Steven Ackerman

Mechanism of action of DRB‡: III. Effect on specific in vitro initiation of transcription

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole, an adenosine analogue, has been used previously as an inhibitor of heterogeneous nuclear and messenger RNA synthesis. In an in vitro transcriptional system, we have detected inhibition of synthesis of full-length runoff RNAs at concentrations at which in vivo mRNA synthesis is inhibited. By hybridization of RNA synthesized in vitro to single-stranded DNA and gel analysis, we were able to reduce the background of the transcription reaction, detect DRB-induced inhibition of full-length runoff RNAs and DRB-insensitive transcription of short RNAs. To establish further the effect of DRB on initiation of transcription, preincubation experiments with template, whole cell extract and two initial nucleotides of the transcript were performed. Elongation was then measured as discrete-sized RNAs transcribed from the truncated template after addition of the other triphosphates (one of them labeled), in the presence or absence of DRB. An effect on initiation but not on elongation or termination was detected. Fingerprint analysis of these runoff RNAs indicates that the labeling of U in the presence of DRB is uniform throughout the molecule. A model to explain a novel interpretation of the action of DRB is presented.

Partial purification of plant transcription factors. II. An in vitro transcription system is inefficient

Crude wheat germ nuclear extracts contain many inhibitors of transcription which need to be removed before an active system can be developed. Using ion exchange column chromatography to resolve RNA polymerase II transcription components we can identify at least four fractions required for transcription by their ability to interact with, or substitute for, particular HeLa fractions. Inhibitors can be removed by a second or third chromatographic process applied to each fraction. Two plant fractions can each effectively replace the corresponding fraction in a HeLa transcription system, and the wheat fractions can work together and replace two HeLa fractions. These plant factors chromatograph identically to HeLa factors on ion exchange columns. The third fraction does not fully substitute for the corresponding HeLa fraction, but can complement this HeLa fraction when both are added at half-optimal levels. An in vitro plant system consisting of four plant chromatographic fractions will selectively transcribe a gene, but only at very low efficiency. The apparent block to greater efficiency is in elongation of the RNA past the 20–30n size.

Coactivators and TAFs of transcription activation in wheat

Transcription regulation often activates quiescent genes in a tissue-specific or developmental manner. Activator proteins bind to a DNA sequence upstream of the promoter, interact with the general transcription proteins via bridging proteins, and elevate transcription levels. One group of bridging proteins, the coactivators, have been characterized in animals as polypeptides tightly associated with the general transcription factor TATA-binding protein (TBP). They are referred to as TAFs (TBP-associated factors), and together with TBP comprise general transcription factor IID. We provide biochemical evidence that wheat IID contains coactivators. An activator protein with an acidic activation domain facilitates the binding of IID to the template, and potentiates activated in vitro transcription with wheat IID, but not with wheat TBP. Using antibodies to wheat TBP, we demonstrate that wheat IID also contains TAFs. This is the first demonstration that a plant contains coactivators and TAFs.

Partial purification of plant transcription factors. I. Initiation.

Crude plant cell protein extracts prepared from wheat germ are inactive for in vitro transcription by RNA polymerase II. These extracts do, however, have correct initiation of transcription by RNA polymerase II. Initiation is monitored by measuring the formation of transcription complexes in vitro. A nuclear extract produces more initiation events than a whole cell extract or a cytosol extract. Some factors necessary for initiation can be separated from other proteins, including inhibitors, by ion exchange column chromatography. One specific fraction is sufficient for the formation of transcription complexes and several other fractions may be stimulatory or accessory factors.

A wheat-germ nuclear fraction required for selective initiation in vitro confers processivity to wheat-germ rna polymerase II

Abstract A protein fraction prepared from wheat-germ nuclear extracts, KB, which is required for selective transcription of class II plant genes, inhibits the single-step addition reaction of UTP to UpA dinucleoside monophosphate primer and the productive synthesis of poly[r(A-U)] chains catalysed by purified wheat-germ RNA polymerase II on a poly[d(A-T)] template. The extent of inhibition is strongly dependent upon the concentration of template DNA in the transcription assays. However, analysis of the transcription products by high-resolution gel electrophoresis reveals that in the presence of wheat-germ fraction KB, the RNA polymerase becomes much more processive than in its absence.

RNA polymerase II transcription complexes are destabilized by ATP or GTP

In vitro transcription by RNA polymerase II requires hydrolysis of the beta-gamma bond of ATP after the transcription complex forms, prior to RNA synthesis. It was observed that the presence of ATP during transcription complex formation inhibits subsequent transcription when the remaining 3 rNTPs are added. We now report that ATP or GTP inhibits transcription if either is present during transcription complex formation to added to preformed complexes. This inhibition is not due to purine rNTP degradation and occurs if as little as 2 mM ATP or 50 mM GTP is added to forming or preformed complexes. Deoxy derivatives of ATP inhibit similarly. AMP-PNP, a beta-gamma imido derivative, neither satisfies the energy requirement nor inhibits transcription if added to incubations of forming or of preformed transcription complexes.

