A conserved RNA structure (thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria

Abstract

The thiCOGE genes of Rhizobium etli code for enzymes involved in thiamin biosynthesis. These genes are transcribed with a 211-base untranslated leader that contains the thi box, a 38-base sequence highly conserved in the 5' regions of thiamin biosynthetic and transport genes of Gram-positive and Gram-negative organisms. A deletion analysis of thiC-lacZ fusions revealed an unexpected relationship between the degree of repression shown by the deleted derivatives and the length of the thiC sequences present in the transcript. Three regions were found to be important for regulation: (i) the thi box sequence, which is absolutely necessary for high-level expression of thiC; (ii) the region immediately upstream to the translation start codon of thiC, which can be folded into a stem-loop structure that would mask the Shine-Dalgarno sequence; and (iii) the proximal part of the coding region of thiC, which was shown to contain a putative Rho-independent terminator. A comparative phylogenetic analysis revealed a possible folding of the thi box sequence into a hairpin structure composed of a hairpin loop, two helices, and an interior loop. Our results show that thiamin regulation of gene expression involves a complex posttranscriptional mechanism and that the thi box RNA structure is indispensable for thiCOGE expression.

Figure 1

(A) Schematic map of thiC gene of R. etli. The stippled box represents the thiC coding sequence. The lines above represent the location of the probes used in the Northern experiment. (B) Deletion constructions fused to lacZ. The fragments obtained by PCR were cloned into pMP220 vector to generate lacZ transcriptional fusions for determination of β-galactosidase activities from cultures grown in MM with or without thiamin as indicated. The letters alongside the arrows represent the names of the oligonucleotides used to obtain the different PCR fragments. The nucleotide positions are given with reference to the tss (position +1) of thiC. The black box represents the thi box sequence, and the potential stem-loop structures in the transcript that comprises the S-D sequence and the attenuator are marked by an inverted U. β-galactosidase activity is expressed as nmol of o-nitrophenol produced min⁻¹⋅mg protein⁻¹. Representative results of three experiments are shown. RR is the repression ratio, the β-galactosidase activity observed in media without thiamin divided by the activity observed in media supplemented with thiamin.

Figure 2

(A) Proposed secondary structure for the thi box sequence. Capital bold letters represent conserved bases in all of the thi box sequences analyzed. The dashed lines represent hydrogen bonds that can be formed only in the R. etli thi box. The capital letters alongside the R. etli thi box represent the changes in sequence observed in the other thi box sequences. The boxed capital letters represent changes that would not permit the formation of base pairs. Subscripts represent the thi box sequences in front of genes: E1, E2, and E3, thiC, thiB and thiM genes of E. coli; M1 and M2, thiO and thiC genes of M. tuberculosis; B1, thiA gene of B. subtilis; S, Synechocystis; St, Salmonella enterica; T, Thiocystis violacea; A, Aeromonas hydrophila. The u marked with a # is absent in M2 thi box (see C). The G in outlined style is inserted between the As in S thi box. (B and C) Hairpin structures formed by the thi box sequences in front of the thiO and thiC genes of M. tuberculosis, respectively. The sequence AGGGA that is highly conserved in the 3′ end of the thi box sequences analyzed is represented in bold, underlined letters. The translation start codon of both genes is boxed.

Figure 3

(A) Proposed hairpin structure in the region surrounding the S-D sequence of the thiC gene of R. etli. The presumptive S-D sequence is represented by bold, capital letters and the translation start of thiC by bold, lowercase letters. (B) Putative Rho-independent terminator found in the coding region of thiC.

Figure 4

thiC gene expression. RNA from the CE3 strain grown in MM with (+) or without (−) thiamin was slot-blotted and hybridized with probe A (a DNA fragment from nucleotide +6 to +217), probe B (an intra thiC fragment from nucleotide +1044 to +1494), and with the 16S rRNA gene of E. coli.

Figure 5

Model for R. etli thiCOGE expression. (Left) The structural and functional organization of the regulatory and coding regions of the genes thiCOGE is represented. Transcription is constitutive from the thiC promoter. Depending on the availability of thiamin in the growth medium, this transcript will be elongated up to the end of the thiE gene (−thiamin condition) or would be prematurely terminated at the putative attenuator located at position +522 to +547 (+thiamin condition). The hairpin structures that can be formed by the thi box, the S-D sequence, and the putative attenuator are marked. thiCOGE genes are not drawn to scale. For details see the text.