allows a real-time observation of bacterial RNAP backtracking. A Cy3 fluorescence probe, when incorporated into a specific site in the nontemplate strand near the site of backtracking, allows RNAP movements to be monitored near the probe because of a robust enhancement of fluorescence caused by protein proximity. Using this approach, we showed that binding of NTP to the active site prior to phosphodiester bond formation inhibited backtracking, consistent with the coupling of NTP binding to translocation. The extent and the kinetics of backtracking did not show a simple correlation with the instability of the DNA-RNA hybrid, indicating a more complex dependence of backtracking on DNA template sequence. Experiments with transcription through an abasic site in DNA template or neutravidin bound to biotinylated template strand base illustrated an important role of backtracking in defining how RNAP reacts to such obstacles in the DNA template. The described approach will be a useful tool in deciphering the mechanism of backtracking and in studying factors that affect the backtracking.

Figure 1

(A) Design of fluorescent construct (P27 promoter) for real-time detection of RNAP backtracking. The star denotes Cy3 label at the +2 position of the nontemplate strand. (B) Calculated relative duplex stabilities (ratio of duplex free energy of the 9 bp hybrid to the average of free energies of all possible sequence combinations for 9 bp long duplexes) of the 9 bp RNA:DNA hybrids along the sequence of the first 27 transcribed nucleotides of P27. (C) Fluorescence intensity changes of P27. At time 0 the P27 was added to a solution of RNAP in a fluorometer cuvette. At the time point marked by a star, the transcription was started by adding ATP, UTP, GTP and heparin (grey) or ATP, UTP, GTP, CTP and heparin (black).

Figure 2

RNAP complexes that accumulate during fluorescence signal recovery phase of the reaction are transcriptionally inactive. The star denotes addition of ATP, UTP, GTP and heparin to the sample. The missing NTP (CTP) was added (as indicated by an arrow) at various delay times after the start of transcription with 3 NTP’s: (A) 15 s, (B) 2 min, (C) 5 min and (D) 20 min.

Figure 3

The effect of GreB on the time course of fluorescence intensity changes of P27. The star denotes initiation of transcription. (A) Transcription was initiated by the addition of 3 NTP’s and heparin and at a time point marked by an arrow GreB was added (150 nM). (B) Transcription was initiated by the addition of 3 NTP’s and heparin together with 150 nM GreB.

Figure 4

(A) Comparison of calculated relative 9 bp duplex stabilities for P27-31 (cyan), P27-32 (pink), P27-33 (blue), P27-34 (green), and P27-35 (red) constructs with P27 construct (black). (B) Fluorescence intensity changes of constructs from panel A (the color coding is the same as in panel A). At time 0 the promoter was added to a solution of RNAP in a fluorometer cuvette. At the time point marked by a star the transcription was started by adding ATP, UTP, GTP and heparin. The arrow indicates addition of CTP. (C) Comparison of calculated relative 9 bp duplex stabilities for P27-36 (red), P27-37 (green), with P27 construct (black). (D) Fluorescence intensity changes of constructs from panel C (the color coding is the same as in panel D). At time 0 the DNA construct was added to a solution of RNAP in a fluorometer cuvette. At the time point marked by a star the transcription was started by adding ATP, UTP, GTP and heparin.

Figure 5

The effect of nonhydrolizable analogue of CTP (CpCpp) on RNAP backtracking on P27 construct. (A) Transcription reactions were initiated by adding ATP, UTP, GTP, heparin and no CpCpp (a), 50 μM CpCpp (b), 150 μM CpCpp (c) or 300 μM CpCpp (d). Curve (e) corresponds to transcription initiated by adding ATP, UTP, GTP, CTP and heparin. (B) Transcription reactions were initiated by adding ATP, UTP, GTP, heparin. CpCpp (150 μM) was added together with NTP’s (a) or after 2 min (b) or 5 min (c) delay after addition of NTP’s. Delayed addition of CpCpp is indicated by the arrows.

Figure 6

(A) Design of fluorescent construct (P54) for real-time detection of RNAP backtracking away from the promoter. The star denotes Cy3 label at +29 position of the nontemplate strand. (B) Fluorescence intensity changes of P54 promoter. At time 0 the P54 was added to a solution of RNAP in a fluorometer cuvette. At the time point marked by a star the transcription was started by adding ATP, UTP, GTP and heparin (red), ATP, UTP, GTP, heparin, and GreB (green) or ATP, UTP, GTP, CTP and heparin (blue). The arrow denotes time points where the missing NTP (CTP) was added.

Figure 7

(A) Design of fluorescent construct (P27A) containing an abasic site at +28 position of the template strand. The star denotes Cy3 label at +2 position of the nontemplate strand. (B) Fluorescence intensity changes of P27A construct. At the time point marked by a star transcription was started by adding ATP, UTP, GTP and heparin (grey) followed by addition of GreB and CTP (indicated by the arrows) or by adding ATP, UTP, GTP, CTP and heparin (black). (C) Design of fluorescent construct (P37B) with neutravidin bound at position +37 of the template strand. The star denotes Cy3 label at +2 position of the nontemplate strand. (D). Fluorescence intensity changes of P37B construct. At the time point marked by a star transcription was started by adding ATP, UTP, GTP and heparin (green), ATP, UTP, GTP, CTP and heparin (pink), ATP, UTP, GTP, heparin and GreB (red) or ATP, UTP, GTP, CTP, heparin and GreB (blue). The arrows denote time points where the missing NTP (CTP) was added.

Figure 8

RNAP backtracking during multiple rounds of transcription from P27. At time 0 the P27 was added to a solution of RNAP in a fluorometer cuvette. The star denotes addition of ATP, UTP, GTP and CTP. Heparin was added (as indicated by an arrow) at various delay times after the start of transcription with 4 NTP’s: (A) 15 s, (B) 2 min, (C) 5 min and (D) 20 min. (E) Effect of GreB on the amounts of full-length transcript produced from P27. Transcription was allowed to proceed for 30 min. The data were normalized to the amount of transcript made by P27 under single round of transcription condition (in the presence of heparin).