AraL sugar phosphatase from B. subtilis L. M. Godinho and I. de Sa´ -Nogueira A B Fig. 5. Site-directed mutagenesis at the 5¢-end of the araL gene and overproduction of recombinant AraL-His6. (A) The secondary structure of the araL mRNA in pLG12 (left) and pLG13 (right), which bears a single nucleotide change. An arrowhead highlights the mutated nucleotide located at the beginning of the araL coding region. The ribosome-binding site, rbs, and the initiation codon (ATG) are boxed. The position relative to the transcription start site is indicated. The free energy of the two secondary structures, cal- culated by DNASIS, version 3.7 (Hitachi Software Engineering Co. Ltd, Tokyo, Japan), is shown. (B) Overproduction of recombinant AraL-His6. Analysis of the soluble (S) and insoluble (P) protein frac- tion (20 lg of total protein) of induced cultures of E. coli Bl21(DE3) pLysS harboring pLG12 (AraL-His6) and pLG113 (AraL-His6 G ﬁ A). The proteins were separated by SDS ⁄ PAGE 12.5% gels and stained with Coomassie blue. A white arrowhead indicates AraL- His6. The sizes (kDa) of the broad-range molecular mass markers (Bio-Rad Laboratories) are indicated. Regulation and putative role of AraL in B. subtilis In B. subtilis, the formation of a similar hairpin struc- ture at the same location is possible and displays a free energy of )21.4 kcalÆmol)1 (Fig. 6A). To determine its role in the regulation of araL expression, a transla- tional fusion of the 5¢-end of the araL gene to the lacZ reporter gene from E. coli was constructed and inte- grated into the B. subtilis chromosome, as a single copy, at an ectopic site. The construct comprises the araL ribosome-binding site, the initiation codon and a fusion between codon 10 of araL and codon 7 of E. coli lacZ. The araL¢-¢lacZ translational fusion is under the control of the strong promoter (Para) of the araABDLMNPQ-abfA operon (Fig. 6B). However, expression from the araL¢-¢lacZ fusion in the presence of arabinose (inducer) is very low, as determined by measuring the levels of accumulated b-galactosidase activity in strain IQB847 (Fig. 6B). By contrast, strain IQB849 carrying a single-base substitution C ﬁ A introduced in the hairpin region displayed an augment in araL¢-¢lacZ expression of 30-fold in the presence of inducer (Fig. 6B). This point mutation increased the free energy of the mRNA secondary structure from )21.4 kcalÆmol)1 to )15.4 kcalÆmol)1 (6 kcalÆmol)1; Fig. 6B). Furthermore, a double point mutation, C ﬁ A and G ﬁ T, introduced a compensatory T in the other part of the stem (Fig. 6A), thus regenerating the stem-loop structure in strain IQB857 and drasti- cally reducing the expression of araL¢-¢lacZ (Fig. 6B). In addition, as described above, a single-point muta- tion C ﬁ G was designed in the same position and the effect was analyzed in strain IQB855 (Fig. 6B). How- ever, no signiﬁcant effect was detected in the expres- sion of the translational fusion, suggesting that the increase of 3 kcalÆmol)1 is insufﬁcient for disrupting this particular RNA secondary structure. Similarly, no translation was measured in strain IQB853 carrying a double point mutation, C ﬁ G and G ﬁ C, which introduced a compensatory C in the other part of the stem (Fig. 6). These results clearly show that the hair- pin structure play an active role in the control of araL expression. The regulatory mechanism operating in this situation is most probably sequestration of the ribo- some binding by the mRNA secondary structure, con- sequently preventing translation, although the possibility of premature transcription termination by early RNA polymerase release cannot be excluded. Translational attenuation by mRNA secondary struc- ture comprising the initiation region is present in many systems of Bacteria, including B. subtilis [32]. As a result the nature of the NagD family members display- ing low speciﬁcity and catalytic activities and lacking 2518 FEBS Journal 278 (2011) 2511–2524 ª 2011 The Authors Journal compilation ª 2011 FEBS