Transcriptional control in the prereplicative phase of T4 development

Control of transcription is crucial for correct gene expression and orderly development. For many years, bacteriophage T4 has provided a simple model system to investigate mechanisms that regulate this process. Development of T4 requires the transcription of early, middle and late RNAs. Because T4 does not encode its own RNA polymerase, it must redirect the polymerase of its host, E. coli, to the correct class of genes at the correct time. T4 accomplishes this through the action of phage-encoded factors. Here I review recent studies investigating the transcription of T4 prereplicative genes, which are expressed as early and middle transcripts. Early RNAs are generated immediately after infection from T4 promoters that contain excellent recognition sequences for host polymerase. Consequently, the early promoters compete extremely well with host promoters for the available polymerase. T4 early promoter activity is further enhanced by the action of the T4 Alt protein, a component of the phage head that is injected into E. coli along with the phage DNA. Alt modifies Arg265 on one of the two α subunits of RNA polymerase. Although work with host promoters predicts that this modification should decrease promoter activity, transcription from some T4 early promoters increases when RNA polymerase is modified by Alt. Transcription of T4 middle genes begins about 1 minute after infection and proceeds by two pathways: 1) extension of early transcripts into downstream middle genes and 2) activation of T4 middle promoters through a process called sigma appropriation. In this activation, the T4 co-activator AsiA binds to Region 4 of σ70, the specificity subunit of RNA polymerase. This binding dramatically remodels this portion of σ70, which then allows the T4 activator MotA to also interact with σ70. In addition, AsiA restructuring of σ70 prevents Region 4 from forming its normal contacts with the -35 region of promoter DNA, which in turn allows MotA to interact with its DNA binding site, a MotA box, centered at the -30 region of middle promoter DNA. T4 sigma appropriation reveals how a specific domain within RNA polymerase can be remolded and then exploited to alter promoter specificity

The Bacteriophage T4 Inhibitor and Coactivator AsiA Inhibits Escherichia coli RNA Polymerase More Rapidly in the Absence of σ(70) Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ(70) and Core

The N-terminal region (region 1.1) of σ(70), the primary σ subunit of Escherichia coli RNA polymerase, is a negatively charged domain that affects the DNA binding properties of σ(70) regions 2 and 4. Region 1.1 prevents the interaction of free σ(70) with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of σ(70)-dependent transcription from promoters that require an interaction between σ(70) region 4 and the −35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with σ(70) region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that σ(70) region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter P(uvsX). However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with σ(70) that lacks region 1.1 is less stable than polymerase with full-length σ(70). Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free σ(70) and then the AsiA/σ(70) complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free σ(70) plus core, yielding more free σ(70) at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free σ(70). Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using an E. coli two-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free σ(70) arises through an indirect effect

Escherichia coli RNA Polymerase Recognition of a σ(70)-Dependent Promoter Requiring a −35 DNA Element and an Extended −10 TGn Motif

Escherichia coli σ(70)-dependent promoters have typically been characterized as either −10/−35 promoters, which have good matches to both the canonical −10 and the −35 sequences or as extended −10 promoters (TGn/−10 promoters), which have the TGn motif and an excellent match to the −10 consensus sequence. We report here an investigation of a promoter, P(minor), that has a nearly perfect match to the −35 sequence and has the TGn motif. However, P(minor) contains an extremely poor σ(70) −10 element. We demonstrate that P(minor) is active both in vivo and in vitro and that mutations in either the −35 or the TGn motif eliminate its activity. Mutation of the TGn motif can be compensated for by mutations that make the −10 element more canonical, thus converting the −35/TGn promoter to a −35/−10 promoter. Potassium permanganate footprinting on the nontemplate and template strands indicates that when polymerase is in a stable (open) complex with P(minor), the DNA is single stranded from positions −11 to +4. We also demonstrate that transcription from P(minor) incorporates nontemplated ribonucleoside triphosphates at the 5′ end of the P(minor) transcript, which results in an anomalous assignment for the start site when primer extension analysis is used. P(minor) represents one of the few −35/TGn promoters that have been characterized and serves as a model for investigating functional differences between these promoters and the better-characterized −10/−35 and extended −10 promoters used by E. coli RNA polymerase

A Mutation within the β Subunit of Escherichia coli RNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters ▿

