Mycobacterial RNA polymerase requires a U-tract at intrinsic terminators and is aided by NusG at suboptimal terminators

Abstract

Intrinsic terminators, which encode GC-rich RNA hairpins followed immediately by a 7-to-9-nucleotide (nt) U-rich "U-tract," play principal roles of punctuating and regulating transcription in most bacteria. However, canonical intrinsic terminators with strong U-tracts are underrepresented in some bacterial lineages, notably mycobacteria, leading to proposals that their RNA polymerases stop at noncanonical intrinsic terminators encoding various RNA structures lacking U-tracts. We generated recombinant forms of mycobacterial RNA polymerase and its major elongation factors NusA and NusG to characterize mycobacterial intrinsic termination. Using in vitro transcription assays devoid of possible mycobacterial contaminants, we established that mycobacterial RNA polymerase terminates more efficiently than Escherichia coli RNA polymerase at canonical terminators with imperfect U-tracts but does not terminate at putative terminators lacking U-tracts even in the presence of mycobacterial NusA and NusG. However, mycobacterial NusG exhibits a novel termination-stimulating activity that may allow intrinsic terminators with suboptimal U-tracts to function efficiently. IMPORTANCE Bacteria rely on transcription termination to define and regulate units of gene expression. In most bacteria, precise termination and much regulation by attenuation are accomplished by intrinsic terminators that encode GC-rich hairpins and U-tracts necessary to disrupt stable transcription elongation complexes. Thus, the apparent dearth of canonical intrinsic terminators with recognizable U-tracts in mycobacteria is of significant interest both because noncanonical intrinsic terminators could reveal novel routes to destabilize transcription complexes and because accurate understanding of termination is crucial for strategies to combat mycobacterial diseases and for computational bioinformatics generally. Our finding that mycobacterial RNA polymerase requires U-tracts for intrinsic termination, which can be aided by NusG, will guide future study of mycobacterial transcription and aid improvement of predictive algorithms to annotate bacterial genome sequences.

FIG 1

Overexpression and purification of rMboRNAP. (A) pAC22, containing M. bovis rpoA, rpoZ, rpoB, and rpoC genes (encoding the α, ω, β, and β′ subunits of rMboRNAP, respectively). β and β′ were fused with a 10-amino-acid (aa) linker (LARHVGGSGA) and a C-terminal His₈ tag. (B) Purified rMboRNAP and EcoRNAP analyzed by SDS-PAGE. Smearing of the β-β′ fusion polypeptide is likely an electrophoretic artifact. (C) Formation of rMboRNAP holoenzyme (holo) analyzed by nondenaturing PAGE. rMboRNAP core was incubated with M. bovis σ⁷⁰ or E. coli σ⁷⁰ at 37°C for 15 min, electrophoresed on a 4% to 15% gradient of native polyacrylamide gel, and stained with Imperial protein stain (Pierce). Core RNAP migrates as a collection of aggregates, as observed previously for EcoRNAP (55, 56). Incubation with M. bovis σ^A, but not with E. coli σ⁷⁰, converts most rMboRNAP core to a holoenzyme that migrates as a discrete, monomeric species. (D) Transcript elongation and lack of noncanonical intrinsic termination by rMboRNAP. Transcript elongation was assayed at successive times after addition of 100 µM concentrations of all 4 NTPs to halted U26 ECs on a linear DNA template from pAC74 containing M. smegmatis t_gyrA* (19) (Materials and Methods). Termination was evident at the E. coli rrnBT2 terminator (Term) near the end of the template but not at the proposed position of noncanonical termination in t_gyrA*. Position of runoff transcript is indicated (RO).

