Strippable coal resources of Illinois : part 7-Vermilion and Edgar Counties

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Architecture of the RNA polymerase II–TFIIF complex revealed by cross-linking and mass spectrometry

Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes

Stress-Induced C/EBP Homology Protein (CHOP) Represses MyoD Transcription to Delay Myoblast Differentiation

When mouse myoblasts or satellite cells differentiate in culture, the expression of myogenic regulatory factor, MyoD, is downregulated in a subset of cells that do not differentiate. The mechanism involved in the repression of MyoD expression remains largely unknown. Here we report that a stress-response pathway repressing MyoD transcription is transiently activated in mouse-derived C2C12 myoblasts growing under differentiation-promoting conditions. We show that phosphorylation of the α subunit of the translation initiation factor 2 (eIF2α) is followed by expression of C/EBP homology protein (CHOP) in some myoblasts. ShRNA-driven knockdown of CHOP expression caused earlier and more robust differentiation, whereas its constitutive expression delayed differentiation relative to wild type myoblasts. Cells expressing CHOP did not express the myogenic regulatory factors MyoD and myogenin. These results indicated that CHOP directly repressed the transcription of the MyoD gene. In support of this view, CHOP associated with upstream regulatory region of the MyoD gene and its activity reduced histone acetylation at the enhancer region of MyoD. CHOP interacted with histone deacetylase 1 (HDAC1) in cells. This protein complex may reduce histone acetylation when bound to MyoD regulatory regions. Overall, our results suggest that the activation of a stress pathway in myoblasts transiently downregulate the myogenic program

Transcriptional elongation by purified RNA polymerase II is blocked at the trans-activation-responsive region of human immunodeficiency virus type 1 in vitro

It has previously been shown that the human immunodeficiency virus type 1 (HIV-1) trans-activation-responsive region (TAR) is contained in a stem-loop RNA structure. Moreover, the interaction of the RNA secondary structure with Tat, the trans-activator protein, seems to play a role in activation of transcription initiation and in preventing transcription attenuation. In this work, we have studied the ability of the HIV-1 TAR stem-loop to act as a specific attenuation signal for highly purified RNA polymerase II. We developed an in vitro system using dC-tailed DNA fragments of HIV-1 to study transcriptional control in the HIV-1 LTR. We have found that transcription in this system yields an attenuator RNA whose 3' end maps to the end of the TAR stem-loop, approximately 60 to 65 nucleotides downstream of the in vivo initiation site. Furthermore, transcription attenuation occurs only under conditions which cause displacement of the nascent transcript from the template DNA strand, thus allowing the RNA to fold into secondary structure. Evidence is provided that the purified polymerase II indeed recognizes stable RNA secondary structure as an intrinsic attenuation signal. The existence of this signal in the TAR stem-loop suggests that in vivo an antiattenuation factor, probably Tat, alone or in combination with other factors, acts to relieve the elongation block at the HIV-1 attenuation site.</jats:p