tRNA requirements for glyQS antitermination: A new twist on tRNA

Abstract

Transcription antitermination of the Bacillus subtilis glyQS gene, a member of the T box gene regulation family, can be induced during in vitro transcription in a minimal system using purified B. subtilis RNA polymerase by the addition of unmodified T7 RNA polymerase-transcribed tRNAGly. Antitermination was previously shown to depend on base-pairing between the glyQS leader and the tRNA at the anticodon and acceptor ends. In this study, variants of tRNAGly were generated to identify additional tRNA elements required for antitermination activity, and to determine the effect of structural changes in the tRNA. We find that additions to the 3′ end of the tRNA blocked antitermination, in agreement with the prediction that uncharged tRNA is the effector in vivo, whereas insertion of I nucleotide between the acceptor stem and the 3′ UCCA residues had no effect. Disruption of the D-loop/T-loop tertiary interaction inhibited antitermination function, as was previously demonstrated for tRNATyr-directed antitermination of the B. subtilis tyrS gene in vivo. Insertion of a single base pair in the anticodon stem was tolerated, whereas further insertions abolished antitermination. However, we find that major alterations in the length of the acceptor stem are tolerated, and the insertions exhibited a pattern of periodicity suggesting that there is face-of-the-helix dependence in the positioning of the unpaired UCCA residues at the 3′ end of the tRNA for interaction with the antiterminator bulge and antitermination.