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Fig. S1. Alignment between MbRpoE/F and MjRpoE/F. Secondary structure elements (α-helices, helix; β-sheet, arrows) in the MjRpoE and MjRpoF subunits are shown above the amino acid sequence, with regions proposed to be involved in nucleic acid binding colored in red.

Fig. S2. Stern–Volmer plots of fluorescence quenching by acrylamide for MbRpoE/F and MjRpoE/F. Plots at 10°C (A) and at 40°C (B) for MbRpoE/F (●) and MjRpoE/F (○). Variation of fluorescence quenching between 10°C and 40°C obtained by subtracting the regression lines of Stern–Volmer plots at individual temperatures (C). The quenching constant KSV values, corresponding to the plot slopes, are 0.015 and 0.011 mM−1 at 10°C and 0.0416 and 0.0204 mM−1 at 40°C for MbRpoE/F and MjRpoE/F respectively.

Fig. S3. EMSA profiles of MbRpoE/F and MjRpoE/F with polyC 25-mer ssDNA. The radiolabelled probe was incubated with indicated quantities (µg) of purified MbRpoE/F and MjRpoE/F as described in Experimental procedures. Bound and free probes were resolved by electrophoresis in a 5% polyacrylamide gel containing 10% glycerol and 0.5× TBE. Bands formed from free DNA (lower band) and protein-bound DNA (highlighted by arrows) were detected by autoradiography.

Fig. S4. EMSA profiles of MbRpoE/F and MjRpoE/F with polyG or poly A 25-mer ssDNA. The radiolabelled probes for polyG (left panels) and polyA (right panels) were incubated with indicated quantities (µg) of purified MbRpoE/F and MjRpoE/F as described in Experimental procedures. Bound and free probes were resolved by electrophoresis in a 5% polyacrylamide gel containing 10% glycerol and 0.5× TBE. Bands formed from free DNA (lower band) and protein-bound DNA (highlighted by arrows) were detected by autoradiography.

Fig. S5. EMSA profiles of MbRpoE/F and MjRpoE/F with polyT 25-mer ssDNA. The radiolabelled probe was incubated with indicated quantities (µg) of purified MbRpoE/F and MjRpoE/F as described in Experimental procedures. Bound and free probes were resolved by electrophoresis in a 5% polyacrylamide gel containing 10% glycerol and 0.5× TBE. Bands formed from free DNA (lower band) and protein-bound DNA (highlighted by arrows) were detected by autoradiography.

Fig. S6. Transcripts bound by MbRpoE/F involved in purine and pyrimidine biosynthesis in M. burtonii. Enzymes in the pathway corresponding to mRNA species bound by MbRpoE/F are shown in orange font. Inosine monophosphate (IMP) is the first purine derivative to be synthesized in the purine de novo biosynthesis pathway. Thestarting substrate for biosynthesis of the ribosyl moiety is ribose-5-phosphate (R5P) which in archaea is a product of the ribulose monophosphate pathway (RuMP). This substrate is first activated by phosphorylation to form phosphoribosyl pyrophosphate (PRPP) and converted to IMP through a series of enzymatic reactions. IMP is converted to adenosine monophosphate (AMP) or guanosine monophosphate (GMP). The pyrimidine de novo biosynthesis pathway is centered on the formation of orotic acid and its subsequent reaction with PRPP to yield orotidine-5-monophosphate (OMP). OMP is decarboxylated to form uridine monophosphate (UMP), which is the substrate for the formation of UTP that leads also to CTP. Enzymes represented by the transcripts bound by MbRpoE/F are: Fae/hps bifunctional enzyme (HPS) and ribose-5-phosphate isomerase (RpiA) that catalyse the two consecutive reactions that produce R5P from the RuMP; adenylosuccinate lyase (ADSL) and phosphoribosylformylglycinamidine synthase II (PurL) that are involved in the synthesis of IMP, with the first also involved in the synthesis of AMP from IMP; adenylate kinase (AK) which converts AMP to ADP which is a crucial intermediate step in the formation of ATP; dihydroorotate dehydrogenase subunits K and D (PyrK and PyrD respectively) which are essential for the formation or orotic acid. MbRpoE/F also binds to the transcript for cytidylate kinase, which catalyses the conversion of CMP to CDP. Other abbreviations are: RPPK, ribose phosphate pyrophosphokinase; PurA, adenylosuccinate synthase; PyrE, orotate phosphoribosyl tranferase.

Fig. S7. Transcripts bound by MbRpoE/F involved in the synthesis of vitamin B12, coenzyme F430 and sirohaem from glutamic acid in M. burtonii. Enzymes in the pathway corresponding to mRNA species bound by MbRpoE/F are shown in orange font. The tetrapyrrole compounds, sirohaem, vitamin B12 (cobalamin) and F430 are all essential cofactors deriving from the precursor uroporphyrinogen III. The first stage of this pathway is the synthesis of ALA through decarboxylation of glutamate. The second stage is the conversion of ALA to uroporphyrinogen III (the first tetrapyrrole in the pathway). Uroporphyrinogen III is methylated to form precorrin-2, the precursor of cobalamin, sirohaem, and coenzyme F430. At this point in the pathway, cobalamin, haem, sirohaem and coenzyme F430 are synthesized from precorrin-2 via separate pathways. Each of these cofactors has a different metal ion in the centre of the prosthetic group: cobalt for cobalamin, iron for haem and sirohaem and nickel for F430. Enzymes represented by the transcripts bound by MbRpoE/F are: glutamate-1-semialdehyde aminomutase (HemL), the protein involved in the final step in the synthesis of ALA; porphobilinogen deaminase (HemC), a key enzyme in the production of uroporphyrinogen III, and uroporphyrinogen-III C-methyltransferase (CysG) that synthesizes precorrin-2 from uroporphyrinogen-III. MbRpoE/F also binds to the transcripts coding for two components of a cobalt-transport complex involved in uptake of Co necessary for cobalamin biosynthesis (CbiM and CbiN), and a ferrous iron transport protein B (FeoB). For cobalamin biosynthesis, the CbiM/N heterodimer binds to the transcript for N1-α-phosphoribosyltransferase gene (cobT), which is responsible for the assembly of the nucleotide loop in the final steps of the pathway. Abbreviations are: ALA, 5-aminolevulinic acid; PBG, porphobilinogen; GltX, glutamyl-tRNA synthetase; HemA, glutamyl-tRNA reductase; HemB, porphobilinogen synthetase; HemD, uroporphyrinogen III synthase.

Table S1. Summary of the binding of MbRpoE/F and MjRpoE/F to 25-mer ssDNA sequences using EMSAs. Summary of EMSA results (examples provided in Figs S3–S5) for the capacity of RpoE/F to bind (yes) or not bind (no) under the conditions tested (see Experimental procedures).

Table S2. mRNA abundance data.

Table S3. COG representation for genes encoding mRNA species bound by MbRpoE/F and those containing motif 1 or motif 2 versus the whole genome.

FilenameFormatSizeDescription
EMI_2385_sm_FigS1-7-TableS1.pdf690KSupporting info item
EMI_2385_sm_tS2.xls4111KSupporting info item
EMI_2385_sm_tS3_Rev.xls38KSupporting info item

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