Cloning and functional characterization of PTRF, a novel protein which induces dissociation of paused ternary transcription complexes.

Termination of transcription by RNA polymerase I (Pol I) is a two-step process which involves pausing of elongating transcription complexes and release of both pre-rRNA and Pol I from the template. In mouse, pausing of elongation complexes is mediated by the transcription termination factor TTF-I bound to the 'Sal box' terminator downstream of the rDNA transcription unit. Dissociation of paused ternary complexes requires a cellular factor, termed PTRF for Pol I and transcript release factor. Here we describe the molecular cloning of a cDNA corresponding to murine PTRF. Recombinant PTRF is capable of dissociating ternary Pol I transcription complexes in vitro as revealed by release of both Pol I and nascent transcripts from the template. Consistent with its function in transcription termination, PTRF interacts with both TTF-I and Pol I. Moreover, we demonstrate specific binding of PTRF to transcripts containing the 3' end of pre-rRNA. Substitution of 3'-terminal uridylates by guanine residues abolishes PTRF binding and impairs release activity. The results reveal a network of protein-protein and protein-nucleic acid interactions that governs termination of Pol I transcription.

RNA polymerase I transcription termination: similar mechanisms are employed by yeast and mammals.

Termination of RNA polymerase I (Pol I) transcription requires the interaction of a specific DNA binding factor with terminator elements downstream of the pre-rRNA coding region. Both the terminator elements and the respective termination factors are distinct in yeast and mammals, and differences in the mechanism of transcription termination have been postulated. We have compared in vitro transcription termination of yeast and mouse Pol I using both the murine factor TTF-I, and the yeast homolog Reb1p. We show that, similar to TTF-I, Reb1p was sufficient for pausing of Pol I from either species, but was unable to cause release of the nascent transcripts from the paused ternary complex. The deficiency of Reb1p to mediate transcript release from Pol I of either species was complemented by the recently characterized murine release factor. Thus, both yeast and mouse Pol I termination requires a trans-acting factor that, in conjunction with the T-rich flanking sequence, releases the transcripts and Pol I from the template. The observation that the murine factor causes dissociation of ternary transcription complexes arrested by Reb1p suggests that the mechanism of Pol I termination is highly conserved from yeast to mammals.

Identification of a transcript release activity acting on ternary transcription complexes containing murine RNA polymerase I.

Termination of mammalian ribosomal gene transcription by RNA polymerase I (Pol I) requires binding of the nucleolar factor TTF-I (transcription termination factor for Pol I) to specific rDNA terminator elements. We have used recombinant murine TTF-I in an immobilized tailed template assay to analyze individual steps of the termination reaction. We demonstrate that, besides the TTF-I-DNA complex which stops elongating Pol I, an additional activity is required to release both the nascent transcript and Pol I from the template. Moreover, transcript release, but not TTF-I-directed pausing, depends on upstream sequences directly flanking the terminator element. Together, complete termination of Pol I transcription requires TTF-I bound to the terminator DNA, a stretch of thymidine residues upstream of the TTF-I-mediated pause site and an activity which releases the RNA transcript and Pol I from the DNA template.

The amino-terminal domain of the transcription termination factor TTF-I causes protein oligomerization and inhibition of DNA binding.

The transcription termination factor TTF-I binds specifically to an 18 bp DNA element in the murine ribosomal gene spacer and mediates termination of RNA polymerase I transcription. In this study, we have compared DNA binding and termination activity of recombinant full-length TTF-I (TTF-Ip130) with two deletion mutants lacking 184 and 322 N-terminal amino acids, respectively. All three proteins exhibit similar termination activity, but the DNA binding of TTF-Ip130 is at least one order of magnitude lower than that of the deletion mutants, indicating that the N-terminus represses the interaction of TTF-I with DNA. The inhibitory effect of the N-terminus can be transferred to a heterologous DNA binding domain and is separable from other activities of TTF-I. We show by several methods that TTF-Ip130, the N-terminal domain alone, and fusions of the N-terminus with the DNA binding domain of Oct2.2 form stable oligomers in solution. Thus, in contrast to previous studies suggesting that activation of TTF-I occurs through proteolysis, we demonstrate that full-length TTF-I mediates termination of rDNA transcription in vivo and in vitro and that the oligomerization state of TTF-I may influence its DNA binding activity.

Effect of radical scavengers and hyperbaric oxygen on smoke-induced pulmonary edema.

Respiratory complications, especially pulmonary edema, account for over 50% of mortalities in inhalation injuries. This study was conducted to determine the effect of free radical scavengers and hyperbaric oxygen (HBO) in vivo on reducing pulmonary edema. Adult New Zealand rabbits were allowed to breath cooled, cotton smoke until a significant inhalation lung injury was produced. Five percent of body weight lactated Ringer's solution was then administered i.v. over 2 h. The following free radical scavengers were given as bolus infusions at the beginning of fluids resuscitation: superoxide dismutase, catalase, butylated hydroxytoluene/piperonyl butoxide, and mannitol. At the completion of fluid administration, half of the subjects were given HBO treatment. Pulmonary edema was then measured as extravascular lung water and wet/dry lung weight. Results indicate that free radical scavengers or HBO reduce pulmonary edema. Free radical scavengers in conjunction with HBO showed no significant improvement over HBO or free radical scavengers alone.

