I I An RNA Polymerase-Binding Protein That Is Required for Communication Between an Enhancer and a Promoter DANIEL R. HERENDEEN, KELLY P. WILLIAMS, GEORGE A. KASSAVETIS, E. PETER GEIDUSCHEK Although bacteriophage T4 late promoters are selectively recognized by Escherichia coli RNA polymerase bearing a single protein encoded by T4 gene 55 (gp55), efficient transcription at these promoters requires enhancement by the three T4 DNA polymerase accessory proteins, bound to distal "mobile enhancer" sites. Two principles are shown to govern this transcriptional enhancement: (i) Promoter recognition and communication between the enhancer and the promoter require separate phage-coded proteins. Only RNA polymerase that has the T4 gene 33 protein (gp33) bound to it is subject to enhancement by the three DNA replication proteins. (ii) Transcriptional enhancement in this prokaryotic system is promoter- specific. Promoter specificity is generated by a direct competition of phage T4 gp33 and gp55 with the E. coli promoter recognition protein, cr70, for binding to the E. coli RNA polymerase core. Thus, polymerase that con- tains Cr70 is competent to transcribe T4 early and middle genes, but lacks the ability to be enhanced by the DNA replication proteins, while polymerase that contains gp55 and gp33 is capable of enhancement via gp33, but its activity is restricted to T4 late promoters by gp55. W HEN VIRUSES MULTIPLY IN THEIR HOSTS, THEY FRE- quently withhold the expression of certain genes, particu- larly those that code for proteins of the mature virus particle, until a late stage of the multiplication cycle. In certain cases, expression of such genes depends at least in some measure on viral genome replication. These relationships were first uncovered through studies of the bacteriophage T4 (1), but other examples of replication-dependent gene expression are known and involve not only bacterial viruses but also such diverse animal viruses as SV40, adenovirus, and herpes simplex virus 1 (2). Our analysis of a mechanism for connecting DNA replication with regulated gene expression focuses on the late genes of bacteri- ophage T4. These genes, which cover approximately 40 percent of the viral genome, are turned on just after the start of viral DNA replication, and their expression diminishes if ongoing DNA repli- 4 MAY I990 cation is shut down (3, 4). In a recently devised in vitro system for analyzing this connection between replication and late gene expres- sion, three T4 DNA polymerase accessory proteins that are encoded by T4 genes 44, 62, and 45 (gp44, gp62, and gp45) have been shown to stimulate initiation of transcription at a T4 late promoter (5). These three replication proteins bind to DNA primer-template junctions (6) and have a DNA-dependent adenosine triphosphatase (ATPase) activity (7). They serve as the sliding clamp of the DNA polymerase, and greatly increase the processivity of DNA chain elongation by that enzyme (8). Transcriptional activation by the three DNA polymerase accessory proteins requires a DNA-binding site that acts like an enhancer, since it can be located at considerable distance upstream or downstream from a promoter. The enhancer and the proteins that bind to it activate T4 late transcription by increasing the rate of promoter opening (5). In our experiments, the enhancer is a simple break in the DNA, to which the activating proteins bind; in other words, it is a structure rather than a specific sequence. We have proposed that, in vivo, it is the viral DNA replication fork that acts as the "mobile enhancer" of T4 late gene transcription and that the three DNA polymerase accessory proteins are part of the replisome when they activate transcription (5). In contrast to the complexity of transcriptional enhancement, late promoter recognition can be executed in vitro by Escherichia coli RNA polymerase core supplemented only with the RNA polymer- ase-binding protein encoded by T4 gene 55 (gp55) (9, 10), a member of the a family of proteins (11). Late promoters are extremely simple, consisting only of TATAAATA located approxi- mately 10 base pairs (bp) upstream of transcriptional start sites. The recognition sequences that are located 16 to 18 bp further upstream in most E. coli and other prokaryotic promoters are lacking in T4 late transcription units (12, 13). There are indications for the existence of one further level of complexity in this system of transcriptional activation. RNA poly- merase from T4-infected cells supplemented with the T4 late promoter recognition-specific gp55 can be transcriptionally en- hanced in vitro, but RNA polymerase core from uninfected E. coli, also supplemented with gp55, cannot (5). The host's RNA polymer- ase undergoes multiple covalent and noncovalent modifications after infection by phage T4 (14); the above-cited evidence implies that one of these modifications is required for interaction with the transcription-enhancing proteins. We have now identified the small RNA polymerase-binding protein encoded by T4 gene 33 (gp33) as the required component: gp33 is the link between replication proteins bound at the enhancer and the transcription initiation RESEARCH ARTICLE 573 The authors are in the Department of Biology and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093. on November 28, 2012 www.sciencemag.org Downloaded from