Translation Limits Synthesis of an Assembly-Initiating Coat Protein of Filamentous Phage IKe

Abstract

Translation is shown to be downregulated sharply between genes V and VII of IKe, a filamentous bacteriophage classed with the Ff group (phages f1, M13, and fd) but having only 55% DNA sequence identity to it. Genes V and VII encode the following proteins which are used in very different amounts: pV, used to coat the large number of viral DNA molecules prior to assembly, and pVII, used to serve as a cap with pIX in 3 to 5 copies on the end of the phage particle that emerges first from Escherichia coli. The genes are immediately adjacent to each other and are represented in the same amounts on the Ff and IKe mRNAs. Ff gene VII has an initiation site that lacks detectable intrinsic activity yet through coupling is translated at a level 10-fold lower than that of upstream gene V. The experiments reported reveal that by contrast, the IKe gene VII initiation site had detectable activity but was coupled only marginally to upstream translation. The IKe gene V and VII initiation sites both showed higher activities than the Ff sites, but the drop in translation at the IKe V-VII junction was unexpectedly severe, ∼75-fold. As a result, gene VII is translated at similarly low levels in IKe- and Ff-infected hosts, suggesting that selection to limit its expression has occurred.

Translation at higher than an optimal level interferes with coupling at an intercistronic junction

“…The inefficient coupling of IKe gene VII translation to gene V could, in principle, reflect an intrinsic property of the gene VII initiation site, the gene V termination site or some other feature of translation through the pair of genes. To (Blumer et al, 1987;Madison-Antenucci and Steege, 1998). The V -VII-lacZ fusions pI5-7 and pf5-7 contain IKe (shaded) or f1 (unshaded) sequence.…”

“…Expression of the gene thus depends completely on translational coupling, which in this case captures 10% of upstream translation from gene V (Blumer et al, 1987;Ivey-Hoyle and Steege, 1989). Investigation of the IKe gene V-VII pair (Madison-Antenucci and Steege, 1998) revealed that translation at the gene V-VII junction is downregulated to a level very similar to that observed in f1-infected hosts. However, other features of translation were found to be different.…”

“…The common region preceding each insert includes the 5 0 -terminal 19 nt of the lacI mRNA and linker sequences. Inserts are as follows: pI5, gene V initiation site and first 15 codons; pI7, gene VII initiation site and first four codons; pI5-7, VII site -lacZ fusion as in pI7, but preceded by intact gene V. These plasmids are identical to the IKe -lacZ fusions described previously (Madison-Antenucci and Steege, 1998). In this paper, these plasmids and the equivalent f1 -lacZ fusions (Blumer et al, 1987) have been given names (Table 4) that indicate whether the phage inserts are of IKe (I) or f1(f) origin.…”

“…Recent study of the gene V-VII pair encoded by the N pilus-specific filamentous phage IKe revealed a puzzling case of translational coupling (Madison-Antenucci and Steege, 1998). The phage IKe and f1 genomes share only 55% sequence identity, but have the same genes and gene order (Hill and Petersen, 1982;Peeters et al, 1985).…”

“…C. Sequences of the IKe and f1 gene VII initiation sites. Toeprinting data and mutational analysis determined that the AUG 2 nt beyond the IKe stop codon serves as the initiator codon (Madison-Antenucci and Steege, 1998). does show ribosome binding capability in toeprinting experiments and weak but detectable independent activity in lacZ fusions ( Fig. 1B, pI7; 260 b-galactosidase units).…”