In Vitro Transcription of Adenovirus Genes

Before the development of recombinant DNA technology, DNA tumor vi–ruses provided a convenient source of DNA for analysis of a limited number of genes in eukaryotic cells. The virus can be purified in large amounts and the DNA extracted from the virions is free of cellular DNA sequences. The lytic viral cycle affects cellular metabolism in a dramatic way, but does not alter the levels of cellular RNA polymerases I, II, and III or induce a virus-coded one (WEINMANN et al. 1976). The genetics of adenovirus was well developed, with deletion and temperature-sensitive mutants (For review see SHENK and WILLIAMS, Vol. ILL, (in press). The advent of restriction enzymes allowed the direct correlation of mutations with gene products and specific DNA regions. The transcribed regionds of the genome were first identified by hybridization kinetic analysis of RNA products. Combined with size analysis and hybridization to separated DNA strands, the polarities of the 3’ and 5’ ends of mRNAs were determined. At least half of the late-infected cell viral transcripts are derived from the major late promoter. A common undecanucleotide present at the 5’ end of all adenovirus structural protein mRNAs was the first suggestion for a common transcriptional start site (Gelinas and Roberts 1977).

Effects of Environmental Factors on DNA: Damage and Mutations

Chemicals in the environment pose myriad challenges to organisms, principally via toxicity or mutagenesis. Mutations are the result of changes in the DNA base sequence or the chemical addition of adducts onto the bases, which prevent correct DNA replication and/or transcription of the DNA into RNA. Additionally, spontaneous mutation of DNA bases can also occur by tautomerization, base deletions or additions, deamination, etc. Every human cell experiences about 10,000 “insults” every day, most of which are repaired by one of the multiple DNA repair systems of the cell. DNA replication itself results in a few base changes every cycle. Io protect itself from accumulating mutations, cells typically divide (mitosis) between 40 and 70 times (the so-called Hayflick number) before they undergo programmed cell death (apoptosis). Hence, it is the accumulation of mutations in the DNA due to chemicals in the environment that seems to be a causative agent of maladies, including cancer. (It is currently hypothesized that multiple DNA base changes, DNA rearrangements, etc., are necessary for the induction of cancer, which is uncontrolled and unregulated cell growth and division). Environmental chemicals that are linked to DNA damage include (but are not limited to) alkylating agents, Agent Orange, intercalating agents, dichlorodiphenyltrichloroethane, triclosan, and plasticizers (including polyvinyl chloride, phthalates, and bisphenol A and its derivatives). Some of the environmental chemicals may not cause DNA damage directly; they can cause epigenetic changes. This means that the DNA base sequence is not altered but the base is chemically modified so that the genetic information is expressed in a manner abnormal to correct cellular function. Moreover, it has become evident that posttranslational modifications of proteins associated with DNA (e.g., histones) can also lead to mutation via incorrect regulation of gene expression. Results of contemporary studies on animals (including human) have also indicated that epigenetic changes can occur in response to environmental distress (e.g., famine, toxic chemicals) and cause an ailment(s) in the individual due to altered gene expression, and that these epigenetic changes, altered gene expression, and ailment(s) are passed on to at least two and maybe three generations of offspring. The gene sequence, however, remains unchanged! This is referred to as transgenerational inheritance.

Transcription factor IIA of wheat and human interacts similarly with the adenovirus-2 major late promoter

Transcription factor IIA (TFIIA) is a necessary component of many RNA polymerase II transcription complexes. Assembly of the transcription complex begins when TFIIA interacts with the promoter. We have previously purified wheat germ TFIIA to homogeneity and demonstrated that it substitutes for human TFIIA in a human in vitro transcription system which utilizes the adenovirus-2 major late promoter (Ad-2 MLP). We now show, by gel retardation assays, that wheat TFIIA interacts with the Ad-2 MLP. Extensively purified human (HeLa) TFIIA interacts with the Ad-2 MLP similarly. Both wheat and human TFIIA interact with a DNA fragment comprising the minimal promoter region (-51/+32) but not with upstream or downstream regions. With both TFIIAs multiple complexes form; the fastest wheat TFIIA/DNA complex appears to be larger than the corresponding human TFIIA/DNA complex. Limited point mutation analysis of the Ad-2 MLP demonstrates that changes at -30 (TATAA region), +1, and -1 diminish TFIIA binding, but a change at -40 does not. DNA footprint analysis of this region is not definitive, but does indicate that following TFIIA binding there are changes in the pattern of hypersensitive sites.