FIG 2

Termination by EcoRNAP and rMboRNAP at known and predicted intrinsic terminator sequences. (A) Terminator sequences are shown as RNA with horizontal arrows and half-ovals to indicate RNA stem-loop structures and vertical arrows to indicate points of termination. Alternative stem-loops and termination points mapped in this study are blue. The 8-to-10-nt segment immediately 3′ of the stem-loop, corresponding to the intrinsic terminator U-tract, is red (for previously proposed terminator hairpins) or blue (for alternative terminator hairpins). Sequence 3′ of termination points is shown as DNA. The M. tuberculosis t_tuf and t_Rv1324 and M. smegmatis t_gyrA* sequences are reported to terminate transcription by M. smegmatis RNAP despite lacking canonical U-tracts (19). t_gyrA from GenBank accession no. CP000480.1 differs in sequence at positions colored green. M. tuberculosis t_Mtu-rpoC and t_Rv3444c and M. bovis t_asnB and t_fadD23 are predicted to be noncanonical mycobacterial terminators (20). t_synA and t_synB are synthetic terminators designed for use in mycobacterial expression vectors (32). (B) EcoRNAP (left panel) and rMboRNAP (right panel) were used to transcribe DNA templates containing the indicated terminators or predicted terminators ligated between the BamHI and NcoI sites of the template depicted in Fig. 1D (see the sequence in Fig. S1 in the supplemental material). Transcription of 25 nM linear DNA by 50 nM RNAP was conducted at 37°C, and the products were separated on a 6% denaturing polyacrylamide gel (see Materials and Methods). Terminated products are marked with a red circle. Termination efficiencies with standard deviations (s.d.) from ≥3 experimental replicates are shown below the gel lanes (*, single determination with no error determination). (C) Detection of released RNA products in rMboRNAP reactions. In cases in which termination was detected, transcription reaction mixtures with RNAP immobilized on Ni²⁺-NTA agarose beads were separated into supernatant (s) and washed-bead (wb) fractions for comparison to total reaction products (r) (Materials and Methods). Appearance of all RNAs in the supernatant fraction indicates transcript release, which was verified using halted ECs as controls (see Fig. S3 in the supplemental material).

FIG 3

rMboRNAP exhibits a greater termination proclivity than EcoRNAP at suboptimal U-tracts. (A) RNA structures and sequences of derivatives of the predicted M. bovis t_asnB terminator containing increasing numbers of U residues in the U-tract segment. The positions of termination are indicated by vertical arrows either as previously reported for t_rrnBT1 (3) or as estimated by comparison on high-resolution denaturing polyacrylamide gels of terminated transcripts to RNA sequence ladders generated using 3′ deoxyNTPs (see Fig. S4 in the supplemental material and Materials and Methods). (B and C) Termination by EcoRNAP and termination by rMboRNAP were compared using the assay shown in Fig. 2. Data represent EcoRNAP (B) and rMboRNAP (C) transcription products of templates shown in panel A. Terminated products are marked with a red circle. (D) Termination efficiency (TE) for t_asnB-5 was determined at various NTP concentrations to produce different rates of average transcript elongation (gel images for the six data points shown here are in Fig. S5 in the supplemental material). TE is plotted as a function of the average elongation rate for EcoRNAP and rMboRNAP. (E) Calculation of apparent termination rate (EC dissociation rate or rate at which ECs commit to the termination pathway) from average elongation rate and TE. (F) Calculated termination (Term.) rates. Except at very low NTP and elongation rates, rMboRNAP exhibited a significantly higher termination rate.

FIG 4

NusA and NusG effects on termination by rMboRNAP. (A) Structures of MtuNusA (pdb 1k0r; see reference 36), TthRNAP EC (adapted from pdb 2o5i; see reference 9), and EcoNusG (pdb 2k06 and 2jvv; see reference 41). Key domains are labeled, and known interactions are indicated with arrows and, for NusG, color coded on the surface of NusG and the EC (clamp helices, green; β gate loop, yellow; nontemplate DNA strand, black). (B) Effects of mycobacterial NusA and NusG on termination by rMboRNAP at a set of possible terminators. NA, no addition. NusG, but not NusA, consistently enhanced TE, but neither NusA nor NusG allowed termination at possible noncanonical terminator sequences. A representative gel of the termination assay for t_Rv1342 is shown in Fig. S6 in the supplemental material. (C) Structure of EcoNusG colored as described for panel A with the backbone shown as a ribbon, key residues shown as spheres, and insertions present in mycobacteria shown as orange (Mboi1) or blue (Mboi2) sequences. (D) Effect of EcoNusG, mycobacterial NusG, and deletion proteins derived from mycobacterial NusG on TE at t_asnB-5 and t_Rv1324. NA, no addition.