tRNA-mediated transcription antitermination in vitro: codon-anticodon pairing independent of the ribosome

Abstract

Uncharged tRNA acts as the effector for transcription antitermination of genes in the T box family in Bacillus subtilis and other Gram-positive bacteria. Genetic studies suggested that expression of these genes is induced by stabilization of an antiterminator element in the leader RNA of each target gene by the cognate uncharged tRNA. The specificity of the tRNA response is dependent on a single codon in the leader, which was postulated to pair with the anticodon of the corresponding tRNA. It was not known whether the leader RNA-tRNA interaction requires additional factors. We show here that tRNA-dependent antitermination occurs in vitro in a purified transcription system, in the absence of ribosomes or accessory factors, demonstrating that the RNA-RNA interaction is sufficient to control gene expression by antitermination. The tRNA response exhibits similar specificity in vivo and in vitro, and the antitermination reaction in vitro is independent of NusA and functions with either B. subtilis or Escherichia coli RNA polymerase.

Fig 1.

Proposed T box antitermination mechanism. The arrow indicates the transcription initiation site. The black rectangle represents the coding region of the regulated gene. Uncharged tRNA is postulated to interact with the nascent transcript at both the specifier sequence and the antiterminator bulge, stabilizing the antiterminator and preventing formation of the competing terminator. RNA polymerase (RNAP) then continues past the terminator region, and the full-length transcript is synthesized. “Factor?” indicates putative factor(s) that could modulate the leader RNA–tRNA interaction in vivo.

Fig 2.

Secondary structure model of the B. subtilis glyQS leader RNA. Sequence is shown from the transcription start site (+1) through the end of the leader region terminator; the alternate antiterminator is shown above the terminator. The structure is based on the covariation model of T box family leaders (2, 5). Major conserved features are labeled, and conserved primary sequence elements are denoted with asterisks. The specifier sequence residues are boxed. The glyQS sequence was obtained from the B. subtilis genome sequence (11); DNA sequencing of this region revealed a substitution of A for U at position +6. The residues in brackets (113–122) are replaced by the stem II and IIA/B elements in most T box family leaders, including B. subtilis tyrS.

Fig 3.

In vitro transcription of the glyQS and tyrS leader regions. Lanes 1–5, glyQS DNA; lanes 6–11, tyrS DNA. Lanes 1, 3, and 6, no tRNA added; lanes 2, 4, and 7, tRNA^Gly (T7 transcript); lanes 5, 8, and 11, tRNA^Tyr (T7 transcript); lane 9, E. coli tRNA^Tyr (modified, purchased from Sigma); lanes 3–9, NusA added; lanes 1, 2, 10, and 11, no NusA. T, terminated transcript; RT, read-through transcript. Percent read-through is indicated at the bottom of each lane.

Fig 4.

Specificity of the glyQS–tRNA^Gly interaction. (A) Interaction of the glyQS leader (black) in the antiterminator conformation with tRNA^Gly (green). Substitutions at the specifier sequence and antiterminator regions of the leader, and at the anticodon and acceptor end of the tRNA, are shown with arrows. (B–E) In vitro transcription reactions using different combinations of variants of glyQS templates and tRNA^Gly. (B) Wild-type glyQS template DNA (GGC specifier sequence, A158 antiterminator). (C) glyQS-UGC template DNA (cysteine specifier sequence, A158 antiterminator). (D) glyQS-A158→U template DNA (GGC specifier, U158 antiterminator). (E) glyQS-UGC/A158→U template DNA (cysteine specifier sequence, U158 antiterminator). Lane 1, no tRNA; lane 2, wild-type tRNA^Gly (GCC anticodon, U73 discriminator); lane 3, tRNA^Gly-GCA (GCA anticodon, U73 discriminator); lane 4, tRNA^Gly-U73→A (GCC anticodon, A73 discriminator); lane 5, tRNA^Gly-GCA/U73→A (GCA anticodon, A73 discriminator). T, terminated transcript; RT, read-through transcript. Percent read-through is indicated at the bottom of each lane.