Analysis of elements involved in pseudoknot-dependent expression and regulation of the repA gene of an IncL/M plasmid

Abstract

Replication of the IncL/M plasmid pMU604 is controlled by a small antisense RNA molecule (RNAI), which, by inhibiting the formation of an RNA pseudoknot, regulates translation of the replication initiator protein, RepA. Efficient translation of the repA mRNA was shown to require the translation and correct termination of the leader peptide, RepB, and the formation of the pseudoknot. Although the pseudoknot was essential for the expression of repA, its presence was shown to interfere with the translation of repB. The requirement for pseudoknot formation could in large part be obviated by improving the ribosome binding region of repA, either by replacing the GUG start codon by AUG or by increasing the spacing between the start codon and the Shine-Dalgarno sequence (SD). The spacing between the distal pseudoknot sequence and the repA SD was shown to be suboptimal for maximal expression of repA.

FIG. 1

Sequence of the replication control regions of IncL/M and IncB. The promoter regions for RNAI (Pri) are labelled, and transcription start sites are indicated by asterisks. Arrows above the sequences represent stem-loop structures (SLI, SLII, and SLIII). The distal and proximal pseudoknot sequences are in boldface type and underlined. SD of the repA and repB genes are in boldface type, and translational start (open) and termination (hatched) codons are boxed.

FIG. 2

Putative stem-loop structures I and II (SLI and SLII) depicting substitutions made to the proximal and distal pseudoknot bases (boldface and underlined nucleotides). The repA SD and start codon are in boldface type, and the repB termination codon is boxed.

FIG. 3

The leader region of the repA mRNA of pMU604 showing putative SLI and SLII structures, the latter indicated by arrows below the sequence. Individual mutations are indicated by arrows or labelled with solid bars coming off the sequence such that the first base of the repB stop codon is the first base on the right of the bar. SD are in boldface type, start codons are boxed, and the wild-type repB termination codon is hatched. Proximal and distal pseudoknot bases are in shaded print. The effect of moving the repB termination codon on repA expression is summarized in the graph in the top right-hand corner of the figure. Bases altered to introduce new repB stop codons and the position of termination relative to G692 of the repA start codon (+1) are shown on the bottom axis of the graph. WT, wild type.

FIG. 4

Partial nucleotide sequence of the replication control region of pMU604 showing mutations affecting the spacing between distal pseudoknot sequence (boldface and underlined) and TIR of repA. The mutations are labelled as those which (i) insert bases between distal sequence and repA SD (PSD+1 to PSD+6); (ii) delete bases between distal sequence and repA SD (PSD−1, PSD−2) and (iii) insert bases between the distal sequence and repA start codon (PST+1, PST+2, PST+3). Compensatory insertions or deletions in the repB coding region are linked to the appropriate mutations by stippled arrows. The promoter region for RNAI is overlined, and the transcriptional start site is indicated. SD are in boldface type, translational start codons are boxed, and the repB stop codon is hatched.