The L box regulon: lysine sensing by leader RNAs of bacterial lysine biosynthesis genes - PubMed (original) (raw)
The L box regulon: lysine sensing by leader RNAs of bacterial lysine biosynthesis genes
Frank J Grundy et al. Proc Natl Acad Sci U S A. 2003.
Abstract
Expression of amino acid biosynthesis genes in bacteria is often repressed when abundant supplies of the cognate amino acid are available. Repression of the Bacillus subtilis lysC gene by lysine was previously shown to occur at the level of premature termination of transcription. In this study we show that lysine directly promotes transcription termination during in vitro transcription with B. subtilis RNA polymerase and causes a structural shift in the lysC leader RNA. We find that B. subtilis lysC is a member of a large family of bacterial lysine biosynthesis genes that contain similar leader RNA elements. By analogy with related regulatory systems, we designate this leader RNA pattern the "L box." Genes in the L box family from Gram-negative bacteria appear to be regulated at the level of translation initiation rather than transcription termination. Mutations of B. subtilis lysC that disrupt conserved leader features result in loss of lysine repression in vivo and loss of lysine-dependent transcription termination in vitro. The identification of the L box pattern also provides an explanation for previously described mutations in both B. subtilis and Escherichia coli lysC that result in lysC overexpression and resistance to the lysine analog aminoethylcysteine. The L box regulatory system represents an example of gene regulation using an RNA element that directly senses the intracellular concentration of a small molecule.
Figures
Fig. 1.
L box lysine biosynthesis genes. (A) Lysine biosynthesis pathway in B. subtilis. Genes found to contain L box leaders in any organism are labeled (see Table 2, which is published as supporting information on the PNAS web site,
), and the corresponding enzyme product is shown in parentheses. Three genes encoding aspartokinase have been identified in B. subtilis; lysC encodes aspartokinase II, the lysine responsive form of the enzyme. Alternative roles of compounds in this pathway are shown with dashed arrows. (B) Structures of lysine and related compounds.
Fig. 2.
Structural model of the B. subtilis lysC leader. Numbering is relative to the transcription start point (10). Helices 1–5 are labeled with boxed numbers. T, terminator; AT, antiterminator; AAT, anti-antiterminator. The antiterminator is formed by pairing of residues from positions 191–217 with residues from positions 223–255. Red residues are found in 100% of the sequences, and blue residues are found in >80% of the sequences (Fig. 5); green residues and dashed lines show a possible tertiary interaction between the loops of helices 2 and 3. Hollow lowercase letters indicate mutations previously shown to cause resistance to AEC and/or constitutive lysC expression (refs. and and H. Paulus, personal communication). Mutations constructed in this study (Xba-1, Xba-2, Xba-3) are labeled with solid lowercase letters. Sequence alterations in the mutants are: Xba-1, A31U/A32C/G33U/U35G; Xba-2, G108U/C110U/G111A/A112G; Xba-3, A198C/C200A/U201G/C202A. Putative S-turn and GA motif elements are labeled.
Fig. 3.
In vitro transcription of B. subtilis lysC. Arrows show positions of the read-through (RT) and terminated (T) transcripts. Percent termination (%T) is shown at the bottom of each lane. DNA templates (lysC or ykrW) were transcribed with B. subtilis RNAP. (A) Lysine-dependent transcription termination. Lysine (lys), DAP, AEC, and SAM were added at 3 mM. (B) In vitro transcription of mutant lysC templates. Lysine was added at 3 mM (+). WT, WT lysC; Xba-1, Xba-1 mutation; Xba-2, Xba-2 mutation; Xba-3, Xba-3 mutation; Xba-1/3, Xba-1 and Xba-3 double mutant. Mutations are shown in Fig. 2.
Fig. 4.
Lysine-dependent structural transition in the lysC RNA. (A) Structural model of the B. subtilis lysC leader in the read-through (-lysine) and termination (+lysine) conformations. Positions of pairing of antisense oligonucleotides a and b, positions of Xba-1 and Xba-2 mutations (Fig. 2), and endpoints of transcription products are shown. (B) Antisense oligonucleotide a-directed RNase H cleavage of transcripts containing lysC sequences from +17 to +228 (211-nt transcript, containing the 5′ side of the antiterminator, but not the 3′ side). Transcription was in the presence (+) or absence (-) of lysine (3 mM). Arrows indicate the RNase H cleavage products, which were observed only when the oligonucleotide was added. (C) Antisense oligonucleotide b-directed RNase H cleavage of transcripts containing lysC sequences from +17 to +253 (236-nt transcript, containing the entire antiterminator, but lacking the 3′ side of the terminator). Arrows indicate the RNase H cleavage products, which were observed only when the oligonucleotide was added.
References
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