Kinetic analysis of tRNA-directed transcription antitermination of the Bacillus subtilis glyQS gene in vitro

Abstract

Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNA(Gly)-dependent antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed. We found that addition of tRNA(Gly) can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.

FIG. 1.

Structural model of the B. subtilis glyQS leader RNA. The structure is shown in the terminator conformation. The antiterminator conformation results from pairing of sequences on the 5′ side of the terminator with upstream sequences. Stem I and stem III represent major conserved structural elements in all T box family leaders; glyQS leader sequences are exceptional in that they lack the stem II and stem IIA/B pseudoknot elements found in most other leaders. Major pause sites are labeled with arrows; bold arrows indicate the most stable sites, G138 and G220 (T, the termination site). The sequence alterations in the stem III loop (Eco1 mutation, U136G, U137A, and G138A) are indicated with hollow arrows and lowercase letters. Numbers are relative to the glyQS transcription initiation site. Residues in the specifier loop and in the antiterminator bulge that pair with the anticodon and acceptor ends of the tRNA, respectively, are boxed. The glyQS construct shown contains a substitution of C at position +2 to allow transcription initiation with ApC.

FIG. 2.

Effect of NTP concentration and tRNA^Gly on glyQS transcription in vitro. Transcription was carried out at 5, 10, 30, 100, or 400 μM NTPs in the elongation reaction mixture, in the presence (filled bars) or absence (open bars) of tRNA^Gly. Percent readthrough was calculated as the fraction of product in the readthrough band compared to the total product in the terminated and readthrough bands.

FIG. 3.

Pausing during glyQS transcription in vitro. Transcription was halted by omission of CTP, and elongation was carried out in the presence of 10 μM NTPs and in the presence or absence of tRNA^Gly, as indicated. Samples were removed at the indicated time points after restart, and the reactions were terminated by phenol extraction. The positions of the major pause sites and the terminated (T) and readthrough (RT) products were identified using an RNA sequencing ladder and are labeled. Lanes 1 and 10, 0 s; lanes 2 and 11, 20 s; lanes 3 and 12, 40 s; lanes 4 and 13, 60 s; lanes 5 and 14, 80 s; lanes 6 and 15, 100 s; lanes 7 and 16, 120 s; lanes 8 and 15, 5 min; lanes 9 and 16, 10 min. (A) B. subtilis RNAP, wild-type template; (B) E. coli RNAP, wild-type template; (C) B. subtilis RNAP, Eco1 mutant template.

FIG. 4.

Effect of tRNA addition during the transcription reaction. Transcription was halted by omission of CTP and restarted in the presence of various NTP concentrations. Samples were taken at intervals during the elongation reactions and were either terminated by phenol extraction, to show the position of the TEC at that time point (“pause”; open symbols), or were added to tRNA and allowed to incubate for an additional 15 min before termination, to determine the readthrough efficiency (“readthrough”; filled symbols). G, GTP; A, ATP; U, UTP; C, CTP.