Supplementary Information

Transcription

1 Supplementary Information Supplementary Figures Figure S1. Study of mgtl translation in vitro. (A) Detection of 5 LR RNA using wild-type and anti-sd (91-95) substituted templates in a transcription-translation coupled reaction supplemented with 1 mm EDTA (i.e., low Mg 2+ ) and without EDTA (i.e., high Mg 2+ ). Fulllength transcripts (264 nt) were detected in all tested conditions, while truncated transcripts (220

2 nt) were detected at much higher levels in high Mg 2+ in both the wild-type and substituted templates. The ladder (M) corresponds to a PCR product generated with primers 241 and 32 P- labeled primer 201 and degraded by the Maxam and Gilbert reaction. FL represents full-length mgta 5 LR premade from a T7 promoter-mgta 5 LR template. (B) Detection of translation products using P lac1-6 -lacz, P lac LR, P lac LR-91-95, and P lac LR-lacZ templates in a transcription-translation coupled reaction in low Mg 2+ and high Mg 2+. As a control, truncated LacZ (~14.8 kda) was detected in similar levels from the P lac1-6 -lacz template, however, in higher levels in low Mg 2+ from the P lac LR-lacZ template. MgtL peptide was not detected in any tested condition. Low-range Rainbow Molecular Weight Marker (GE Healthcare) was used as a ladder. Proteins were separated in a Novex 4-20% Tris-Glycine Gel (Invitrogen). (C) In vitro transcription of DNA templates harboring the P lac1-6 promoter and the full-length wild-type mgta 5 LR or derivatives with substitutions in anti-sd sequence (91-95) or the anti-sd and antianti-sd sequences (91-95, ) was conducted with 0.35 mm (L) and 3.5 mm (H) Mg 2+. The percent of readthrough product was calculated by the formula (intensity of readthrough transcript at a given Mg 2+ concentration)/(intensity of readthrough transcript at a given Mg 2+ concentration + intensity of truncated transcript in such Mg 2+ concentration) X 100. The ladder used was the same as in A.

3 Figure S2. Autoradiograph of the loop A region after treatment with RNase T1 following denaturation and then renaturation in 0.3 mm (L) or 3 mm (H) Mg 2+. 6% polyacrylamide gel was used to separate the products. Lane M corresponds to Maxam-Gilbert reaction using DNA fragment amplified from pys1010 with primers 220 and 32 P-labeled 201. Lane G corresponds to T1-treated RNA that was previously denatured. Quantification was conducted using Quantity One software (Bio-Rad). After all bands in a lane were normalized, cleavage ratio was calculated using quantified band derived from high Mg 2+ divided by band from low Mg 2+.

4 Figure S3. Characterization of wild-type and auxotrophs harboring pys1010 and its derivatives. (A) β-galactosidase activity determined in Salmonella wild-type and proc harboring pys1010 (mgtl + ), and the triple- (Pro-5,7,9) and tetra- (Pro-3,5,7,9) substituted plasmids. (B) Colony forming units (CFU) determined from wild-type and proc. (C) Intracellular proline concentration determined in wild-type and proc using acid-ninhydrin. As described in Materials

5 and Methods in a recent study (Park et al, 2010), Salmonella cells in A, B and C were grown in modified N-minimal medium with mm (low) or 0.5 mm (high) Mg 2+ in the presence of 1 mm proline for 1 hr, washed and then grown for 15 min and 2 h in medium containing or lacking proline. (D) β-galactosidase activity determined in E. coli wild-type BW25113, prob, proc, argb, and arge harboring wild-type plasmid pys1010. β-galactosidase activity was determined from two groups: the first: BW25113, prob and proc grown for 4 h in N medium supplemented with 0, 0.1, 1, or 10 mm proline, and 0.01 mm (low), 0.5 mm, or 10 mm (high) Mg 2+ ; and the second: BW25113, argb and arge grown for 4 h in N medium supplemented with 0, 0.1, 1, or 10 mm arginine, and 0.01 mm (low), 0.5 mm, or 10 mm (high) Mg 2+. Assays were conducted in triplicate. Error bars correspond to the standard deviation.

6 Figure S4. The standard curve relating proline concentration to OD 520 in the acid-ninhydrin method. The proline solutions with known concentrations were incubated with acid-ninhydrin. After stopping the reaction, the mixtures were extracted with toluene. The toluene phase was separated and the absorbance was read at 520 nm using SmartSpec spectrophotometer (Bio-Rad).

