The relationship between the general transcription factor TFIIF and Gdown1 in promoter proximal pausing and the properties of TFIIFs truncated versions 1-217 and 1-356 within that relationship

RNA Polymerase II (Pol II) requires several additional factors, known as general transcription factors (GTFs), to recognise promoters and initiate the synthesis of pre-mRNA. It is now appreciated that a post-initiation step, involving extended pausing of Pol II at ~50 base pairs downstream of transcription start, is also a key regulatory step in gene expression. It is not well understood how the GTFs relate to the factors that control this pausing. Of particular interest, is the relationship between one GTF, TFIIF, and one of the pausing factors, Gdown1. Gdown1 associates very tightly with Pol II and it can be thought of as a 13th, substoichiometric subunit of polymerase. Surprisingly, Gdown1 shares binding sites on Pol II with TFIIF, blocks TFIIF association with free Pol II, and displaced TFIIF from Pol II during transcript elongation. An important question thus arises: How can transcription begin if a factor necessary in pausing blocks the association of a factor necessary for transcription to start? Here I show, using a well-characterized in vitro transcription system: 1) that once a pol II pre-initiation complex (PIC) is formed, Gdown1 cannot displace TFIIF from it and 2) that the truncated versions of TFIIF are less effective at rescuing transcription than full length TFIIF

An 8 nt RNA triggers a rate-limiting shift of RNA polymerase II complexes into elongation

To better understand the critical conversions that RNA polymerase II complexes undergo during promoter escape, we determined in vitro the precise positions of the rate-limiting step and the last step requiring negative superhelicity or TFIIE and TFIIH. We found that both steps occur after synthesis of an 8 nt RNA during the stage encompassing translocation of the polymerase active site to the ninth register. When added to reactions just before this step, TFIIE and TFIIH overcame the requirement for negative superhelicity. The positions at which both steps occur were strictly dependent on RNA length as opposed to the location of the polymerase relative to promoter elements, showing that the transcript itself controls transformations during promoter escape. We propose a model in which completion of promoter escape involves a rate-limiting conversion of early transcribing complexes into elongation complexes. This transformation is triggered by synthesis of an 8 nt RNA, occurs independent of the general transcription factors, and requires under-winding in the DNA template via negative superhelicity or the action of TFIIE and TFIIH

Ethanol sensitizes skeletal muscle to ammonia-induced molecular perturbations.

Ethanol causes dysregulated muscle protein homeostasis while simultaneously causing hepatocyte injury. Since hepatocytes are the primary site for physiological disposal of ammonia, a cytotoxic cellular metabolite generated during a number of metabolic processes, we determined if hyperammonemia aggravates ethanol-induced muscle loss. Differentiated murine C2C12 myotubes, skeletal muscle from pair-fed or ethanol-treated mice and human patients with alcoholic cirrhosis and healthy controls were used to quantify protein synthesis, mTORC1 signaling and autophagy makers. Alcohol metabolizing enzyme expression and activity in mouse muscle and myotubes and ureagenesis in hepatocytes were quantified. Expression and regulation of the ammonia transporters, RhBG and RhCG, were quantified by real time PCR, immunoblots, reporter assays, biotin tagged promoter pulldown with proteomics, and loss of function studies. Alcohol and aldehyde dehydrogenases were expressed and active in myotubes. Ethanol exposure impaired hepatocyte ureagenesis, induced muscle RhBG expression and elevated muscle ammonia concentrations. Simultaneous ethanol and ammonia treatment impaired protein synthesis and mTORC1 signaling and increased autophagy with consequent decreased myotube diameter to a greater extent than either treatment alone. Ethanol treatment and withdrawal followed by ammonia exposure resulted in greater impairment in muscle signaling and protein synthesis than ammonia treatment in ethanol-naïve myotubes. Of the 3 transcription factors that were bound to the RhBG promoter in response to ethanol and ammonia, DR1/NC2 indirectly regulated transcription of RhBG during ethanol and ammonia treatment. Direct effects of ethanol was synergistic with increased ammonia uptake in causing dysregulated skeletal muscle proteostasis and signaling perturbations with a more severe sarcopenic phenotype.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

RNA polymerase is poised for activation across the genome

Regulation of gene expression is integral to the development and survival of all organisms. Transcription begins with the assembly of a pre-initiation complex at the gene promoter1, followed by initiation of RNA synthesis and the transition to productive elongation2–4. In many cases, recruitment of RNA polymerase II (Pol II) to a promoter is necessary and sufficient for activation of genes. However, there are a few notable exceptions to this paradigm, including heat shock genes and several proto-oncogenes, whose expression is attenuated by regulated stalling of polymerase elongation within the promoter-proximal region5–13. To determine the importance of polymerase stalling for transcription regulation, we carried out a genome-wide search for Drosophila melanogaster genes with Pol II stalled within the promoter-proximal region. Our data show that stalling is widespread, occurring at hundreds of genes that respond to stimuli and developmental signals. This finding indicates a role for regulation of polymerase elongation in the transcriptional responses to dynamic environmental and developmental cues