Group IIC intron mobility into attC sites involves a bulged DNA stem-loop motif

Abstract

Bacterial group IIC introns are a subclass of group II intron ribozymes that are typically located downstream from transcriptional terminators. Class IIC-attC introns constitute a monophyletic subset of subgroup IIC, which preferentially insert into site-specific recombination sequences for integron integrases (attC). attCs are a diverse family of nucleotide sequences composed of conserved inverted repeats that flank a variable, but palindromic, central region. In this study, we used both PCR and colony patch hybridization methods to determine the basis for recognition of the attC(aadA1) stem-loop motif by the Serratia marcescens intron (S.ma.I2) in vivo. The quantitative results showed that mobility into the wild-type site occurs at a frequency of 18%, and is strongly biased by the orientation of the homing site relative to the direction of DNA replication. S.ma.I2 mobility results into mutant attC(aadA1) sites are consistent with recognition of stem-loop motifs in unwound DNA. The homing frequency results showed that, while the entire attC sequence is not necessary for recognition of the insertion site, short deletions of the attC stem-loop motif inhibited the intron mobility. Moreover, our data show that S.ma.I2 requires a bulged base in the folded attC stem for high homing frequency. We demonstrate that the IBS1/IBS3 motifs and two bulge bases conserved among attCs determine S.ma.I2 homing specificity for the attC bottom strand. These results suggest that class IIC-attC introns tolerate attC variation by recognition of a bulged hairpin DNA motif rather than a specific sequence.

FIGURE 1.

Integron structure and attC recombination sites. (A) General schematic representation of an integron. intI, integrase gene; P_i, integrase promoter; P_c, cassette promoter; attI, integrase recombination site (integrase specific); ORF, cassette open reading frame; attC, integrase recombination site (cassette specific). (B) Sequence of the attC_aadA1 site. 2L-2R and 1L-1R boxes are inverted repeats; arrows, integrase recombination site and intron insertion site; Vienna output (dots and parentheses between the top-strand [ts] and bottom-strand [bs] sequences), base pairings from a potential single-stranded secondary structure based on MFOLD prediction. (C) Predicted stem–loop structures of the attC top- and bottom-strand sequences based on MFOLD predictions. IBS1 and IBS3, intron binding sites 1 and 3; asterisk (*), extrahelical bases T38 and G45 upon folding of the attC bottom strand.

FIGURE 2.

Schematic of the two-plasmid mobility assay in E. coli (see the Materials and Methods section for a more detailed protocol). E₁ and E₂, exon sequences; IEP, S.ma.I2 intron encoded protein; Plac, lac promoter; Ap^R and Cm^R, ampicillin and chloramphenicol resistance genes; pMB1 and p15A, plasmid replication origins.

FIGURE 3.

Sequence alignments of the attC_aadA1 mutants tested as homing sites in this study. (A) Mutation of the stem lengths; (B) mutation of the stem pairings; (C) mutation of IBS1 or IBS3; (D) deletion of the extrahelical bases; (E) mutation of the extrahelical bases; and (F) inversion of the intron binding sites with or without the extrahelical bases. The figure shows only the bottom-strand sequences of the wild-type (WT) and mutant attC_aadA1 sites. Capital letters, attC sequences; lower case letters, pACYC184 sequences; gray shading and Vienna outputs, nucleotide pairings from a potential single strand stem–loop structure determined using MFOLD. Asterisk (*), intron homing site; underlined bases, IBS1 (TTGT) and IBS3 (T); dot, sequence identity to WT; dash, base deletion.

FIGURE 4.

S.ma.I2 in vivo mobility results into attC_aadA1 mutants. Agarose gels of the PCR products using primer pair “a” or “b” in order to detect the orientation of intron insertion. The figure shows the 5′ intron–exon integration junction (see Fig. 2 for primer position). “WT,” “IBS ts,” and “IBS + bulges ts” attC_aadA1 were cloned in both orientations (LAG or LEAD) relative to the direction of DNA replication in pACYC184 (see Materials and Methods).

FIGURE 5.

S.ma.I2 in vivo mobility results into attC_aadA1 mutants. Agarose gels of the PCR products using PACYC-5′ and PACYC.rev primers in order to detect variations in the degree of intron insertion for each mutant compared with the WT site (see Fig. 2 for primer position). The Figure shows attCs that were and were not targeted by S.ma.I2.

FIGURE 6.

(A) Alignment of the sequenced PCR products from the S.ma.I2 mobility assays into the WT, “IBS ts,” and “IBS + bulges ts” attC_aadA1 sites. Arrows indicate the intron homing site determined by PCR (Fig. 4). The thickness of the arrows corresponds to the mobility frequencies observed in Table 1. Asterisk (*), intron homing site; underlined bases, intron binding site; highlighted bases, difference from the WT sequence; dash, base deletion. (B) Predicted stem–loop structures of the attC_aadA1 mutant top- and bottom-strand sequences based on MFOLD predictions. IBS1 and IBS3, intron binding sites 1 and 3; asterisk (*), extrahelical bases T38 and G45 upon folding of the attC bottom strand.