The process by which RNA polymerase II elongates RNA chains remains poorly understood. Elongation factor SII is known to be required to maximize readthrongh at intrinsic termination sites in vitro. We found that SII has the additional and unanticipated property of facilitating transcript cleavage by the ternary complex. We first noticed that the addition of SII caused a shortening of transcripts generated by RNA polymerase II at intrinsic termination sites during transcription reactions in which a single NTP was limiting. Truncation of the nascent transcript was subsequently observed using a series of ternary complexes artificially paused after the synthesis of 15-, 18-, 20-, 21-, and 35-nucleotide transcripts. Transcripts as short as 9 or 10 nucleotides were generated in 5-min reactions. All of these shortened RNAs remained in active ternary complexes because they could be chased quantitatively. Continuation of the truncation reaction produced RNAs as short as 4 nucleotides; however, once cleavage had proceeded to within 8 or 9 bases of the 5' end, the resulting transcription complexes could not elongate the RNAs with NTP addition. Transcript cleavage requires a divalent cation, appears to proceed primarily in 2-nucleotide increments, and is inhibited by ~-amanitin. The catalytic site of RNA polymerase II is repositioned after transcript cleavage such that polymerization resumes at the proper location on the template strand. The extent and kinetics of the transcript truncation reaction are affected by both the position at which RNA polymerase is halted and the sequence of the transcript.

The process by which RNA polymerase II elongates RNA chains in vivo, where the template is at least partially in a nucleosomal configuration, remains poorly understood. To approach this question we have partially purified RNA polymerase lI transcription complexes paused early in elongation. These complexes were then used as substrates for chromatin reconstitution. Elongation of the nascent RNA chains on these nucleosomal templates is severely inhibited relative to elongation on naked DNA templates. Elongation on the nucleosomal templates results in a reproducible template-specific pattern of transcripts generated by RNA polymerase pausing. The RNA polymerases are not terminated because the large majority will resume elongation upon the addition of Sarkosyl or 400 mM KCI. The effectiveness of RNA polymerase II pause/termination sites is enhanced by the presence of nucleosomes. For example, a pause site similar in sequence to the c-myc gene exon 1 terminator is used four to seven times more effectively in reconstituted templates. A comparison of elongation on templates bearing phased nucleosomes and on reconstituted templates that show no predominant phasing pattern indicates that the locations of pause sites are not related to the positions of the nucleosomes. Rather, the major determinant of RNA polymerase pausing on the nucleosomal templates appears to be the underlying DNA sequence.[Key Words: RNA polymerase II; nucleosome; chromatin] Received November 8, 1990; revised version accepted February 5, 1991.A number of studies have indicated that transcriptionally active regions may continue to be packaged in nucleosomes while transcription occurs (Nacheva et al. 1989;Chen et al. 1990;Ericsson et al. 1990;Pederson and Morse 1990). This raises the question of how RNA polymerase II can effectively elongate RNA chains on these nucleoprotein templates. Studies with model systems using bacteriophage RNA polymerases and reconstituted templates indicate that nucleosomes need not be insurmountable barriers to RNA synthesis. These single-subunit RNA polymerases can efficiently elongate on both short fragments bearing one or two nucleosomes (Lorch et al. 1987;Losa and Brown 1987;Morse 1989) and on templates with extended arrays of nucleosomes (Pfaffle et al. 1990). RNA polymerase II will efficiently traverse a single nucleosome (Lorch et al. 1987), but the effect of long nucleosomal arrays on polymerase II elongation has not been established.The necessity for approaching this question biochem-ically has been emphasized by the growing realization that control of elongation by RNA polymerase II is an important aspect of gene regulation. Premature termination or pausing by RNA polymerase II has been observed in vivo for a number of genes, such as the proto-oncogenes c-myc and c-myb, the adenosine deaminase gene, and the hsp70 gene of Drosophila melanogaster (for review, see Spencer and Groudine 1990 and references therein). In vitro systems have been employed that reproduce some of the transcriptional pausing/termination events...

