Saturation mutagenesis of the TATA box and upstream activator sequence in the haloarchaeal bop gene promoter

Abstract

Degenerate oligonucleotides were used to randomize 21 bp of the 53-bp minimal bop promoter in three 7-bp segments, including the putative TATA box and the upstream activator sequence (UAS). The mutagenized bop promoter and the wild-type structural gene and transcriptional terminator were inserted into a shuttle plasmid capable of replication in the halophilic archaeon Halobacterium sp. strain S9. Active promoters were isolated by screening transformants of an orange (Pum- bop) Halobacterium mutant for purple (Pum+ bop+) colonies on agar plates and analyzed for bop mRNA and/or bacteriorhodopsin content. Sequence analysis yielded the consensus sequence 5'-tyT(T/a)Ta-3', corresponding to the promoter TATA box element 30 to 25 bp 5' of the transcription start site. A putative UAS, 5'-ACCcnactagTTnG-3', located 52 to 39 bp 5' of the transcription start site was found to be conserved in active promoters. This study provides direct evidence for the requirement of the TATA box and UAS for bop promoter activity.

FIG. 1

Organization of the bop gene region and promoter mutagenesis. (A) Organization of the bop gene region (bop, brp, bat, and blp genes) shown in boxes with the promoters indicated above by arrows. (B) Minimum bop promoter and a few surrounding nucleotides. The sequence is numbered starting with the transcription start point (indicated by an arrow) as +1. The UAS, TATA box, and R-Y box regions are boxed, and the translational start codon is underlined. The locations of osmium tetroxide and S1 nuclease cleavages are indicated by vertical arrows (36). (C) E. coli-Halobacterium shuttle vector (pNB series), which contains the bop gene (black arrow) and promoter (white box). The E. coli replication origin (ori ColE1) and selectable marker (bla [shaded arrow]) are indicated, as are the Halobacterium replication origin (ori pNRC100), repH gene (white arrow), and selectable marker (mev [shaded arrow]).

FIG. 2

Analysis of TATA box mutants at the transcriptional (A) and translational (B) levels. (A) Primer extension analysis of bop mRNA in four TATA box mutants using crude RNA with the bop2 primer. Strain designations are above the lanes. The controls used in this experiment were Pum⁻ (bop) host strain SD23, strain 100E (SD23 containing a plasmid with an unmutated promoter), and TATA box mutants (1B2, 2B12, 2H10, and 1D2). A sequencing reaction (lane C) performed with the bop2 primer on a pMS1 template (plasmid containing the cloned bop gene) is shown, as is the double-stranded sequence across the transcription start point (the start site and direction are indicated by the arrow). (B) Spectra (absorbance versus wavelength from 400 to 700 nm) of purple membrane preparations for the four TATA box mutant strains and the two control strains (same as in panel A). Relative BR content was quantified by comparison of absorbance at 568 nm.

FIG. 3

Tabulation of strain designations, promoter sequences, phenotypes, and BR contents of TATA box mutants (A) and analysis of promoter sequences (B). In panel A, the strain designations are shown in the first column, and the sequence of nucleotides −31 to −25 (identity to the wild type base is denoted by a dot), the Pum phenotype (−, negative; +, positive; ++, overproducer), and relative BR content are shown to the right. ND, not detectable; ∗, not done. For panel B, the TATA box consensus sequence was determined by tallying individual nucleotides observed at each position in Pum⁺ strains. The consensus sequence is indicated at the bottom.

FIG. 4

Tabulation of strain designations, promoter sequences, phenotypes, and BR contents of UAS mutants (A) and analysis of promoter sequences (B). In panel A, the strain designations are shown in the first column, and sequence of nucleotides −52 to −39 (identity to the wild type base is denoted by a dot), the Pum phenotype (for definitions, see the legend to Fig. 3), and relative BR content are shown to the right. ND, not detectable; ∗, not done. For panel B, the UAS element consensus sequence was determined by tallying individual nucleotides observed at each position in Pum⁺ strains. The consensus sequence is indicated at the bottom.

FIG. 5

Effect of UAS mutagenesis on transcription start site location (A and C) and promoter strength (B and D). For panels A and B, transcription start sites were mapped by comparing the mobilities of primer extension products to that of a sequencing ladder generated with the bop2 primer and the pMS1 template (plasmid with a cloned bop gene). Lane C of the sequencing reaction is shown. The strain designations are shown over the corresponding lanes. For panels C and D, promoter strength was measured by quantifying the bop message by PhosphorImager analysis of the primer extension gels. The strain designations (corresponding to A and C, respectively) are shown below the bars plotted against relative intensity.