Programmed translational frameshift in the bacteriophage P2 FETUD tail gene operon

Abstract

The major structural components of the P2 contractile tail are encoded in the FETUD tail gene operon. The sequences of genes F(I) and F(II), encoding the major tail sheath and tail tube proteins, have been reported previously (L. M. Temple, S. L. Forsburg, R. Calendar, and G. E. Christie, Virology 181:353-358, 1991). Sequence analysis of the remainder of this operon and the locations of amber mutations Eam30, Tam5, Tam64, Tam215, Uam25, Uam77, Uam92, and Dam6 and missense mutation Ets55 identified the coding regions for genes E, T, U, and D, completing the sequence determination of the P2 genome. Inspection of the DNA sequence revealed a new open reading frame overlapping the end of the essential tail gene E. Lack of an apparent translation initiation site and identification of a putative sequence for a programmed translational frameshift within the E gene suggested that this new reading frame (E') might be translated as an extension of gene E, following a -1 translational frameshift. Complementation analysis demonstrated that E' was essential for P2 lytic growth. Analysis of fusion polypeptides verified that this reading frame was translated as a -1 frameshift extension of gpE, with a frequency of approximately 10%. The arrangement of these two genes within the tail gene cluster of phage P2 and their coupling via a translational frameshift appears to be conserved among P2-related phages. This arrangement shows a striking parallel to the organization in the tail gene cluster of phage lambda, despite a lack of amino acid sequence similarity between the tail gene products of these phage families.

FIG. 1.

Genetic map of P2 and physical map of the tail gene region reported in this paper. (A) Linear map of the P2 genome, with cosL at the left. Thin black arrows indicate the direction and extent of the known transcription units. orf, open reading frame. (B) Expanded view of the physical map of the part of the P2 genome characterized in this report and the location of amber mutations in this region. Selected restriction sites used in subcloning and marker rescue are indicated, with coordinates shown in nucleotides from the left end of the P2 genome. Boxed regions delineate the coding regions for each of the genes indicated in the physical map. The extent of DNA carried by each plasmid subclone is aligned below the relevant restriction sites or position on the physical map; gray bars indicate the intervals to which amber mutations were mapped by marker rescue. (C) DNA sequence of the region between the end of gene E and the beginning of gene T, showing the −1 (E′) open reading frame between these two genes. The TGA stop codon for gene E and the two TAA stop codons defining the boundaries of the −1 reading frame are shown in boldface type. The first in-frame Met codon in the −1 reading frame is underlined, and the location of the C-to-A change introduced to create an amber mutation in E′ is indicated.

FIG. 2.

Complementation of P2 E mutants by cloned fragments carrying the E-E′ region. P2 Ets55 was plated at 42°C. Complementation was shown by growth of P2 mutants as follows: +, growth; −, no growth; NT, not tested.

FIG. 3.

Analysis of the region containing a programmed translational frameshift. (A) The nucleotide sequence spanning the end of gene E and the beginning of open reading frame E′ is shown, along with the oligonucleotides used to clone this region into the frameshift detection plasmid p138. The position where a C residue was inserted to allow translation of lacZ by ribosomes that did not shift reading frame is indicated. Below the DNA sequence the predicted amino acid sequences encoded by E and the −1 open reading frame E′ are shown, along with the actual N-terminal sequence determined from the β-galactosidase (βgal) fusion protein purified from cells carrying pTG316. (B) Measurement of frameshifting efficiency in vivo. The β-galactosidase (β-gal) activity was determined in cells carrying a plasmid with the wild-type (wt) E-E′ fragment, in which lacZ must be translated by ribosomes that have undergone a −1 frameshift, and from cells with a plasmid carrying the equivalent fragment in which a C was inserted just upstream of the stop codon for gene E (fs+1), so that lacZ is translated by ribosomes that did not shift reading frame into E′.

FIG. 4.

Synthesis of a MalE fusion protein carrying the frameshifted gpE+E′polypeptide. Protein was affinity purified as described in Materials and Methods and analyzed by electrophoresis on a 10% SDS-polyacrylamide gel stained with Gelcode blue (Pierce). Protein bands corresponding to the expected sizes for the MalE-gpE fusion protein and the MalE-gpE+E′ frameshifted protein are indicated, as are the sizes (in kilodaltons) of molecular mass standards. Densitometry and digital imaging of the gel was performed on a ChemiImager 4000 (Alpha Innotech Corp.), and the figure was compiled using Microsoft PowerPoint.

FIG. 5.

Potential translational frameshift sites in P2-related phages. (A) Alignment of sequences from the region corresponding to E-E′ in related phages that parallel P2 in genome organization. Sequences are from the following phages (GenBank accession numbers are shown in parentheses): E. coli phages 186 (NC_001317) and Wφ (Esposito et al., unpublished); S. enterica phages SopEφ (AF153829), Fels-2 (sequenced as a prophage in the S. enterica serovar Typhimurium LT2 genome; (AE006468) and PSP3 (Christie et al., unpublished); and P. aeruginosa phage 102 φCTX (NC_003278). Boxes indicate the extent of the open reading frame (orf) and overlap in the genes equivalent to P2 E (white) and the −1 reading frame E′ (light gray), as well as show the beginning of T (dark gray, which in all cases is in the same frame as E). The DNA sequences from this region are aligned below the sequence alignment. The codons for the last amino acid read in the gpE reading frame and the first amino acid read in the gpE+E′ reading frame are indicated by an underline and overline, respectively. Nucleotides shown in boldface type are complementary to the 3′ end of 16S rRNA. Arrows indicate complementary sequence encoding potential RNA hairpins predicted by the GCG program MFold. In the case of P2 and Wφ, the most stable of several predicted structures is shown. However, the extensive similarity between these two sequences might also allow formation of a hairpin equivalent to that of P2 in Wφ and vice versa. (B) A similar comparison of open reading frames upstream of the putative tail tape measure protein in the more distantly related P2-like phages from H. influenzae (HP1 [NC_001697] and HP2 [NC_003315]) and V. cholerae (K139 [NC_003313]) (GenBank accession numbers shown in brackets). The DNA sequence of the region of overlap between the genes in locations equivalent to E and E is shown. The TAA stop codon for open reading frame 26 (orf26) (HP1 and HP2) or orf30 (K139) is underlined, as is the TGA stop codon that defines the extent of overlap in the −1 reading frame.