Quantitation of RNA Polymerase II and Its Transcription Factors in an HeLa Cell: Little Soluble Holoenzyme but Significant Amounts of Polymerases Attached to the Nuclear Substructure

Abstract

Various complexes that contain the core subunits of RNA polymerase II associated with different transcription factors have been isolated from eukaryotes; their precise molecular constitution depends on the purification procedure. We estimated the numbers of various components of such complexes in an HeLa cell by quantitative immunoblotting. The cells were lysed with saponin in a physiological buffer; ϳ140,000 unengaged polymerases (mainly of form IIA) were released. Only ϳ4,000 of these soluble molecules sedimented in glycerol gradients as holoenzyme-sized complexes. About 180,000 molecules of polymerases (ϳ110,000 molecules of form IIO) and 10,000 to 30,000 molecules of each of TFIIB, TFIIE␣, TFIIE␤, TFIIF-RAP74, TFIIF-RAP30, and TFIIH-MAT1 remained tightly associated with the nuclear substructure. Most proteins and run-on activity were retained when ϳ50% of the chromatin was detached with a nuclease, but ϳ45,000 molecules of bound TATA binding protein (TBP) were detached. Similar results were obtained after cross-linking living cells with formaldehyde. The results provide little support for the existence of a large pool of soluble holoenzyme; they are consistent with TBP-promoter complexes in nuclease-sensitive chromatin being assembled into preinitiation complexes attached to the underlying structure.

A Conserved Organization of Transcription during Embryonic Stem Cell Differentiation and in Cells with High C Value

“…Band intensities given by two samples of differentiated F9 cells (inevitably a mixed population; Figure 1A) were roughly one-half those given by two samples of undifferentiated cells ( Figure 2B; compare lanes 5 and 6 with 7 and 8). To convert relative numbers to absolute numbers, band intensities were compared with those given by HeLa cells ( Figure 2B, lanes 1-4), which are known to contain ϳ65,000 elongating molecules of polymerase II (Kimura et al, 1999). Quantitative analysis of several blots showed the number of active molecules in F9 and ES cells fell by 40 -50% as they differentiate ( Figure 2C).…”

“…Cells were lysed (10 min; 4°C) in 10 mM Tris-HCl, pH 8.0, 0.5 M NaCl, and 1% Triton X-100; proteins were resolved on SDS-polyacrylamide gels (5-15% for Figure 2A, 6% for Figure 2B; Sambrook et al, 1989) and blotted onto membranes; antigens were detected (Kimura et al, 1999) using an enhanced chemiluminescence kit (GE Healthcare); digital images were collected; and band intensities were measured using Photoshop (Adobe Systems, Mountain View, CA). Primary antibodies were 1) mouse monoclonal anti-Oct-4 (2 g/ml; Santa Cruz Biotechnology, Santa Cruz, CA), 2) goat anti-secreted protein which is acidic and rich in cysteines (SPARC) (2 g/ml; R&D Systems, Minneapolis, MN), 3) rabbit anti-histone H3 (COOH-terminal 130 -135 peptide; 1/1000 -1/3000; Stemmer et al, 1997), and 4) mouse monoclonal antipolymerase (7C2; 1/10,000 dilution; Besse et al, 1995).…”

Isolation of the protein and RNA content of active sites of transcription from mammalian cells

“…First, active enzymes represent only a quarter of the total; the remaining majority form a rapidly-diffusing soluble pool that tends to aggregate in non-isotonic buffers 8,10,11 . Therefore, we use isotonic conditions whilst removing the inactive fraction.…”

“…Therefore, we use isotonic conditions whilst removing the inactive fraction. Second, engaged polymerases plus their templates and transcripts are housed in factories bound to the underlying nuclear scaffold 8,[10][11][12] .…”

Intranuclear Trafficking: Organization and Assembly of Regulatory Machinery for Combinatorial Biological Control

“…Similarly, newly transcribed RNA is exported out of the nucleus in a stringently regulated manner (18 -20). Thus, the nuclear membrane controls the flow of regulatory information between the two compartments.Compartmentalization of nuclear regulatory complexes is illustrated by focal organization of promyelocytic leukemia (PML) 1 bodies (21), Runx (Runt-related factor)/acute myelogenous leukemia (AML)/Cbfa (core binding factor ␣) domains (2, 6, 22), the nucleolus and chromosomes (5), as well as by the punctate intranuclear distribution of sites for replication (23-25), DNA repair (26), transcription (25,(27)(28)(29)(30)(31)(32)(33)(34)(35), steroid and polypeptide modulation of gene expression, and the processing of gene transcripts (36 -38). It is necessary to design experiments that define mechanisms that direct genes and regulatory factors to sites within the nucleus where localization integrates regulatory parameters of gene expression and establishes microenvironments with boundaries between regulatory complexes that are required for fidelity of activity.…”