Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1.

Tight binding of Gdown1 represses RNA polymerase II (Pol II) function in a manner that is reversed by Mediator, but the structural basis of these processes is unclear. Although Gdown1 is intrinsically disordered, its Pol II interacting domains were localized and shown to occlude transcription factor IIF (TFIIF) and transcription factor IIB (TFIIB) binding by perfect positioning on their Pol II interaction sites. Robust binding of Gdown1 to Pol II is established by cooperative interactions of a strong Pol II binding region and two weaker binding modulatory regions, thus providing a mechanism both for tight Pol II binding and transcription inhibition and for its reversal. In support of a physiological function for Gdown1 in transcription repression, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei of early Drosophila embryos but re-localizes to the cytoplasm during zygotic genome activation. Our study reveals a self-inactivation through Gdown1 binding as a unique mode of repression in Pol II function

Transcriptional Organization and In Vivo Role of the Escherichia coli rsd Gene, Encoding the Regulator of RNA Polymerase Sigma D

The regulator of sigma D (Rsd) was identified as an RNA polymerase � 70-associated protein in stationaryphase Escherichia coli with the inhibitory activity of � 70-dependent transcription in vitro (M. Jishage and A. Ishihama, Proc. Natl. Acad. Sci. USA 95:4953–4958, 1998). Primer extension analysis of rsd mRNA indicated the presence of two promoters, � S-dependent P1 and � 70-dependent P2 with the gearbox sequence. To get insight into the in vivo role of Rsd, the expression of a reporter gene fused to either the � 70-or � S-dependent promoter was analyzed in the absence of Rsd or the presence of overexpressed Rsd. In the rsd null mutant, the � 70- and � S-dependent gene expression was increased or decreased, respectively. On the other hand, the � 70-or � S-dependent transcription was reduced or enhanced, respectively, after overexpression of Rsd. The repression of the � S-dependent transcription in the rsd mutant is overcome by increased production of the � S subunit. Together these observations support the prediction that Rsd is involved in replacement of the RNA polymerase � subunit from � 70 to � S during the transition from exponential growth to the stationary phase. The survival of bacterial cells in various environments depends on their abilities to sense the external conditions and adopt their internal metabolic systems by turning on or off the expression of specific sets of genes (17). Bacteria employ severa

Nystrom T: Regulation of or factor competition by the alarmone ppGpp

. Similarly, an in vivo competition assay showed that the ability of both 32 and S to compete with 70 is diminished in cells lacking ppGpp. Consistently, the fraction of S and 32 bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of factors in accordance with cellular demands during physiological stress

Regulation of σ factor competition by the alarmone ppGpp

Many regulons controlled by alternative σ factors, including σ(S) and σ(32), are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of σ(S)-dependent promoters because underproduction of σ(70), specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-σ(70)) restored expression from σ(S)-dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type σ(70) to compete with σ(32) for core binding and the mutant σ(70) proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type σ(70). Similarly, an in vivo competition assay showed that the ability of both σ(32) and σ(S) to compete with σ(70) is diminished in cells lacking ppGpp. Consistently, the fraction of σ(S) and σ(32) bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of σ factors in accordance with cellular demands during physiological stress

Control of the Synthesis and Functions of RNAPolymerase Sigma Subunits in Escherichia coli

Selection of a σ subunit of RNA polymerase is a powerful mechanism for switching transcription of a group of genes. For understanding the regulation of σ subunit utilization in Escherichia coli, I determined the intracellular levels of four members among 8 known σ subunits of E. coli strains, W3110 and MC4100, at various growth phases by a quantitative Western immunoblot analysis. The level of σ70 is rather constant throughout the growth, and those of σ54 and σ28 are maintained at 10 and 50% the level of σ70 in both strains growing at exponential and stationary phases, respectively. At the exponential growth phase σ38 is undetectable but increases to up to 30% of σ70 at the stationary phase, supporting the concept that σ38 plays a key role in transcription of the stationary phase - expressed genes. Stress - coupled change in the intracellular level was observed for two σ subunits: an increase in σ38 level and a decrease in σ28 level upon exposure to heat shock at the exponential phase; and the increase in σ38 level under a high osmolality condition at both the exponential and the stationary phases. I also found that the composition of σ subunits is different between bacterial strains, even under the same strain name. For instance, the compositions of σ28 and σ38 differ among three W3110 strains. The growth transition from exponential to stationary phase is accompanied by the replacement of RNA polymerase - associated σ70 subunit with σ38. She found that the GST - fused σ70 in stationary - phase cell extracts exists as a complex with a new protein, and it was designated Rsd (Regulator of Sigma D). A gene located at 90 min on the E. coli chromosome encodes Rsd, and is transcribed from upstream and downstream promoters with σ38 and σ70 holoenzymes, respectively. Over - expressed and purified Rsd protein can form a complex with σ70 in vitro but not with any other σ subunits; σ54, σ38, σ32, σ28 and σ24. A binding assay with proteolytic fragments of σ70 suggested that Rsd interacts with the conserved region 4, the promoter - 35 recognition domain, and its downstream of σ70. The isolated Rsd inhibited transcription in vitro to various extents depending on the promoters used. The intracellular level of Rsd starts to increase during the transition from growing to stationary phase. Experiments using strains with disrupted rsd gene and those with overproduced Rsd indicated that Rsd is a negative factor for transcription directed by σ70 and a positive factor for that by σ38. The results indicate that Rsd is a stationary - phase E. coli protein regulating the activity of the σ70 function, providing an evidence for the existence of the control of the activities as well as the levels of the σ subunits of RNA polymerase, contributing to a group switching of genes ofE. coli. Taking all the observations together, she discussed possible models for the switching mechanism