During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. Middle genes, whose expression begins about 1 min postinfection, are transcribed both from the extension of early RNA into middle genes and by the activation of T4 middle promoters. Middle-promoter activation requires the T4 transcriptional activator MotA and coactivator AsiA, which are known to interact with σ70, the specificity subunit of RNA polymerase. T4 motA amber [motA(Am)] or asiA(Am) phage grows poorly in wild-type E. coli. However, previous work has found that T4 motA(Am)does not grow in the E. coli mutant strain TabG. We show here that the RNA polymerase in TabG contains two mutations within its β-subunit gene: rpoB(E835K) and rpoB(G1249D). We find that the G1249D mutation is responsible for restricting the growth of either T4 motA(Am)or asiA(Am) and for impairing transcription from MotA/AsiA-activated middle promoters in vivo. With one exception, transcription from tested T4 early promoters is either unaffected or, in some cases, even increases, and there is no significant growth phenotype for the rpoB(E835K G1249D) strain in the absence of T4 infection. In reported structures of thermophilic RNA polymerase, the G1249 residue is located immediately adjacent to a hydrophobic pocket, called the switch 3 loop. This loop is thought to aid in the separation of the RNA from the DNA-RNA hybrid as RNA enters the RNA exit channel. Our results suggest that the presence of MotA and AsiA may impair the function of this loop or that this portion of the β subunit may influence interactions among MotA, AsiA, and RNA polymerase

Neural modulation of temporal encoding, maintenance, and decision processes.

Time perception emerges from an interaction among multiple processes that are normally intertwined. Therefore, a challenge has been to disentangle timekeeping from other processes. Though the striatum has been implicated in interval timing, it also modulates nontemporal processes such as working memory. To distinguish these processes, we separated neural activation associated with encoding, working-memory maintenance, and decision phases of a time-perception task. We also asked whether neuronal processing of duration (i.e., pure tone) was distinct from the processing of identity (i.e., pitch perception) or sensorimotor features (i.e., control task). Striatal activation was greater when encoding the duration than the pitch or basic sensory features, which did not differentially engage the striatum. During the maintenance phase, striatal activation was similar for duration and pitch but at baseline in the control task. In the decision phase, a stepwise reduction in striatal activation was found across the 3 tasks, with activation greatest in the timing task and weakest in the control task. Task-related striatal activations in different cognitive phases were distinguished from those of the supplementary motor area, inferior frontal gyrus, thalamus, frontoparietal cortices, and the cerebellum. Our results were consistent with a model in which timing emerges from context-dependent corticostriatal interactions

Neural Modulation of Temporal Encoding, Maintenance, and Decision Processes

Time perception emerges from an interaction among multiple processes that are normally intertwined. Therefore, a challenge has been to disentangle timekeeping from other processes. Though the striatum has been implicated in interval timing, it also modulates nontemporal processes such as working memory. To distinguish these processes, we separated neural activation associated with encoding, working-memory maintenance, and decision phases of a time-perception task. We also asked whether neuronal processing of duration (i.e., pure tone) was distinct from the processing of identity (i.e., pitch perception) or sensorimotor features (i.e., control task). Striatal activation was greater when encoding the duration than the pitch or basic sensory features, which did not differentially engage the striatum. During the maintenance phase, striatal activation was similar for duration and pitch but at baseline in the control task. In the decision phase, a stepwise reduction in striatal activation was found across the 3 tasks, with activation greatest in the timing task and weakest in the control task. Task-related striatal activations in different cognitive phases were distinguished from those of the supplementary motor area, inferior frontal gyrus, thalamus, frontoparietal cortices, and the cerebellum. Our results were consistent with a model in which timing emerges from context-dependent corticostriatal interactions

Neural systems supporting timing and chronometric counting: an FMRI study.

At least two strategies are available to humans for estimating multisecond intervals. One depends on an interval timing system that is common to many species. The other is the language-based strategy of chronometric counting. These two strategies are easily distinguished by the psychophysical properties of their behavioral correlates: counting supports substantially more precise estimates than are possible using the more general interval timing system. The present study investigates the neural systems that underlie the execution of these different strategies. Eighteen adults reproduced a 16-s interval either by internally timing or covertly counting the duration. Comparison of counting and timing to a resting baseline suggested that these strategies engage some nonoverlapping neural systems. Counting, but not timing, strongly activated Broca's area, primary motor cortex in the mouth region, and right cerebellum, all of which are associated with internal speech. Counting also activated parts of the medial premotor circuit, including the putamen, supplementary motor area (SMA) proper, and cingulate motor area (CMA), that have been associated with reproducing isochronous and syncopated rhythms of elements lasting hundreds of milliseconds. During timing, only a portion of this circuit, the SMA proper and CMA, was engaged. Both timing and counting interfered with semantic processing during the resting state, evidenced by task-related decreases in the left inferior and middle frontal gyri, right superior frontal gyrus, left angular gyrus, and bilateral posterior cingulate cortex. This study suggests that counting activates a corticostriatal network associated with millisecond, rhythmic timing. In contrast, timing long durations without the benefit of linguistic strategies for subdividing counts reduces activity in this circuitry