Netropsin specifically enhances RNA polymerase II termination at terminator sites in vitro.

Abstract

We describe an in vitro system that emulates the specific and efficient transcriptional termination associated with the human gastrin gene terminator in vivo. The system involves a dC-tailed DNA template containing the gastrin gene terminator sequence, purified RNA polymerase II, and purified elongation factor TFIIS. In this system, the basal level of termination by RNA polymerase II at the gastrin gene terminator is specifically enhanced by netropsin, an (A+T)-rich minor groove-binding peptide. This enhanced termination is maintained even with TFUS, which normally suppresses termination at this site. In vitro termination is terminator sequence-specific. Mutant sequences that reduce or abolish termination in vivo show corresponding reductions in activity in the in vitro system. This in vitro emulation of in vivo activities of wild-type and mutant terminators strongly suggests that netropsin and a putative termination factor may share some aspects of their biochemical mechanisms. The general applicability of this system to the study of RNA polymerase II elongation and termination is suggested by the enhancement of termination seen at both the gastrin and human histone H3.3 gene terminators.In eukaryotes, the 3' ends of mRNAs are generally formed by posttranscriptional processing events, such as addition of a poly(A) tail and removal of a 3' extended region from a specific RNA secondary structure. Thus the mature 3' termini do not correspond to the position of the DNA template where the RNA polymerase II (pol II) actually ceases RNA synthesis (1,2). pol II is known to synthesize RNA well beyond such processing sites [except in situations where it encounters premature termination sites (3, 4)], usually terminating at sites that lie between 100 and 4000 base pairs (bp) downstream of the processing site(s) (1, 2). Several biochemical pathways may regulate the termination process. For instance, termination in the globin gene depends on a functional poly(A) addition signal (5), whereas the U2 RNA gene requires elements within the U2 promoter itself (6). A terminator that is functional in any sequence context, regardless of its promoter or poly(A) signal elements, is found downstream of the poly(A) addition signal in the human gastrin gene (7,8). This apparent diversity in termination mechanisms in eukaryotes is reminiscent of the alternative means of termination in prokaryotes. Indeed, the biochemical complexity of termination pathways in eukaryotes may exceed that of their prokaryotic counterparts.Based on available information, termination signals can be broadly classified into two categories: signals within or near regions with stable RNA secondary structures (9, 10) and signals that are rich in adenosine in their transcribed strand but that lack significant RNA secondary structure (7, 11). Studies in this laboratory have focused on the gastrin gene terminator (gt), which belongs to the deoxyadenosine-rich class of terminators (7). This terminator is highly effective in vivo (7) but weakly functional in an i...

Sequence-Specific DNA Minor Groove Binders. Design and Synthesis of Netropsin and Distamycin Analogues

“…Computational studies have contributed information that provides a rational explanation for the selective fit of these crescent-shaped ligands into the minor groove of DNA (26,27). Thermodynamic studies of Net and Dst binding to DNA, and their effects on the formation of protein/DNA complexes, have also been thoroughly investigated (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). Since the pioneer discoveries of the AT-selective binding of Net and Dst to DNA (41), a considerable number of physicochemical, biochemical, and biological studies have been reported to increase ous understanding of the details of the mechanisms by which these two antibiotics bind to and recognize double-stranded DNA squence (42).…”

Improved Mammalian Expression Systems by Manipulating Transcriptional Termination Regions

“…The 3′‐end region of the human gastrin gene (GeneBank accession no. M12929) has been extensively studied (7 − 9), and the positions of three important DNA elements (polyadenylation signal, cleavage site, and transcriptional terminator) were identified (Figure 1A). To examine the effects of the transcriptional terminator element on the expression of the cloned genes, a series of expression plasmids were constructed using the commercially available vector, pSV‐β‐gal, as a basic vector (Figure 1B; see Materials and Methods) and transfected into HeLa cells.…”

“…However, little information is available on the transcriptional terminator elements where RNA polymerases actually stop synthesizing mRNAs. Although a consensus sequence has been identified in the terminal region of nine eukaryotic genes (6), the only transcription terminator functionally identified so far is the one existing at the 3′‐end region of the human gastrin gene (7 − 9). Here we report that the application of the transcriptional terminator element of the human gastrin gene to a mammalian expression system enhances the transient expressions of the cloned genes.…”

Linked Lexitropsins and the in Vitro Inhibition of HIV-1 Reverse Transcriptase RNA-Directed DNA Polymerization: A Novel Induced-Fit of 3,5 m-Pyridyl Bisdistamycin to Enzyme-Associated Template-Primer

“…It has been demonstrated in Vitro that these compounds inhibit many DNA-dependent enzymatic functions associated with DNA replication or RNA transcription. Enzymes affected include DNA and RNA polymerases and topoisomerases (Puschendorf & Grunicke, 1969;Zimmer et al, 1971b;Wahnert et al, 1975;Woynarowski et al, 1989;Mortensen et al, 1990;McHugh et al, 1990;Ueno et al, 1992), as well as DNA gyrase and helicase (Bachur et al, 1993;Storl et al, 1993). Recent studies, moreover, have illustrated the ability of these groove-binding drugs to prevent the binding of transcription factors (Gambari et al, 1991;Dorn et al, 1992;Chiang et al, 1994;Welch et al, 1994).…”