A homing endonuclease and the 50-nt ribosomal bypass sequence of phage T4 constitute a mobile DNA cassette

Abstract

Since its initial description more than two decades ago, the ribosome bypass (or "hop") sequence of phage T4 stands out as a uniquely extreme example of programmed translational frameshifting. The gene for a DNA topoisomerase subunit of T4 has been split by a 1-kb insertion into two genes that retain topoisomerase function. A second 50-nt insertion, beginning with an inphase stop codon, is inserted near the start of the newly created downstream gene 60. Instead of terminating at this stop codon, approximately half of the ribosomes skip 50 nucleotides and continue translation in a new reading frame. However, no functions, regulatory or otherwise, have been imputed for the truncated peptide that results from termination at codon 46 or for the bypass sequence itself. Moreover, how this unusual mRNA organization arose and why it is maintained have never been explained. We show here that a homing endonuclease (MobA) is encoded in the insertion that created gene 60, and the mobA gene together with the bypass sequence constitute a mobile DNA cassette. The bypass sequence provides protection against self-cleavage by the nuclease, whereas the nuclease promotes horizontal spread of the entire cassette to related bacteriophages. Group I introns frequently provide protection against self-cleavage by associated homing endonucleases. We present a scenario by which the bypass sequence, which is otherwise a unique genetic element, might have been derived from a degenerate group I intron. bacteriophage gene structure | group I intron | horizontal gene transfer | ribosomal frameshifting | mobile genetic element I n most of the phages of the T4 superfamily, the large subunit of DNA topoisomerase is a single polypeptide encoded by gene 39. However, in phage T4 this gene is disrupted by a 1-kb insertion, creating a truncated gene 39 plus a new gene (gene 60) that encodes the remainder of the topoisomerase subunit (1, 2) ( Fig. 1). Furthermore, the C terminus of the truncated gene 39 and the N terminus of the newly created gene 60 each contain additional amino acid residues (43 and 30, respectively) that were not present in the original gene 39 homologs. These two independently translated proteins interact with the small subunit (encoded by gene 52) to create an enzymatically active topoisomerase (4). A second, 50-nt insertion beginning with an inphase stop codon, is inserted into the newly created downstream gene 60 (2). Instead of terminating at this stop codon, approximately half of the ribosomes skip 50 nucleotides and continue translation in a new reading frame (5). Features involved in this remarkable process include: the stop codon, the codon at which translation resumes, a portion of the pre-hop nascent peptide, a stem-loop at the start of the bypassed sequence, a Shine/Dalgarno-like sequence within the bypassed RNA, and the structure of the bypassed mRNA (5-8).The genome sequence of phage T4 (GenBank accession no. NC_000866) indicated that the insertion into gene 39 contains two ORFs: an apparent H-N-H homing endonuclease ps...

High-efficiency translational bypassing of non-coding nucleotides specified by mRNA structure and nascent peptide

“…The take-off codon is 5 0 adjacent to UAG and both are near the top of a stem loop (SL), the 'take-off SL' that is critical for bypassing 4,[7][8][9][10] . The analysis of the mRNA structure suggested that the gap region is structurally autonomous from the upstream and downstream coding regions 10 , consistent with the evolutionary origin of the gap from a mobile element 6 . The presence of the take-off GGA and the matching landing codons, the UAG stop codon next to the take-off GGA and the SL structure in the 5 0 gap are essential for bypassing.…”

“…The interrupted ORF is unique to the T4 genome and is caused by the disruption of gene 39, which encodes the DNA topoisomerase subunit in other T-even bacteriophages 5 , by the insertion of a mobile DNA element 6 . The mobile element splits gene 39 into a sequence coding for the N-terminal part of gp39 and the newly formed gene 60, which codes for the C-terminal portion of the topoisomerase.…”

“…The mobile element splits gene 39 into a sequence coding for the N-terminal part of gp39 and the newly formed gene 60, which codes for the C-terminal portion of the topoisomerase. One consequence of the mobile element insertion is the addition within the gene 60 coding sequence of a 50-base pair foreign sequence 6 , which is copied into the gene 60 mRNA. The non-coding sequence is not spliced but is skipped during translation 3,4 .…”

Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

“…This insertion splits the N-terminal portion of the conserved gene 39 ORF from its C-terminal portion, which is encoded by the new T4 gene 60 (Fig. 1A;Hatfull 2008;Bonocora et al 2011;Cresawn et al 2011). The mobile DNA element encodes the homing endonuclease MobA, which catalyzes the transfer of the mobile DNA element between related phage genomes (Edgell et al 2010;Bonocora et al 2011).…”

“…1A;Hatfull 2008;Bonocora et al 2011;Cresawn et al 2011). The mobile DNA element encodes the homing endonuclease MobA, which catalyzes the transfer of the mobile DNA element between related phage genomes (Edgell et al 2010;Bonocora et al 2011). MobA recognizes and cleaves a specific sequence within the genomic DNA of gene 39 (Bonocora et al 2011) just upstream of the coding region for the highly conserved Toprim motif (Fig.…”