Elongation factor-dependent transcript shortening by template-engaged RNA polymerase II

Abstract

In addition to polynucleotide polymerization, DNA polymerases and bacterial RNA polymerase can also remove nucleotides from the growing end of nucleic acid chains. For DNA polymerases this activity is an important factor in establishing fidelity in DNA synthesis. This report describes a novel in vitro activity of RNA polymerase II whereby it cleaves an RNA chain contained within an active elongation complex. These elongation complexes are arrested at a previously identified, naturally occurring transcriptional pause site in a human gene. The new 3'-end revealed by this cleavage remains associated with an active elongation complex and is capable of being extended by RNA polymerase II. Nascent RNA cleavage is evident after removal of free nucleotides and is dependent upon a divalent metal cation and transcription elongation factor SII. This function of SII could be important in its function as an activator of transcription elongation. It is also possible that the transcript cleavage activity of RNA polymerase II represents a proofreading function of the enzyme.

Fig. 1. Nascent RNA cleavage in the absence of nucleotides is factor dependent

Elongation complexes arrested at a specific site (Ia) in a human histone gene were assembled on a linear DNA template from partially purified rat liver RNA polymerase II and general transcription factors and immunopurified as described under “Materials and Methods.” RNA was pulse labeled with [α-³²P]CTP. Complexes were washed with buffer to remove solutes including unincorporated nucleotides and were made 7 mM in MgCl₂ and 800 μM each in ATP, UTP, GTP, and CTP (lanes 1 and 3). Buffer (lanes 1 and 2) or 2 μg of partially purified SII from bovine brain (lanes 3–5) were added, and complexes were incubated at 28 °C for 30 min. Samples in lanes 1–4 were prepared for electrophoresis. The sample in lane 5 was treated as that in lane 4 except that it was chased with 800 μM each of ATP, UTP, GTP, and CTP for an additional 30 min at 28 °C before stoppage. RNA was analyzed by polyacrylamide gel electrophoresis and autoradiography. Three major RNAs were generated by transcription from pAdTerm-2 (Reines et al., 1989): a runoff RNA of 530 nucleotides (RO) and RNAs resulting from the arrest of RNA polymerase II at site Ia (205 nucleotides) and a site further downstream in the plasmid (325 nucleotides). The major cleavage product is indicated by an asterisk. Arrowheads indicate RNA size markers of 260, 380, 420, and 540 nucleotides synthesized from the plasmid pKK34-121 with E. coli RNA polymerase holoenzyme.

Fig. 2. RNA polymerase II must be in an elongation complex to cleave nascent transcripts

Elongation complexes containing ³²p-labeled runoff RNA (RO), transcript Ia, and the 325-nucleotide RNA were synthesized as described in the legend of Fig. 1. The RNAs were isolated and run on a polyacrylamide gel (lane 1) or mixed with intact, washed elongation complexes (lane 2). This mixture, and washed elongation complexes alone (lanes 3 and 4), were incubated with 7 mM MgCl₂ and 2 μg of partially purified bovine brain SII at 28 °C for 15 min. One reaction (lane 4) also received α-amanitin (1 μg/ml) before incubation. RNA was isolated and analyzed by electrophoresis as described under “Materials and Methods.” Migration positions of RNAs are indicated as described in the legend of Fig. 1.

Fig. 3. Pure calf thymus SII and a divalent metal activate RNA cleavage

Washed elongation complexes were incubated with 36 ng of pure calf thymus SII (+) or buffer (−). Reactions were adjusted to 7 mM in MgCl₂, MnCl₂, or CaCl₂ as indicated and incubated at 28 °C for 30 min. RNA was isolated and separated on a 5% polyacrylamide gel as described under “Materials and Methods.” Migration positions of RNAs are indicated as described for Fig. 1.

Fig. 4. Kinetics of SII-activated transcript cleavage by RNA polymerase II

Washed elongation complexes were incubated with 2 μg of partially purified bovine brain SII and 7 mM MgCl₂. Incubation proceeded at 28 °C for 0, 2, 5, or 60 min (panel A) or 0, 20, 50, 80, 110, and 300 s (panel B). One sample (panel A, lane 1) was made 0.25% (w/v) in sarkosyl before incubation at 28 °C for 30 min. RNA was isolated and analyzed by electrophoresis as described under “Materials and Methods.” Migration positions of RNAs are indicated as described for Fig. 1.

Fig. 5. Ribonucleoside triphosphates do not inhibit nascent transcript cleavage by RNA polymerase II

Washed elongation complexes were incubated with 7 mM MgCl₂ and either buffer (lane 1) or 2 μg of partially purified bovine brain SII (lanes 2–6) for 30 min at 28 °C. Samples also contained 800 μM ATP (lane 3). UTP (lane 4), GTP (lane 5), or CTP (lane 6) or no nucleotide (lanes 1 and 2) during this incubation. Migration positions of RNAs are indicated as described for Fig. 1.

Fig. 6. Model for SII-dependent transcription elongation through histone site Ia possible role of RNA cleavage and DNA unbending

Solid circle, RNA polymerase II; bold parallel lines, DNA duplex; thin solid line, RNA transcript; Ia, transcription arrest site Ia. See “Discussion” for explanation.