Transcription termination control of the S box system: direct measurement of S-adenosylmethionine by the leader RNA

Abstract

Modulation of the structure of a leader RNA to control formation of an intrinsic termination signal is a common mechanism for regulation of gene expression in bacteria. Expression of the S box genes in Gram-positive organisms is induced in response to limitation for methionine. We previously postulated that methionine availability is monitored by binding of a regulatory factor to the leader RNA and suggested that methionine or S-adenosylmethionine (SAM) could serve as the metabolic signal. In this study, we show that efficient termination of the S box leader region by bacterial RNA polymerase depends on SAM but not on methionine or other related compounds. We also show that SAM directly binds to and induces a conformational change in the leader RNA. Both binding of SAM and SAM-directed transcription termination were blocked by leader mutations that cause constitutive expression in vivo. Overproduction of SAM synthetase in Bacillus subtilis resulted in delay in induction of S box gene expression in response to methionine starvation, consistent with the hypothesis that SAM is the molecular effector in vivo. These results indicate that SAM concentration is sensed directly by the nascent transcript in the absence of a trans-acting factor.

Figure 1

B. subtilis yitJ leader structural model. The structural model is based on phylogenetic analyses (8) and is shown in the terminator conformation. Red and blue residues indicate the alternate pairing for formation of the antiterminator, shown above the terminator. Boxed numbers indicate helices 1–4; T, terminator; AT, antiterminator; AAT, anti-antiterminator. Numbering of residues is relative to the predicted transcription start point. Sequence changes in the Pst-1 and Pst-2 alleles are shown with lowercase letters.

Figure 2

In vitro transcription of S box genes. ○, Terminated transcript; ●, read-through transcript. Percent termination (% T) is shown at the bottom of each lane. (A) SAM-dependent transcription termination. DNA templates were transcribed with E. coli (lanes 1–6) or B. subtilis (lanes 7–13) RNAP. Lanes 1 and 2, ykrW DNA; lanes 3, 4, 7, and 8, P_gly-ykrW DNA; lanes 5 and 6, P_tyr-yitJ DNA; lanes 9 and 10, P_gly-yitJ DNA; lanes 11–13, glyQS DNA. SAM was added at 150 μM where indicated (lanes 2, 4, 6, 8, 10, and 13). tRNA^Gly was added at 70 nM for lanes 12 and 13. The P_gly-yitJ constructs contain additional sequences downstream of the terminator, resulting in a larger read-through product than is observed with P_tyr-yitJ DNA. (B) SAM-dependent transcription termination of multiple B. subtilis S box leaders. DNA templates were transcribed with B. subtilis RNAP in the presence (lanes 2, 4, 6, 8, 10, 12, 14, and 16) or absence (lanes 1, 3, 5, 7, 9, 11, 13, and 15) of SAM (150 μM). Gene names are shown above each pair of lanes. (C) Specificity of SAM-dependent transcription termination of the B. subtilis ykrW leader. The ability of SAM-related compounds to stimulate termination by E. coli RNAP, and to block SAM-dependent termination, was tested. MET, methionine; ADO, adenosine; HCY, homocysteine; MTA, methylthioadenosine; SF, sinefungin. All compounds were tested at 1.5 mM except homocysteine (500 μM) and sinefungin, which was tested at both 500 μM (low, lanes 15 and 16) and 1.5 mM (high, lanes 17 and 18). SAM was included at 7.5 μM where indicated. (D) Effect of yitJ leader mutations on SAM-dependent transcription termination. DNA templates were P_gly-yitJ constructs; mutations are shown in Fig. 1. Transcription was with E. coli RNAP, in the presence (lanes 2, 4, and 6) or absence (lanes 1, 3, and 5) of SAM (150 μM). Templates were yitJ (lanes 1 and 2), yitJ-Pst-1 (lanes 3 and 4), and yitJ-Pst-2 (lanes 5 and 6).

Figure 3

Interaction of SAM with S box leader RNAs. (A) Binding of ¹⁴C-SAM. ¹⁴C-SAM (8 μM) was incubated with T7 RNAP-transcribed leader RNA (8 μM) in 1× transcription buffer in the presence or absence of unlabeled SAM or SAH (400 μM). SAM binding is expressed as the fraction of ¹⁴C-SAM retained after filtration through a Nanosep 3K filter and washing with 1× transcription buffer relative to the amount added to the binding reaction. (B) Antisense oligonucleotide-dependent RNase H cleavage. The cartoon shows the structural models in the absence or presence of SAM. T, terminator; AT, antiterminator; AAT, anti-antiterminator. Positions of pairing of oligonucleotides 1–3 are shown. The 154-nt RNA was generated by transcription with E. coli RNAP (lanes 1–10) in the presence or absence of SAM (150 μM). The 140-nt RNA was generated by T7 RNAP transcription, heated to 65°C and slow-cooled to 40°C before addition of SAM (lanes 11–18). Oligonucleotides were added at a 100-fold molar excess before digestion with RNase H, which specifically targets RNA–DNA hybrids. Positions of the uncut RNA and RNase H cleavage products are indicated by brackets. Cleavage products that change in response to SAM are marked with asterisks. The higher-molecular-weight band observed with E. coli RNAP in the presence of oligo 2 (●, lanes 3 and 8) is likely to be the result of template switching during transcription, because it was absent when oligo 2 was added to premade RNA (lanes 13 and 17). The leftmost lane contains ³²P-labeled pBR322 DNA digested with MspI used as a molecular weight standard.

Figure 4

Expression of a yitJ-lacZ transcriptional fusion. Fusions were introduced into strains 168 (wild type; squares) and SA29 (metK overexpression strain; circles). Cells were grown in defined medium containing methionine, collected by centrifugation, and resuspended in the same medium (filled symbols) or in medium without methionine (open symbols). Samples were taken at 1-h intervals and assayed for β-galactosidase activity, expressed in Miller units.