J. Barry Egan

Systematic method for the detection of potential λ Cro-like DNA-binding regions in proteins

We have developed and tested a systematic method for the location and statistical evaluation of potential DNA-binding regions of the lambda Cro type in protein sequences. Using this approach to examine proteins expected to contain such regions, we have been able to compile a statistically homogeneous master set of 37 lambda Cro-like DNA-binding domains. Examination of a protein database revealed other prokaryotic proteins that are similar to this lambda Cro-like group. There are also many DNA-binding proteins that are not found to be significantly similar to the lambda Cro group, consistent with previous suggestions that different types of protein sequence may be able to achieve a similar mode of binding and that there exist other modes of sequence-specific DNA-binding. A useful feature of the method is that it can be applied without a computer.

Potent transcriptional interference by pausing of RNA polymerases over a downstream promoter

Elongating RNA polymerases (RNAPs) can interfere with transcription from downstream promoters by inhibiting DNA binding by RNAP and activators. However, combining quantitative measurement with mathematical modeling, we show that simple RNAP elongation cannot produce the strong asymmetric interference observed between a natural face-to-face promoter pair in bacteriophage lambda. Pausing of elongating polymerases over the RNAP-binding site of the downstream promoter is demonstrated in vivo and is shown by modeling to account for the increased interference. The model successfully predicts the effects on interference of treatments increasing or reducing pausing. Gene regulation by pausing-enhanced occlusion provides a general and potentially widespread mechanism by which even weak converging or tandem transcription, either coding or noncoding, can bring about strong in cis repression.

Transcriptional interference by RNA polymerase pausing and dislodgement of transcription factors.

Transcriptional interference is the in cis suppression of one transcriptional process by another. Mathematical modeling shows that promoter occlusion by elongating RNA polymerases cannot produce strong interference. Interference may instead be generated by (1) dislodgement of slow-to-assemble pre-initiation complexes and transcription factors and (2) prolonged occlusion by paused RNA polymerases.

Control of gene expression in the P2-related template coliphages. III: DNA sequence of the major control region of phage 186

The PstI fragment (65.5% to 77.4%) of coliphage 186, known genetically to encode the major control genes, has been sequenced, and an analysis performed to assess coding capacity, transcription-translation signals, and to identify any other significant features. Our analysis indicates that the region encodes: seven genes, including the int and cI genes, which overlap, the late control gene B, and two genes, named CP75 and CP76, encoding potential DNA-binding proteins; a promoter pB and terminator tB for the rightward transcription of the B gene, and we predict the existence of this transcript in a lysogen; a promoter pL and terminator tL for leftward transcription that encodes the int and cI genes, and represents the presumed lysogenic transcript; a promoter pR for rightward transcription to give the presumed (early) lytic transcript that is overlapping and convergent with the lysogenic transcript; and finally, a potential operator site for repressor binding in the region of the pR promoter. Preliminary evidence is presented to support this analysis.

Characterization of the DNA from bacteriophage P2-186 hybrids and physical mapping of the 186 chromosome

SummaryThe DNA from two P2-186 hybrid phages and three 186 insertion mutants have been characterized by heteroduplex analysis and denaturation mapping. The results allow the orientation of the physical and genetic maps of bacteriophage 186 DNA and put physical limits on the chromosomal locations of the phage attachment sites, immunity genes and tail genes.

The Cro‐like Apl repressor of coliphage 186 is required for prophage excision and binds near the phage attachment site

The Apl protein of the temperate coliphage 186 represses transcription of the immunity repressor gene and down‐regulates lytic transcription. It is shown here that an apl‐ mutant is competent for lytic development and establishes lysogeny normally but is defective in excision of the prophage. The Apl protein binds between the lytic and lysogenic promoters and also near the phage attachment site, suggesting that its role in excision is direct. Apl thus appears to act as an excisionase as well as a repressor. The pattern of Apl‐induced DNase I enhancements indicates that the DNA is bent by Apl. Potential Apl recognition sequences are identified; these sequences are directly repeated several times across each binding region and are spaced 10 or 11 bases apart, suggesting that Apl binds to one face of the DNA helix.

Functional Alignment of Regulatory Networks: A Study of Temperate Phages

The relationship between the design and functionality of molecular networks is now a key issue in biology. Comparison of regulatory networks performing similar tasks can provide insights into how network architecture is constrained by the functions it directs. Here, we discuss methods of network comparison based on network architecture and signaling logic. Introducing local and global signaling scores for the difference between two networks, we quantify similarities between evolutionarily closely and distantly related bacteriophages. Despite the large evolutionary separation between phage λ and 186, their networks are found to be similar when difference is measured in terms of global signaling. We finally discuss how network alignment can be used to pinpoint protein similarities viewed from the network perspective.

The dual role of Apl in prophage induction of coliphage 186

In the present study we show that the Apl protein of the temperate coliphage 186 combines, in one protein, the activities of the coliphage lambda proteins Cro and Xis. We have shown previously that Apl represses both the lysogenic promoter, pL, and the major lytic promoter, pR, and is required for excision of the prophage. Apl binds at two locations on the phage chromosome, i.e. between pR and pL and at the phage‐attachment site. Using an in vivo recombination assay, we now show that the role of ApI in excision is in the process itself and is not simply a consequence of repression of pR or pL. To study the repressive role of Apl at the switch promoters we isolated Apl‐resistant operator mutants and used them to demonstrate a requirement for Apl in the efficient derepression of the lysogenic promoter during prophage induction. We conclude that Apl is both an excisionase and transcriptional repressor.

Isolation of phage P2-186 intervarietal hybrids and 186 insertion mutants.

SummaryIntervarietal hybrids formed between coliphages P2 and 186 have been isolated and their preliminary genetic characterization described. Three insertion mutants of 186 have also been isolated.

Control of gene expression in the P2-related temperate coliphages: IV. Concerning the late control gene and control of its transcription

In this paper we have sequenced four amber mutants and thereby confirmed the gene D (CP65) and gene B (CP67) assignments made in the accompanying paper (Kalionis et al., 1986). We have also studied, by gel electrophoresis, the transcription patterns of gene B in vivo. In a lysogen, gene B is present on a short transcript under autogenous (negative) control. Upon prophage induction, this transcript is amplified, but later in the cycle gene B is present on a larger transcript that originates in the late region. We have detected two copies of an inverted repeat in the promoter region of the B gene that we predict is recognized by the B protein. One arm of this repeat is associated with three of four P2 late promoters, downstream from the start point of transcription. The repeat is not present in the promoter region of P2 ogr. We describe the considerable homology in amino acid sequence seen with the late control proteins 186 gpB, P4 gp delta and P2 gpOgr, and present a working model for control of late gene transcription.