Regulation of pyr gene expression in Mycobacterium smegmatis by PyrR-dependent translational repression

Abstract

Regulation of pyrimidine biosynthetic (pyr) genes by a transcription attenuation mechanism that is mediated by the PyrR mRNA-binding regulatory protein has been demonstrated for numerous gram-positive bacteria. Mycobacterial genomes specify pyrR genes and contain obvious PyrR-binding sequences in the initially transcribed regions of their pyr operons, but transcription antiterminator and attenuation terminator sequences are absent from their pyr 5' leader regions. This work demonstrates that repression of pyr operon expression in Mycobacterium smegmatis by exogenous uracil requires the pyrR gene and the pyr leader RNA sequence for binding of PyrR. Plasmids containing the M. smegmatis pyr promoter-leader region translationally fused to lacZ also displayed pyrR-dependent repression, but transcriptional fusions of the same sequences to a lacZ gene that retained the lacZ ribosome-binding site were not regulated by PyrR plus uracil. We propose that PyrR regulates pyr expression in M. smegmatis, other mycobacteria, and probably in numerous other bacteria by a translational repression mechanism in which nucleotide-regulated binding of PyrR occludes the first ribosome-binding site of the pyr operon.

FIG. 1.

A. Schematic diagram of the chromosomal organization of the pyrimidine biosynthetic (pyr) genes in M. smegmatis. The pyrRBC-orf3045-carAB-pyrF operon is shown at the left; the pyrD and pyrE genes are unlinked and located away from the pyr operon. MSMEG3045 denotes an open reading frame of unknown function. The bent arrow indicates the start of transcription of the operon. A portion of the promoter-leader region of the transcript shown below illustrates the predicted secondary fold of the PyrR-binding loop (BL; shaded) in the RNA, the Shine-Dalgarno sequence of a putative ribosome binding site (SD), and start codon (shaded AUG) for pyrR, the first gene of the operon. B. DNA sequence on the pyr operon promoter-leader region; the nontemplate strand is shown in capital letters. The sequence is numbered with the first base of the pyrR start codon as +1. The sequence specifying the PyrR-binding loop is enclosed in a box and shaded, except for a box designating the putative Shine-Dalgarno sequence. Putative −35 and −10 promoter sequences are shown in boldface. MSMEG3041 indicates an open reading frame of unknown function (annotated as a member of the thiopurine S-methyltransferase family) that is transcribed in an opposite direction to the pyr operon. Regions deleted from the binding loop in pyr′-lacZ fusion plasmids pFS20 and pFS22 are denoted by Δ1; those deleted in pyr′-lacZ fusion plasmids pFS21 and pFS23 are denoted by Δ2.

FIG. 2.

Construction of pyr′-lacZ fusion plasmids for characterization of regulation. A. Schematic diagrams showing the set of pJEM13-derived translational fusions in which the pyr leader, including the PyrR-binding loop region, pyrR ribosome-binding site (RBS), and first three codons of pyrR were fused in frame via a five-codon linker (Gly-Thr-Lys-Leu-Ala) to lacZ at codon 13 (left), and the set of pJEM15-derived transcriptional fusions in which the same pyr segments were fused to 56 bp of the lacZ leader so that the lacZ ribosome-binding site lies at the normal distance from the lacZ open reading frame (right). B. Diagram showing the locations of PCR primers (F1, F2, F3, R1, and R2) and segments of M. smegmatis DNA that were inserted upstream of lacZ to generate the plasmids described in the text and in Tables 2 and 3. Numbering of the sequence is as in Fig. 1B. C. Proposed secondary structures of the pyr leader RNA regions in the pyr′-lacZ fusion plasmids from which segments of the PyrR-binding loop were deleted (Table 3). Arrows denote the site of deletion. SD denotes the Shine-Dalgarno sequence of the presumed pyr ribosome-binding site.

FIG. 3.

Proposed mechanism of PyrR-mediated regulation of the M. smegmatis pyr operon by translational repression.