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In Vitro Analysis of a Transcription Termination Site for RNA Polymerase II DEBRA K

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In Vitro Analysis of a Transcription Termination Site for RNA Polymerase II DEBRA K MOLECULAR AND CELLULAR BIOLOGY, Nov. 1990, p. 5782-5795 Vol. 10, No. 11 0270-7306/90/115782-14$02.00/0 Copyright © 1990, American Society for Microbiology In Vitro Analysis of a Transcription Termination Site for RNA Polymerase II DEBRA K. WIEST' 2 AND DIANE K. HAWLEY' 3* Institute of Molecular Biology' and Departments ofBiology2 and Chemistry, University of Oregon, Eugene, Oregon 97403 Received 11 June 1990/Accepted 8 August 1990 Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination. Regulation of transcription is known to be an important tion system to study the mechanism of transcription termi- mechanism for altering gene expression during differentia- nation in vitro downstream of the adenovirus major late tion and development in eucaryotic cells. Much of this (ML) promoter. In vivo, transcription from the ML pro- regulation occurs at the level of initiation of transcription by moter pauses or terminates within intron 1 of the major late RNA polymerase II (pol II), the nuclear enzyme that tran- transcription unit at late but not early times during adenovi- scribes protein-encoding genes. Recently, research in a rus infection (29, 32). In vitro, extended pausing or termina- number of laboratories has demonstrated that pol II is also tion at this site is not observed under standard reaction subject to regulation after initiation by selective arrest of conditions when the ML promoter is transcribed in nuclear transcription elongation at specific sites within genes (re- extracts prepared from uninfected human cells. However, in viewed in reference 50). Perhaps the best evidence for such the of more than the regulation has been obtained through the study of expression presence Sarkosyl, 80% of transcripts of the c-myc gene in humans and mice. In those systems, initiated at the ML promoter indefinitely paused or termi- changes in the ability of transcription elongation to proceed nated at this site (16). past the 3' end of exon 1 have been shown to correlate with The finding that Sarkosyl promoted termination at a spe- changes in c-myc expression that occur during differentiation cific site downstream ofthe ML promoter was an outcome of and with the abnormal expression of c-myc in some Burkitt's experiments originally designed to test the usefulness of lymphoma cells (reviewed by C. A. Spencer and M. Grou- Sarkosyl as a means of blocking transcription initiation (16, dine, Adv. Cancer Res., in press). Evidence for regulated 17). Sarkosyl had previously been shown to prevent initia- blocks to transcription elongation has also been obtained for tion but to allow elongation by purified pol II in vitro at other cellular genes, including the mouse c-myb, hamster concentrations that dissociated most DNA-binding proteins c-fos, Drosophila hsp70, and human and mouse adenosine (14). Consistent with that observation, Sarkosyl was found deaminase genes and viral genes encoded by simian virus 40, to inhibit pol II transcription initiation in a nuclear extract polyomavirus, human adenovirus, minute virus of mice, and system as well but not to inhibit elongation generally (16). human immunodeficiency virus (2, 7, 11, 13, 18, 22, 27, 29, These initial observations have now been confirmed and 38, 41, 48). extended by a number ofgroups studying initiation in vitro at To date, most of the studies of potential attenuation sites the ML promoter, as well as at other class II promoters, and for pol II transcription have relied primarily on nuclear the usefulness of this approach for limiting transcription by run-on assays and isolation of prematurely terminated or pol II to a single round of initiations is well documented (4, processed transcripts from injected oocytes or virus-infected 5, 8, 15, 28, 47). Moreover, other groups have now demon- cells. Neither approach is amenable to detailed mechanistic strated Sarkosyl-dependent termination or pausing by pol II studies. We used a cell-free, promoter-dependent transcrip- at other sites corresponding to transcriptional attenuation sites in vivo (37, 38, 40). The method may, therefore, have a general usefulness in characterizing the elongation reaction * Corresponding author. as well. However, it remains to be determined whether the 5782 VOL. 10, 1990 TERMINATION IN VITRO BY RNA POLYMERASE II 5783 mechanism of induction of termination by Sarkosyl is the from pAdTr to the HindIII site ofplasmids pPT+ and pP+T, same at these various sites. respectively. To make plasmids pP+TT.2 and pP+T+T, we Because it is an unusually strong promoter in vitro, the first digested pAdT and pAdT+, respectively, with SmaI ML promoter has been studied extensively and was used in and PvuII to generate a DNA fragment containing all the the original isolation and characterization of transcription adenovirus sequences from each plasmid flanked by vector factors (10, 31, 42). For these reasons, and because of the DNA. The DNA fragment was isolated and inserted between high efficiency of termination we have observed, this homol- the promoter and terminator sites in pP+T, using a BamHI ogous system is uniquely suited for the study of the proteins site that had first been filled in with the Klenow fragment of and DNA sequences responsible for termination by pol II in DNA polymerase I to make it compatible with the blunt- vitro. As a first step, we began to define the specific DNA ended restriction fragment. sequences that contribute to termination downstream of the To make the plasmid pMPT+, we first constructed the ML promoter and to test several simple models for the plasmid pM,0, which contains mouse 3-globin DNA from mechanism of Sarkosyl-induced termination. the ClaI site at -110 to the BamHI site at +475 ligated to the AccI and BamHI sites of pTZ18U. These 1-globin sequences MATERIALS AND METHODS were obtained from the plasmid pMH,8 (33; gift from R. Myers, University of California, San Francisco) and con- Plasmid constructions. The plasmid pMLH1, which con- tained an 8-bp BglII linker at +26. To construct pMPT+, tains adenovirus DNA sequences from -260 to +536 with pMIO was restricted at a PstI site immediately upstream of respect to the ML cap site, has been described previously the ,-globin sequences, and the 3' overhanging ends were (17). pMLH3, which was used as a source of promoter- or removed with T4 DNA polymerase. This DNA was then terminator-containing DNA during construction of several of restricted with BglII, and the resulting DNA fragment con- the plasmids described below, contains ML promoter se- taining ,B-globin sequences from -110 to +26 was ligated to quences from -260 to + 198 and was made by ligating a pAdT+ restricted with SmaI and BamHI. PstI-HindIII DNA fragment from pMLH1 to the PstI and Plasmids pPC28T and pPC246T were derivatives of plas- HindIII sites in the vector pTZ19U (U.S. Biochemicals). mids we obtained in making a series of 3' deletions of the The plasmid pPT was made in two steps. First, a DNA G-less cassette sequence in pMLC2AT (45). The construc- fragment containing adenovirus sequences from the HaeIII tion of those plasmids will be presented in greater detail site at + 153 to the HindIII site at + 193 was purified and elsewhere. The parent plasmids used in this study contained ligated to the vector pTZ18U (U.S. Biochemicals) restricted ML promoter DNA from -260 to + 10 followed by 28 or 246 with HincII and HindIII. The resulting plasmid was called bp of the original G-less cassette sequence inserted into the pAdT. Next, a 337-base-pair (bp) fragment was purified after vector pTZ18U. A HindIII fragment containing adenovirus digestion of pMLH3 with EcoRI and PvuII. The EcoRI site sequences from pAdTr was ligated to each parent plasmid at was located upstream of adenovirus sequences, and the a HindIII site located 4 bp downstream of the end of the PvuII site was at +33 with respect to the ML cap site. This G-less cassette. fragment was ligated to pAdT restricted with EcoRI and The dideoxy-chain termination DNA-sequencing method SmaI to create pPT. To make the plasmid pP+T, we of Sanger et al.
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