7 Supplementary Methods Construction of chromosomal mutations and strain with FLAG sequence inserted in the mgta 5 LR region Oligonucleotides used in this study are described in Table 1. Deletion of the coding region of a gene was generated as described previously (Datsenko & Wanner, 2000). Inactivation of the proc gene was carried out using primers 1607 and 1608 to amplify the Cm R cassette from pkd3 and integrate the resulting PCR product into the chromosome. The Cm R cassette was removed by pcp20. The arga mutant was constructed as above with primers 768 and 769. A Salmonella strain harboring mgtl-flag fusion in the chromosome was generated as following: Briefly, the Cm R resistance gene was introduced upstream of the mgta promoter using a PCR product generated with primers 811 and 812 and plasmid pkd3. The resulting PCR product was integrated into the chromosome to make strain up-mgta. The chromosomal DNA from the upmgta strain was used as template to produce the PCR-generated DNA fragment for insertion of the FLAG tag in frame with the mgtl coding region with primers 811 and 794. The resulting PCR product was integrated into the wild-type chromosome to generate mgtl-flag strain. Plasmid construction Plasmid pys1475 was constructed as follows: a DNA fragment containing the full-length mgta 5 leader fragment, generated by PCR with primers 298 and 299 and the mgtl-flag strain chromosomal DNA as template, was digested with SalI and XhoI and cloned into pys1000 [rep p15a Cm R P lac1 6 lacz t his, see Ref. (Kong et al, 2008)] that had been digested with the same enzymes. Derivatives of pys1010 and pys1475 with nucleotide substitutions were constructed

8 using the Gene Tailor Site Directed Mutagenesis System (Invitrogen) with Platinum Taq Polymerase High Fidelity (Invitrogen). pys1010 and pys1475 DNAs were purified with the GeneJET Plasmid Miniprep Kit (Fermentas) and the following primer pairs were used: 913 and 1553 for pys1010-a71c; 912 and 913 for pys1475-a71c; 913 and 1554 for pys1010-a71g; 1243 and 1244 for pys1010-g73c; 1243 and 1245 for pys1010-g74c; 1154 and 1557 for pys1010-pro3; 1559 and 1560 for pys1010-pro5; 1578 and 1579 for pys1010-pro3,5; 1559 and 1562 for pys1010-pro7; 1559 and 1564 for pys1010-pro9; 1609 and 1610 for pys1010- Pro5,7,9; 1611 and 1612 for pys1010-pro3,5,7,9; 1154 and 1555 for pys1010-g80t; 1361 and 1362 for pys1010-c98a; 1600 and 1601 for pys ; 1600 and 1602 for pys , puhe-mgtl was constructed as follows: a PCR fragment containing the mgta 5 leader region generated by PCR with primers 903 and 904 and wild-type strain chromosomal DNA as template was digested with BamHI and HindIII and cloned into puhe21 [rep pmb1 Ap R laci q, Ref. (Kong et al, 2008)] digested with the same enzymes. In vitro transcription The in vitro transcription assay was carried out as described in a previous study (Cromie et al, 2006). Linear DNA templates containing the P lac1-6 promoter region and full-length mgta 5 leader fragment were generated by PCR with primers 201 and 241 and template pys1010 or its derivatives. When necessary, ribosomes from the PURExpress Ribosome Kit (New England Biolabs) were added to a final concentration 2.4 mm as recommended by the manufacturer. In vitro transcription-translation coupling reaction The in vitro transcription-translation assay was carried out using PURExpress In Vitro Protein

9 Synthesis system (New England Biolabs) and E. coli RNA polymerase σ 70 holoenzyme (Epicentre) according to the manufacturer s specifications. Linear DNA templates containing the P lac1-6 promoter region and full-length mgta 5 leader fragment were generated by PCR with primers 1613 and 241 and plasmid pys1010 or its derivative pys DNA templates containing the P lac1-6 promoter, full-length mgta 5 leader region and partial lacz gene was generated with primers 1614 and 241 and plasmid pys1010. DNA templates containing the P lac1-6 promoter region and partial lacz gene (lacz ) was generated with primers 1614 and 241 and plasmid pys1000. The RNA and protein products were labeled with α- 32 P-UTP (Perkin Elmer) and 35 S-methionine (Perkin Elmer) respectively, and detected by autoradiography. Supplementary References Cromie MJ, Shi Y, Latifi T, Groisman EA (2006) An RNA sensor for intracellular Mg 2+. Cell 125(1): Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97(12): Kong W, Weatherspoon N, Shi Y (2008) Molecular mechanism for establishment of signaldependent regulation in the PhoP/PhoQ system. J Biol Chem 283(24): Park SY, Cromie MJ, Lee EJ, Groisman EA (2010) A bacterial mrna leader that employs different mechanisms to sense disparate intracellular signals. Cell 142(5):

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