In the presence of elongation factor SII, arrested RNA polymerase II ternary complexes cleave 7-17 nucleotides from the 3'-ends of their nascent RNAs. It has been shown that transcription of linear templates generates apparent run-off RNAs, which are nevertheless truncated upon incubation with SII. By using high resolution gels, we demonstrate that transcription of blunt or 3'-overhung templates with RNA polymerase II generates two populations of ternary complexes. The first class pauses 5-10 bases prior to the end of the template strand. These complexes respond to SII by cleaving approximately 9-17 nucleotide RNAs from their 3'-ends and therefore may be termed arrested. A second class of complexes, which fail to respond to SII, transcribe to within 3 bases of the end of the template strand. These complexes appear to have run off the template since they have released their nascent RNAs. Run-off transcription occurs on all types of templates, but it is the predominant reaction on DNAs with 5'-overhung ends. Thus, RNA polymerase II ternary complexes that retain 5-10 bases of contact with the template strand down-stream of the catalytic site become arrested. Further reduction of downstream template contacts can lead to termination. We also show that the addition of Sarkosyl to the elongation reactions significantly changes the pattern of transcriptional arrest near the end of linear templates.

RNA polymerase II may become arrested during transcript elongation, in which case the ternary complex remains intact but further RNA synthesis is blocked. To relieve arrest, the nascent transcript must be cleaved from the 3' end. RNAs of 7-17 nt are liberated and transcription continues from the newly exposed 3' end. Factor SU increases elongation efficiency by strongly stimulating the transcript cleavage reaction. We show here that arrest relief can also occur by the addition of pyrophosphate. This generates the same set of cleavage products as factor SiU, but the fragments produced with pyrophosphate have 5'-triphosphate termini. Thus, the active site of RNA polymerase II, in the presence of pyrophosphate, appears to be capable of cleaving phosphodiester linkages as far as 17 nt upstream of the original site of polymerization, leaving the ternary complex intact and transcriptionanly active.RNA polymerase II may become arrested during transcript elongation, in which case the polymerase remains in ternary complex but cannot continue RNA synthesis. To recover from this condition, cleavage ofthe transcript from the 3' end is necessary (1, 2). Elongation factor SII facilitates this cleavage reaction, which occurs 7-17 nt from the transcript 3' end (1, 2). The 3'-OH generated by transcript cleavage is accessible to the catalytic site of the RNA polymerase and elongation resumes from the point of cleavage (1-7). Elongation-competent ternary complexes that have stopped transcription because an NTP is missing from the reaction mixture (which we refer to as stalled complexes) can also undergo SII-facilitated transcript cleavage, but the products in this case are predominantly dinucleotides (5).The necessity for transcript cleavage in recovery from arrest suggested to us that in the arrested state the catalytic site of the polymerase has lost contact with the 3' end of the transcript. If this were true, then the cleavage reaction can be understood as a means ofproducing a new substrate for chain elongation. Pyrophosphorolysis is the reversal of normal RNA synthesis; NTPs are regenerated by successively rejoining NMPs from the 3' end of the RNA to pyrophosphate (PPi; see refs. 8 and 9). Such a reaction requires access of the catalytic site to the 3' end of the nascent RNA. Thus, we predicted from our initial model that arrested complexes would be unable to undergo pyrophosphorolytic cleavage. However, two groups have recently reported that arrested RNA polymerase II ternary complexes do cleave their nascent transcripts in the presence of PPi (1,6). As the initial cleavage products were not identified in these experiments, it remained unclear whether PPi mediates transcript cleavage in arrested complexes from the 3' end of the transcript or from internal sites as is the case with SII-facilitated cleavage. To address this important issue, we have treated arrested complexes with PPi and directly measured the increment of transcript cleavage by resolution of the released RNA fragments on high-percentage polyacrylamide...