Genome sequencing projects are predicting large numbers of novel proteins, whose interactions with other proteins must mediate the function of cellular processes. To analyse these networks, we used the yeast two-hybrid system on a genome-wide scale to identify 25 interactions among the proteins of Escherichia coli bacteriophage T7. Among these is a set of six interactions connecting proteins that function in DMA replication and DMA packaging. Remarkably, two genes, arranged such that one entirely overlaps the other and uses a different reading frame, encode interacting proteins. Several of the interactions reflect intramolecular associations of different domains of the same polypeptide, suggesting that the two-hybrid assay may be useful in the analysis of protein folding. This global approach to protein–protein interactions may be applicable to the analysis of more complex genomes whose sequences are becoming available.

A protein linkage map of Escherichia coli bacteriophage T7.

Addition of acetate to a stationary phase culture of Escherichia coli in glycerol mineral salts medium containing phosphorus-32-labeled orthophosphate results in rapid loss of isocitrate dehydrogenase activity and concomitant incorporation of phosphorus-32 into the enzyme. This is the first example of protein phosphorylation in a bacterium in which the endogenous substrate for the protein kinase has been identified.

Phosphorylation of Isocitrate dehydrogenase of Escherichia coli

The hok (host killing) and sok (suppressor of killing) genes (hok/sok) efficiently maintain the low-copy-number plasmid R1. To investigate whether the hok/sok locus evolved as a phage-exclusion mechanism, Escherichia coli cells that contain hok/sok on a pBR322-based plasmid were challenged with T1, T4, T5, T7, and lambda phage. Upon infection with T4, the optical density of cells containing hok/sok on a high-copy-number plasmid continued to increase whereas the optical density for those lacking hok/sok rapidly declined. The presence of hok/sok reduced the efficiency of plating of T4 by 42% and decreased the plaque size by approximately 85%. Single-step growth experiments demonstrated that hok/sok decreased the T4 burst size by 40%, increased the time to form mature phage (eclipse time) from 22 to 30 min, and increased the time to cell lysis (latent period) from 30 to 60 min. These results further suggest that single cells exhibit altruistic behavior.

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Exclusion of T4 phage by the hok/sok killer locus from plasmid R1.

Molecular Evolution of Multisubunit RNA Polymerases: Sequence Analysis

In this chapter I shall not try to review in detail the genetics and molecular biology of phage T7, since this topic has been covered repeatedly (Studier, 1972; Hausmann, 1976; Rabussay and Geiduschek, 1977; Studier and Dunn, 1983). Besides, Dunn and Studier (1983) have exhaustively analyzed the genome structure of T7, giving the full sequence of the 39,936 base pairs of its DNA and a complete survey of its 50-odd genes and its functions (where known), in addition to a full analysis of its signal sequences, such as the terminal repeats, promoters, terminators, processing sequences for RNase III, and origins of DNA replication. Rather, I shall here recapitulate some of the basic findings with regard to structure and life cycle of T7 in order to allow the reader to follow my thought with regard to the main problem I have in mind (but which I am unable to solve): How did this surprising biological entity come about? Unless we find satisfactory answers to the questions regarding origins and evolution of this phage, we cannot really claim to have understood it, notwithstanding the full grasp we might come to have of all molecular details of the workings of that self-replicating machine.

F. W. STUDIERt

Papers

Protein Kinase Induction in Escherichia coli by Bacteriophage T7 (early T7 protein/serylphosphate/UV mapping/ribosomal proteins)