Transcriptional antitermination.

Antiterminator proteins control gene expression by recognizing control signals near the promoter and preventing transcriptional termination which would otherwise occur at sites that may be a long way downstream. The N protein of bacteriophage lambda recognizes a sequence in the nascent RNA, and modifies RNA polymerase by catalysing the formation of a stable ribonucleoprotein complex on its surface, whereas the lambda Q protein recognizes a sequence in the DNA. These mechanisms of antitermination in lambda provide models for analysing antitermination in viruses such as HIV-1 and in eukaryotic genes.

Elongation factor NusG interacts with termination factor rho to regulate termination and antitermination of transcription.

NusG is a transcriptional elongation factor in Escherichia coli that aids transcriptional antitermination by the phage lambda N protein. By using NusG affinity chromatography, we found that NusG binds directly and selectively to termination factor rho. NusG was shown previously to be needed for termination by rho in vivo, and we show here that NusG increases the efficiency of termination by rho at promoter-proximal sites in vitro. The rho026 mutation makes termination by rho less dependent on NusG. It also makes antitermination by N at rho-dependent terminators and the binding of rho to NusG temperature sensitive. Therefore, the interaction of NusG with rho is important both for rho-dependent termination and for antitermination by N at rho-dependent terminators.

Host factor requirements for processive antitermination of transcription and suppression of pausing by the N protein of bacteriophage lambda.

The N protein of phage lambda prevents termination of transcription by Escherichia coli RNA polymerase at Rho-dependent and -independent terminators in the lambda early operons. The modification of RNA polymerase by N requires an N-utilization (nut) site, present in each lambda early operon, and involves the E. coli factors NusA, NusB, NusG, and ribosomal protein S10. We show that, in the presence of NusA, N inhibits pausing by RNA polymerase and Rho-dependent termination in vitro at three sites in the lambda terminator tR1 which are located less than 100 base pairs downstream from nutR. NusA is also sufficient for partial antitermination at sites located farther downstream from nutL and nutR if there is a high concentration of N in the reaction. At low concentrations of N, the additional factors NusB, S10, and NusG are essential for antitermination at distal sites. In these conditions, the presence of NusA, NusB, S10, and NusG in the reaction enables N-modified RNA polymerase to elongate efficiently and processively through Rho-dependent and -independent terminators over distances as great as 7 kilobases downstream from the lambda nut sites. This substantial processivity of antitermination in vitro also occurs in vivo and probably reflects the stable association of N, NusA, NusB, S10, and NusG with RNA polymerase and nut site RNA in elongation complexes transcribing the lambda chromosome.

Direct interaction between two Escherichia coli transcription antitermination factors, NusB and ribosomal protein S10.

The Escherichia coli proteins NusB and ribosomal protein S10 are important for transcription antitermination by the bacteriophage lambda N protein. We have used sucrose gradient co-sedimentation and affinity chromatography with immobilized ribosomal protein S10, a glutathione S-transferase-S10 fusion protein, and NusB to show that NusB binds directly and very selectively to S10. The interaction is non-ionic and has an estimated Kd value of 10(-7) M. We hypothesize that NusB binds to N-modified transcription complexes primarily by interacting with S10.

Assembly of transcription elongation complexes containing the N protein of phage lambda and the Escherichia coli elongation factors NusA, NusB, NusG, and S10.

The transcription antitermination protein, N, of bacteriophage lambda; the Escherichia coli elongation factors NusA, NusB, ribosomal protein S10, and NusG; and a DNA template containing a lambda nut (N-ututilization) site are necessary and sufficient for the highly cooperative formation in vitro of stable transcription complexes containing all five elongation factors. Mutations in the nut site, NusA, or the beta-subunit of RNA polymerase (RNAP) that impair antitermination in vivo also abolish the assembly of a stable complex containing the antitermination factors in vitro. The effects of RNAP mutations on assembly imply that the antitermination factors assemble on the surface of RNAP. We have shown previously that NusA binds directly to transcribing RNAP (Ka approximately 10(7) M-1); Ka = association constant and we show here that S10 also binds directly and specifically to RNAP with an apparent Ka of 10(6) M-1. These observations led to a model for the ordered assembly of the N-modified transcription complex.

Effects of ibuprofen on pulmonary oedema in an animal smoke inhalation model.

The ability of ibuprofen to lower extravascular lung water significantly was examined in an animal smoke inhalation model. Adult New Zealand White rabbits weighing 3-5 kg were anaesthetized and intubated. They were then allowed to breathe cooled cotton smoke until the carboxyhaemoglobin (COHb) reached a level of 60 per cent or higher. Each ibuprofen-treated animal received a dose of 50 mg/kg either intraperitoneally or intravenously. Ibuprofen was administered to animals that received smoke inhalation alone and those that received smoke inhalation combined with a 10 per cent BSA partial skin thickness thermal injury. Control groups were established for both of the above-mentioned groups. Peak carboxyhaemoglobin levels as well as CO half-lives were not significantly different between ibuprofen-treated groups and the controls. Ibuprofen treatment resulted in significantly (P less than 0.05) decreased lung water in both smoke, and smoke plus thermal injury groups as compared to controls. These results suggest that ibuprofen promotes the reduction of early-onset lung water resulting from smoke inhalation injury alone or from smoke inhalation injury plus a thermal injury.