Mycobacterial RNA polymerase forms unstable open promoter complexes that are stabilized by CarD

Abstract

Escherichia coli has served as the archetypal organism on which the overwhelming majority of biochemical characterizations of bacterial RNA polymerase (RNAP) have been focused; the properties of E. coli RNAP have been accepted as generally representative for all bacterial RNAPs. Here, we directly compare the initiation properties of a mycobacterial transcription system with E. coli RNAP on two different promoters. The detailed characterizations include abortive transcription assays, RNAP/promoter complex stability assays and DNAse I and KMnO4 footprinting. Based on footprinting, we find that promoter complexes formed by E. coli and mycobacterial RNAPs use very similar protein/DNA interactions and generate the same transcription bubbles. However, we find that the open promoter complexes formed by E. coli RNAP on the two promoters tested are highly stable and essentially irreversible (with lifetimes much greater than 1 h), while the open promoter complexes on the same two promoters formed by mycobacterial RNAP are very unstable (lifetimes of about 2 min or less) and readily reversible. We show here that CarD, an essential mycobacterial transcription activator that is not found in E. coli, stabilizes the mycobacterial RNAP/open promoter complexes considerably by preventing transcription bubble collapse.

Figure 1.

Sequences of the Mtb AP3 [Mtb rrnA–P3; (25)] and AC50 [-35con of (24)] promoters.

Figure 2.

Dependence of Mbo and Eco RNAP transcription activity on salt concentration for both the AP3 and AC50 promoters. Single round abortive initiation assays measured GpUpU (AP3) or GpGpA (AC50) production. On the left, [³²P]-labeled abortive transcript production was monitored by polyacrylamide gel electrophoresis and autoradiography. On the right, transcript production was quantified by phosphorimagery and plotted versus [KCl] (top) or [KGlu] (bottom) concentration (10–250 mM). On the plots, Mbo RNAP (alone) is shown in red, Mbo RNAP + CarD in green, Eco RNAP in blue. (A) AP3 promoter. (B) AC50 promoter.

Figure 3.

Lifetimes of promoter complexes measured by abortive transcription. On the top, [³²P]-labeled abortive transcript production at times after addition of a large excess of competitor promoter DNA trap was monitored by polyacrylamide gel electophoresis and autoradiography. On the bottom, transcript production was quantified by phosphorimagery and plotted. The lines indicate single-exponential decay curves fit to the data points. The decay half-lives (t_1/2) calculated from the fits are shown to the right of the gel images. The insets show histograms denoting transcription activity at time 0 (before incubation with competitor trap DNA). (A) AP3 promoter: assays were performed in transcription buffer (see Materials and Methods) with 10 mM KGlu. (B) AC50 promoter: assays were performed in transcription buffer (see Materials and Methods) with 150 mM KGlu.

Figure 4.

Weak activity of Mbo RNAP on the AP3 promoter is not due to the suboptimal 18-bp −10/-35 spacer of AP3 nor Mbo σ^A. (A) Sequences of AP3 (18-bp −10/-35 spacer, top) and spacer mutant promoters. AP3Δ23 has a deletion of the −23 bp, giving AP3Δ23 a 17-bp −10/-35 spacer. AP3(AC50sp) has the optimal 17-bp spacer of AC50 (blue) swapped for the AP3 spacer. (B) Single round abortive initiation activity of RNAPs on wt AP3, AP3Δ23 and AP3(AC50sp) was determined in transcription buffer as described (Figure 2 and Materials and Methods) with 10 mM KGlu. Gels show transcription initiation products (GpUpU* synthesis). (C) Single round abortive initiation assays were performed as described in (B) with hybrid holoenzymes. Eco core RNAP was mixed with either Eco σ⁷⁰ or Mbo σ^A and assayed for activity on the AP3 promoter. The reverse experiment was also performed with Mbo core RNAP mixed with Eco σ⁷⁰ or Mbo σ^A. CarD was also tested for effects on transcription where indicated. Graphs below represent relative activities of hybrid holoenzymes normalized to Eco-core/σ⁷⁰ holoenzyme. The inset shows a magnification of Mbo-core/Eco σ⁷⁰, Mbo-core/Eco σ⁷⁰ + CarD and Mbo-core/Mbo σ^A to better visualize the weak activity (<0.1% that of Eco-core/σ⁷⁰).

Figure 5.

DNAse I footprints (template strand) of Eco and Mbo (±CarD) RNAPs on the AP3 and AC50 promoters. In each panel (A and B), lane 1 shows the AG sequencing ladder (assignments shown on the left), lane 2 shows DNAse I cleavage in the absence of any proteins. DNAse I footprints are shown without competitor trap, and with competitor trap incubation prior to cleavage (times as indicated). The colored bars on the right denote the footprint characteristics (blue, DNAse I protection for both Eco and Mbo RNAPs; red, DNAse I hypersensitivity for both RNAPs; orange, protection by Eco RNAP but not Mbo RNAP; cyan, protection by Mbo RNAP but not Eco RNAP). Densitometric traces provided on the right illustrate the protection profiles. Colors of each trace correspond to samples indicated by the colored dots below the gel lanes. (A) AP3 promoter: protection by Mbo RNAP alone is not as apparent as with CarD, therefore, the blue bars only represent protection by Eco RNAP and Mbo RNAP + CarD. (B) AC50 promoter.

Figure 6.

KMnO₄ footprints (template strand) of Eco and Mbo (±CarD) RNAPs on the AP3 and AC50 promoters. (A) Sequence of the AP3 promoter, except the altered sequence (+3 and downstream) of AP3* is boxed (gives rise to the template-strand T at +5 which is absent in AP3, see Figure 1). Template strand (bottom) thymidines rendered KMnO₄ reactive by RNAP are denoted. (B) KMnO₄ footprints. Lane 1, no protein added. (C) Effect of adding initiating NTPs (GpU ribonucleotide dimer and CTP) on the KMnO₄ footprint of Mbo RNAP on the AP3* promoter. (D) Sequence of the AC50 promoter. Template strand (bottom) thymidines rendered KMnO₄ reactive by RNAP are denoted. (E) KMnO₄ footprint of Eco RNAP on the AC50 promoter. (F) KMnO₄ footprints of Mbo RNAP (±CarD as indicated) on the AC50 promoter.

Figure 7.

A non-complementary transcription bubble rescues short half-life of Mbo RPo on the AC50 promoter and renders CarD redundant. The AC50 double-stranded promoter (from −60 to +20) was synthesized and used as a template for abortive transcription assays (AC50-DS). In AC50-bubble, non-complementary mismatches (underlined) were introduced from −11 to +2, generating a non-collapsible transcription bubble (AC50_Bubble). Half-life assays were performed and RPo half-lives calculated as described in Figure 3B.