Volume 37, Issue 1, Pages 21-33 (January 2010) The cspA mRNA Is a Thermosensor that Modulates Translation of the Cold-Shock Protein CspA  Anna Maria Giuliodori, Fabio Di Pietro, Stefano Marzi, Benoit Masquida, Rolf Wagner, Pascale Romby, Claudio O. Gualerzi, Cynthia L. Pon  Molecular Cell  Volume 37, Issue 1, Pages 21-33 (January 2010) DOI: 10.1016/j.molcel.2009.11.033 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 CspA mRNA Undergoes a Temperature-Dependent Structural Change The 5′UTR of cspA mRNA was probed in vivo with DMS: autoradiogram showing the chemical modifications obtained (A) at 37°C or (B) after 15 min cold shock at 10°C, upon treatment of E. coli cells with the amounts of DMS specified in the Experimental Procedures. Lanes N, control without DMS; lanes G, A, T, and C, sequencing ladders. The differences in reactivity are indicated by arrows. Primer extension analysis was performed using a primer (csp1 of Table S1), which annealed starting from 125 nt downstream of the transcriptional start. The transcriptional start site of cspA mRNA is taken as +1 in the numbering at the sides of the autoradiograms. Further details are given in the Experimental Procedures. cspA and cspD mRNAs were analyzed by temperature gradient gel electrophoresis: (C) electrophoretic mobility in a 7.5% acrylamide gel of cspA mRNA (428 nt) and cspD mRNA (372 nt) as a function of a linear (10°C–40°C) temperature gradient applied perpendicular to the direction of the electrophoresis; LcspA corresponds to a transcript (∼100 nt longer than cspA mRNA) produced by readthrough of the natural cspA transcription termination by T7 RNA polymerase. Prior to loading, the RNAs were heated at 90°C for 3 min and cooled to 4°C in 50 mM Na-cacodylate buffer (pH 7.2) containing 100 mM NaCl and 1 mM DTT. After electrophoresis the RNAs were detected by silver staining. Molecular Cell 2010 37, 21-33DOI: (10.1016/j.molcel.2009.11.033) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Schematic Representation of the Secondary Structures of cspA mRNA Fragments of Increasing Length The depicted structures are derived from the results of the enzymatic and chemical probing of 87cspA RNA, 137cspA RNA, 187cspA RNA, and full-length cspA mRNA performed at the indicated temperatures. The SD sequence (red), the start codon (blue), and the putative S1-binding site (green) are indicated. Nucleotides are numbered taking the transcriptional start site as +1. Molecular Cell 2010 37, 21-33DOI: (10.1016/j.molcel.2009.11.033) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 In Vitro Probing Experiments of Full-Length cspA mRNA (A) Short electrophoretic migration of the fragments generated by RNase T1 (T1), RNase T2 (T2), and RNase V1 (V1) digestion of 5′ end 32P-labeled cspA mRNA; (B) long electrophoretic migration of the same samples showing the cleavages in the TIR region. Chemical modifications with DMS and CMCT in the proximal (C) and distal (D) portions of cspA mRNA coding region. Primer extension analysis was performed using a primer (csp3 of Table S1) that annealed to the 3′UTR. The experiments were carried out at 10°C, 20°C, or 37°C with the amounts of enzymes and chemical reagents indicated in Table S2. Lanes N, controls without RNase or modifying agents; lanes T, RNase T1 cleavages under denaturing conditions; lanes L, alkaline ladder; lanes G, A, T, and C: sequencing ladders. Further details are given in the Experimental Procedures. Molecular Cell 2010 37, 21-33DOI: (10.1016/j.molcel.2009.11.033) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Secondary Structure of cspA mRNA at Cold-Shock Temperatures (A) Model of cspA secondary structure at 10°C and 20°C derived from the probing data shown in Figure 3 and Figures S4B, S4C, and S5. The SD sequence (red), the start codon (blue), and the putative S1-binding site (green) are indicated. Asterisks indicate bases modified in vivo by DMS. Identical probing data were obtained from at least two independent experiments. (B) Schematic representation of the cspA mRNA TIR structure at the cold-shock temperature. The secondary structure model of cspA mRNA was drawn using the program PseudoViewer 3 (Byun and Han, 2009). Molecular Cell 2010 37, 21-33DOI: (10.1016/j.molcel.2009.11.033) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Secondary Structure of cspA mRNA at 37°C (A) Model of cspA secondary structure at 37°C derived from the probing data shown in Figure 3 and Figures S4B, S4C, and S5. The SD sequence (red), the start codon (blue), and the putative S1-binding site (green) are indicated. Asterisks indicate bases modified in vivo by DMS. Identical probing data were obtained from at least two independent experiments. (B) Schematic representation of the cspA mRNA TIR structure at 37°C. The secondary structure model of cspA mRNA was drawn using the program PseudoViewer 3. Molecular Cell 2010 37, 21-33DOI: (10.1016/j.molcel.2009.11.033) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Enzymatic Probing of M-1, M-2, M-3, M-4, and M-5 Mutants of cspA mRNAs Electrophoretic separation of the fragments generated at 10°C and 37°C by RNase T1 (T1), RNase T2 (T2), and RNase V1 (V1) digestion of 5′ end 32P-labeled WT and mutated cspA RNA. Shown are the following: (A) M-1, short migration; (B) M-2 and M-3, short migration; (C) M-4, long migration; (D) M-5, short migration; and (E) WT cspA mRNA, long migration. The WT mRNA sample analyzed in (E) was subjected to denaturation at 90°C and renaturation at 15°C or 37°C in buffer A, followed by enzymatic probing at the same temperatures. An aliquot of the 37°C renatured mRNA was subjected to a further incubation at 15°C for 5 min and probed at this temperature. The amounts of enzymes used at the two temperatures are indicated in Table S2. Lanes N, control without RNase; lanes L, alkaline ladder. The long/short migrations of these probing experiments are shown in Figure S5. Further details are given in the Experimental Procedures. Molecular Cell 2010 37, 21-33DOI: (10.1016/j.molcel.2009.11.033) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 Biological Activity of the Different cspA mRNA Conformers Shown are the following: (A) binding to the 30S ribosomal subunits, (B) [35S]-fMet-tRNA binding assay, (C) in vitro translation, and (D) chemical stability of the two cspA mRNA conformers. These experiments were carried out at 15°C in the presence of non-cold-shock 30S ribosomal subunits or non-cold-shock cell extracts. The cold-shock and the 37°C structures are indicated by closed and open squares, respectively. The data points in (A) are the average of three independent experiments; error bars represent the standard deviations. Further details are given in the Experimental Procedures. Molecular Cell 2010 37, 21-33DOI: (10.1016/j.molcel.2009.11.033) Copyright © 2010 Elsevier Inc. Terms and Conditions